Purification and Substrate Specificities of Two α-l-Arabinofuranosidases from Aspergillus awamori IFO 4033

α-l-Arabinofuranosidases I and II were purified from the culture filtrate of Aspergillus awamori IFO 4033 and had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum temperature of 60°C and exhibited stability at pH values from 3 to 7 and at temperatures up to 60°C. The enzymes released arabinose from p-nitrophenyl-α-l-arabinofuranoside, O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose, and arabinose-containing polysaccharides but not from O-β-d-xylopyranosyl-(1→2)-O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. α-l-Arabinofuranosidase I also released arabinose from O-β-d-xylopy-ranosyl-(1→4)-[O-α-l-arabinofuranosyl-(1→3)]-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. However, α-l-arabinofuranosidase II did not readily catalyze this hydrolysis reaction. α-l-Arabinofuranosidase I hydrolyzed all linkages that can occur between two α-l-arabinofuranosyl residues in the following order: (1→5) linkage > (1→3) linkage > (1→2) linkage. α-l-Arabinofuranosidase II hydrolyzed the linkages in the following order: (1→5) linkage > (1→2) linkage > (1→3) linkage. α-l-Arabinofuranosidase I preferentially hydrolyzed the (1→5) linkage of branched arabinotrisaccharide. On the other hand, α-l-arabinofuranosidase II preferentially hydrolyzed the (1→3) linkage in the same substrate. α-l-Arabinofuranosidase I released arabinose from the nonreducing terminus of arabinan, whereas α-l-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan.Recently, it has been proven that l-arabinose selectively inhibits intestinal sucrase in a noncompetitive manner and reduces the glycemic response after sucrose ingestion in animals (33). Based on this observation, l-arabinose can be used as a physiologically functional sugar that inhibits sucrose digestion. Effective l-arabinose production is therefore important in the food industry. l-Arabinosyl residues are widely distributed in hemicelluloses, such as arabinan, arabinoxylan, gum arabic, and arabinogalactan, and the α-l-arabinofuranosidases (α-l-AFases) (EC 3.2.1.55) have proven to be essential tools for enzymatic degradation of hemicelluloses and structural studies of these compounds.α-l-AFases have been classified into two families of glycanases (families 51 and 54) on the basis of amino acid sequence similarities (11). The two families of α-l-AFases also differ in substrate specificity for arabinose-containing polysaccharides. Beldman et al. summarized the α-l-AFase classification based on substrate specificities (3). One group contains the Arafur A (family 51) enzymes, which exhibit very little or no activity with arabinose-containing polysaccharides. The other group contains the Arafur B (family 54) enzymes, which cleave arabinosyl side chains from polymers. However, this classification is too broad to define the substrate specificities of α-l-AFases. There have been many studies of the α-l-AFases (3, 12), especially the α-l-AFases of Aspergillus species (2–8, 12–15, 17, 22, 23, 28–32, 36–39, 41–43, 46). However, there have been only a few studies of the precise specificities of these α-l-AFases. In previous work, we elucidated the substrate specificities of α-l-AFases from Aspergillus niger 5-16 (17) and Bacillus subtilis 3-6 (16, 18), which should be classified in the Arafur A group and exhibit activity with arabinoxylooligosaccharides, synthetic methyl 2-O-, 3-O-, and 5-O-arabinofuranosyl-α-l-arabinofuranosides (arabinofuranobiosides) (20), and methyl 3,5-di-O-α-l-arabinofuranosyl-α-l-arabinofuranoside (arabinofuranotrioside) (19).In the present work, we purified two α-l-AFases from a culture filtrate of Aspergillus awamori IFO 4033 and determined the substrate specificities of these α-l-AFases by using arabinose-containing polysaccharides and the core oligosaccharides of arabinoxylan and arabinan.     

Gene Cloning,Nucleotide Sequencing,and Purification and Characterization of the Low-Specificity l-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictly l specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, including l-β-3,4-dihydroxyphenylserine, l-β-3,4-methylenedioxyphenylserine, and l-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity l-TA from Saccharomyces cerevisiae, l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the l-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.β-Hydroxy-α-amino acids constitute an important class of compounds. They are natural products in their own right and are components of a range of antibiotics, for example, cyclosporin A, lysobactin, and vancomycin (30) and bouvardin and deoxybouvardin (6). 4-Hydroxy-l-threonine is a precursor of rizobitoxine, a potent inhibitor of pyridoxal 5′-phosphate (PLP)-dependent enzymes (32). 3,4,5-Trihydroxyl-l-aminopentanoic acid is a key component of polyoxins (32). l-threo-3,4-Dihydroxyphenylserine is a new drug for Parkinson’s disease therapy (13). However, the industrial production of β-hydroxy-α-amino acids has been limited to chemical synthesis processes, which need multiple steps to isolate the four isomers (l-threo form, d-threo form, l-erythro form, and d-erythro form). Threonine aldolase (EC 4.1.2.5), which stereospecifically catalyzes the retro-aldol cleavage of threonine, is a potentially useful catalyst for the synthesis of substituted amino acids from aldehyde and glycine (27, 31, 32).Two different types of threonine aldolases are known so far. l-allo-Threonine aldolase (l-allo-TA), isolated and purified from Aeromonas jandaei DK-39 (8), stereospecifically catalyzes the reversible interconversion of l-allo-threonine and glycine. Low-specificity l-threonine aldolase (l-TA) catalyzes the cleavage of both l-threonine and l-allo-threonine to glycine and acetaldehyde, as well as the reverse reaction, aldol condensation. The enzymes have been purified and characterized from Candida humicola (9, 34) and Saccharomyces cerevisiae (12). Low-specificity l-TA activity has also been shown to exist in mammals (7, 23, 26) and a variety of other microbial species (2, 4, 17, 35). The enzyme is physiologically important for the synthesis of cellular glycine in yeast (12, 15, 16). Threonine aldolases with distinct stereospecificities are ideal targets for enzymology studies on structural and functional relationships. However, information on the primary structures of threonine aldolases was limited to our recent studies (11, 12). The construction of an overproduction system for threonine aldolase will be indispensable for the industrial biosyntheses of β-hydroxy-α-amino acids.The present work focuses on the cloning, sequencing, and overexpression in Escherichia coli cells of the low-specificity l-TA gene from Pseudomonas sp. strain NCIMB 10558, the purification and characterization of the recombinant enzyme, and the identification of the active-site lysine residue of the enzyme by site-directed mutagenesis. Evidence is presented that Lys207 of low-specificity l-TA probably functions as a catalytic residue, forming an internal Schiff base with the PLP of the enzyme to catalyze the reversible aldol reaction. This is the first report showing a purified enzyme with l-β-3,4-dihydroxyphenylserine aldolase and l-β-3,4-methylenedioxyphenylserine aldolase activities, providing a new route for the industrial production of these important unnatural amino acids.     

Identification of Galacturonic Acid-1-phosphate Kinase, a New Member of the GHMP Kinase Superfamily in Plants, and Comparison with Galactose-1-phosphate Kinase

The process of salvaging sugars released from extracellular matrix, during plant cell growth and development, is not well understood, and many molecular components remain to be identified. Here we identify and functionally characterize a unique Arabidopsis gene encoding an α-d-galacturonic acid-1-phosphate kinase (GalAK) and compare it with galactokinase. The GalAK gene appeared to be expressed in all tissues implicating that glycose salvage is a common catabolic pathway. GalAK catalyzes the ATP-dependent conversion of α-d-galacturonic acid (d-GalA) to α-d-galacturonic acid-1-phosphate (GalA-1-P). This sugar phosphate is then converted to UDP-GalA by a UDP-sugar pyrophosphorylase as determined by a real-time ¹H NMR-based assay. GalAK is a distinct member of the GHMP kinase family that includes galactokinase (G), homoserine kinase (H), mevalonate kinase (M), and phosphomevalonate kinase (P). Although these kinases have conserved motifs for sugar binding, nucleotide binding, and catalysis, they do have subtle difference. For example, GalAK has an additional domain near the sugar-binding motif. Using site-directed mutagenesis we established that mutation in A368S reduces phosphorylation activity by 40%; A41E mutation completely abolishes GalAK activity; Y250F alters sugar specificity and allows phosphorylation of d-glucuronic acid, the 4-epimer of GalA. Unlike many plant genes that undergo duplication, GalAK occurs as a single copy gene in vascular plants. We suggest that GalAK generates GalA-1-P from the salvaged GalA that is released during growth-dependent cell wall restructuring, or from storage tissue. The GalA-1-P itself is then available for use in the formation of UDP-GalA required for glycan synthesis.d-Galacturonic acid (d-GalA)³ is a quantitatively major glycose present in numerous plant polysaccharides including pectins and arabinogalactan proteins (1, 2). The synthesis of these polysaccharides requires a large number of glycosyltransferases and diverse nucleotide-sugar (NDP-sugar) donors (1, 3). Some of these NDP-sugars are formed by interconversion of pre-existing NDP-sugars and by salvage pathways. For example, the main pathway for UDP-GalA formation is the 4-epimerization of UDP-GlcA, a reaction catalyzed by UDP-GlcA 4-epimerase (4–6). However, in ripening Fragaria fruit d-GalA is incorporated into pectin (7). It is likely that a sugar kinase converts the d-GalA to GalA-1-P (8), which is then converted to UDP-GalA by a nonspecific UDP-sugar pyrophosphorylase (9). Myo-inositol may also be a source of GalA for polysaccharide biosynthesis (10).Galacturonic acid is likely to be generated by enzyme-catalyzed hydrolysis of pectic polysaccharides in plant tissues. Polysaccharide hydrolase activities are present in germinating seeds (11, 12), in germinating and elongating pollen (13–15), and in ripening fruit (14). Thus, monosaccharide salvage pathways may be required for normal plant growth and development.Numerous sugar-1-P kinases, including d-Gal-1-P kinase (16), l-Ara-1-P kinase (17), and l-Fuc-1-P kinase (18), have been described (19), but no d-GalA-1-P kinase has been identified in any species to account for the hydrolysis and recycle of pectic polymers. The subsequent pyrophosphorylation of UDP-sugars could be carried out by UDP-sugar pyrophosphorylases (20).Here, we report the identification and characterization of a functional galacturonic acid kinase (GalAK). We compared the activity of GalAK with a previously uncharacterized Arabidopsis GalK and discussed the evolution of these sugar kinase members of the GHMP kinase.     

Role of the gerI Operon of Bacillus cereus 569 in the Response of Spores to Germinants

Bacillus cereus 569 (ATCC 10876) germinates in response to inosine or to l-alanine, but the most rapid germination response is elicited by a combination of these germinants. Mutants defective in their germination response to either inosine or to l-alanine were isolated after Tn917-LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to l-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to l-alanine. These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination. Stimulation of germination in l-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of l-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci. Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome. This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the gerA and related operons of Bacillus subtilis. The individual genes of this gerI operon have been named gerIA, gerIB, and gerIC. The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.Bacillus species have the ability, under certain nutrient stresses, to undergo a complex differentiation process resulting in the formation of a highly resistant dormant endospore (6). These spores can then persist in the environment for prolonged periods until a sensitive response mechanism detects specific environmental conditions, initiating the processes of germination and outgrowth (9, 21, 25). Germination can be initiated by a variety of agents (12), including nutrients, enzymes, or physical factors, such as abrasion or hydrostatic pressure.The molecular genetics of spore germination has been most extensively studied in Bacillus subtilis 168 (21). B. subtilis spores can be triggered to germinate in response to either l-alanine or to a combination (29) of asparagine, glucose, fructose, and potassium ions (AGFK). Mutants of B. subtilis which are defective in germination responses to one or to both types of germinant have been isolated previously (20, 27). Analysis of these mutants suggests that the germinants interact with separate germinant-specific complexes within the spore (21). This in some way leads to activation of components of the germination apparatus common to both responses, such as germination-specific cortex lytic enzymes, leading in turn to complete germination of the spore (10, 22). The mutations within the gerA operon of B. subtilis specifically block germination initiated by l-alanine (34). The predicted amino acid sequences of the three GerA proteins encoded in the operon suggest that these proteins could be membrane associated, and they are the most likely candidates to represent the germinant receptor for alanine (21).The amino acid l-alanine has been identified as a common but not universal germinant in a variety of Bacillus species, often requiring the presence of adjuncts such as electrolytes and sugars. Ribosides, such as inosine, represent another type of common germinant, although many species are unable to germinate rapidly in response to these without the addition of l-alanine (9).The food-borne pathogen Bacillus cereus is a major cause of food poisoning of an emetic and diarrheal type (13, 16). The germination and growth of Bacillus cereus spores during food storage can lead to food spoilage and the potential to cause food poisoning (16). B. cereus has been shown to germinate in response to l-alanine and to ribosides (11, 18, 23). Spore germination can be triggered by l-alanine alone, but at high spore densities this response becomes inhibited by d-alanine, generated by the alanine racemase activity associated with the spores (8, 11). This auto-inhibition of l-alanine germination can be reduced by the inclusion of a racemase inhibitor (O-carbamyl-d-serine) with the germinating spores (11).Inosine is the most effective riboside germinant for B. cereus T, while adenosine and guanosine are less potent (28). The rate of riboside-triggered germination has been reported to be enhanced dramatically by the addition of l-alanine (18). It is unclear whether ribosides can act as a sole germinant, or whether there is an absolute requirement for l-alanine (28).An attempt has been made to analyze genetically the molecular components of the germination apparatus in B. cereus in order to dissect the germination responses of this species and to determine whether riboside-induced germination involves components related to those already described for amino acid and sugar germinants in B. subtilis.     

Purification and Characterization of a Trehalose Synthase from the Basidiomycete Grifola frondosa

A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from d-glucose and α-d-glucose 1-phosphate (α-d-glucose 1-P) was detected in a basidiomycete, Grifola frondosa. TSase was purified 106-fold to homogeneity with 36% recovery by ammonium sulfate precipitation and several steps of column chromatography. The native enzyme appears to be a dimer since it has apparent molecular masses of 120 kDa, as determined by gel filtration column chromatography, and 60 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to d-glucose and α-d-glucose 1-P, in addition to the synthesis of trehalose from the two substrates, the TSase equilibrium strongly favors trehalose synthesis. The optimum temperatures for phosphorolysis and synthesis of trehalose were 32.5 to 35°C and 35 to 37.5°C, respectively. The optimum pHs for these reactions were 6.5 and 6.5 to 6.8, respectively. The substrate specificity of TSase was very strict: among eight disaccharides examined, only trehalose was phosphorolyzed, and only α-d-glucose 1-P served as a donor substrate with d-glucose as the acceptor in trehalose synthesis. Two efficient enzymatic systems for the synthesis of trehalose from sucrose were identified. In system I, the α-d-glucose 1-P liberated by 1.05 U of sucrose phosphorylase was linked with d-glucose by 1.05 U of TSase, generating trehalose at the initial synthesis rate of 18 mmol/h in a final yield of 90 mol% under optimum conditions (300 mM each sucrose and glucose, 20 mM inorganic phosphate, 37.5°C, and pH 6.5). In system II, we added 1.05 U of glucose isomerase and 20 mM MgSO₄ to the reaction mixture of system I to convert fructose, a by-product of the sucrose phosphorylase reaction, into glucose. This system generated trehalose at the synthesis rate of 4.5 mmol/h in the same final yield.Trehalose (1-α-d-glucopyranosyl-α-d-glucopyranoside) is a nonreducing disaccharide with an α,α-1,1 glycosidic linkage and is widely distributed in plants, insects, fungi, yeast, and bacteria (7). Due to the absence of reducing ends in trehalose, it is highly resistant to heat, pH, and Maillard’s reaction (24). In trehalose-producing organisms, this compound may serve as an energy reserve, a buffer against stresses such as desiccation and freezing, and a protein stabilizer (5, 7, 26, 31, 32). If trehalose can be produced economically, then it has potential commercial applications as a sweetener, a food stabilizer, and an additive in cosmetics and pharmaceuticals (6, 25). Recently, trehalose production through fermentation of yeast (17) and Corynebacterium (30), enzymatic processes from starch (18, 34) and maltose (19, 22, 23, 33), and extraction from transformed plants (10) has been reported.Our approach to trehalose production is to use an enzymatic process to produce trehalose from sucrose, one of the least expensive sugars. Since sucrose is efficiently converted to α-d-glucose 1-phosphate (α-d-glucose 1-P) and fructose by sucrose phosphorylase (SPase), we screened microorganisms for an enzyme that converts α-d-glucose 1-P to trehalose on the assumption that the combination of the putative trehalose synthase (TSase) and SPase would convert sucrose into trehalose. Although similar enzyme activities have been reported in the basidiomycete Flammulina velutipes (11) and in the yeast Pichia fermentans (27), these enzymes have not been well characterized.Our objectives were (i) to screen microorganisms, primarily fungi, for TSase activity; (ii) to purify and characterize the TSase; (iii) to identify the enzymatic process by which trehalose is produced from sucrose; and (iv) to identify an enzymatic process for production of trehalose from sucrose in which the fructose component is also converted to trehalose.     

Purification and Characterization of l-Methionine γ-Lyase from Brevibacterium linens BL2

l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase from Pseudomonas putida. Its K_m for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min⁻¹ mg⁻¹. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

Methanethiol is associated with desirable Cheddar-type sulfur notes in good-quality Cheddar cheese (2, 27). The mechanism for the production of methanethiol in cheese is unknown, but it is linked to the catabolism of methionine (1, 15). l-Methionine γ-lyase (EC 4.4.1.11; MGL), also known as methionase, l-methionine γ-demethiolase, and l-methionine methanethiollyase (deaminating), is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the direct conversion of l-methionine to α-ketobutyrate, methanethiol, and ammonia by an α,γ-elimination reaction (26). It does not catalyze the conversion of d enantiomers (24–26). MGL in Pseudomonas putida is a multifunctional enzyme system since it catalyzes the α,γ- and α,β-elimination reactions of methionine and its derivatives (24). In addition, the enzyme also catalyzes the β-replacement reactions of sulfur amino acids (24). Since its discovery in Escherichia coli and Proteus vulgaris by Onitake (19), this enzyme has been found in various bacteria and is regarded as a key enzyme in the bacterial metabolism of methionine. However, this enzyme has not been purified to homogeneity from any food-grade microorganisms.MGL is widely distributed in bacteria, especially in pseudomonads, and is induced by the addition of l-methionine to the culture medium (9, 28). The enzyme has been purified from Pseudomonas putida (25), Aeromonas sp. (26), Clostridium sporogenes (11), and Trichomonas vaginalis (16) and partially purified from and characterized for Brevibacterium linens NCDO 739 (4).B. linens is a nonmotile, non-spore-forming, non-acid-fast, gram-positive coryneform bacterium normally found on the surfaces of Limburger and other Trappist-type cheeses. This organism tolerates salt concentrations ranging between 8 and 20% and is capable of growing in a broad pH range from 5.5 to 9.5, with an optimum pH of 7.0 (20). In Trappist-type cheeses, brevibacteria depend on Saccharomyces cerevisiae to metabolize lactate, which increases the pH of the curd, as well as to produce growth factors that are important for their growth (20). Interest in B. linens has focused around its ability to produce an extracellular protease, which has recently been isolated (21), and its ability to produce high levels of methanethiol (3, 9, 10, 22).B. linens produces various sulfur compounds, including methanethiol, that are thought to be important in Cheddar-like flavor and aroma (3, 9, 10, 22). Ferchichi et al. (9) suggested that MGL is responsible for the methanethiol-producing capability of B. linens but did not provide definitive evidence. Weimer et al. (28) proposed that B. linens BL2 is responsible for Cheddar-type flavor development in low-fat cheese, but again conclusive evidence was lacking. In this study, MGL was purified to homogeneity from B. linens BL2 and its physical and chemical properties were examined.     

Crystal Structure of a Homolog of Mammalian Serine Racemase from Schizosaccharomyces pombe

d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5′-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. The enzyme also catalyzes the dehydration of d- and l-serine. Both reactions are enhanced by Mg·ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 Å resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with l-serine and d-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique “lysino-d-alanyl” residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme.d-Serine, which is present at a high level in the mammalian brain, serves as an endogenous coagonist for the N-methyl-d-aspartate (NMDA)⁵ receptor selectively localized on the postsynaptic membrane of the excitatory synapse (1–5) and is involved in excitatory neurotransmission and higher brain functions such as learning and memory (3, 6, 7). Stimulation of the NMDA receptor requires the binding of d-serine as well as the agonist l-glutamate. The major enzyme for d-serine synthesis from l-serine in the brain is considered to be pyridoxal 5′-phosphate (PLP)-dependent serine racemase (SR) (8–10). d-Serine and SR are localized on protoplasmic astrocytes that have the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor. Glutamate released from presynaptic neurons approaches and activates the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, which in turn induces SR to produce d-serine and is followed by d-serine release from astrocytes that act on the NMDA receptor. Recently, it was shown that not only glia but also neurons synthesize and release d-serine involved in signaling (11). SR also catalyzes α,β-elimination of water from d- or l-serine to form pyruvate and ammonia as well as the conversion of l-serine into d-serine and vice versa and is presumed to link d-serine synthesis and energy metabolism of astrocytes (12) and to control the d-serine level (13). Mg·ATP, which is fully bound to SR under physiological conditions, stimulates racemization and the α,β-elimination reaction catalyzed by SR (12, 14).SR was first discovered in pupae of the silkworm Bombyx mori (15), which was followed by purification of the enzyme from a rat brain and cloning of the mouse and human genes (8, 9). The primary structure of mammalian SR is distinct from those of racemases from prokaryotes but is similar to those of fold-type II PLP-dependent enzymes (16–18). We have cloned and expressed the Schizosaccharomyces pombe gene homologous to human and mouse SRs, the sequence identities being 35.1 and 37.4%, respectively, in Escherichia coli. The protein product is a bifunctional enzyme that catalyzes racemization and the α,β-elimination reaction of D, l-serine as mammalian SR does. SR from S. pombe (spSR) comprises 322 residues (the N-terminal Met is removed in the purified enzyme) and one PLP per subunit, the subunit molecular weight being 34,917. The mammalian SR homolog, spSR, is an interesting target enzyme for the development of a novel therapeutic compound controlling the d-serine level because d-serine is the product of an SR-catalyzed reaction. In our recent report, the active site of spSR was shown to be modified with its natural substrate serine by mass spectroscopic and x-ray studies (19). Interestingly, the catalytic lysine, which originally forms a Schiff base with PLP, is converted to a lysino-d-alanyl residue through the reaction with the substrate, serine (Fig. 1). The modified enzyme exhibits racemase (54% of the wild-type enzyme) and α,β-elimination (68% of the wild-type enzyme) activities with the amino group of the d-alanyl moiety of the lysinoalanyl residue forming a Schiff base with PLP in place of the lysine (19). In addition, the mammalian SR seems to be possibly modified to have a lysinoalanyl residue at the active site, as observed in spSR (20).Open in a separate windowFIGURE 1.Covalent modification of the active site. The catalytic Lys-57 in spSRw is converted to lysino-d-alanyl residue. The α-amino group (indicated with “α”) of the d-alanyl moiety in the residue acts as a catalytic base in spSRm. The circled P is a phosphate group.Although the structure of modified spSR (spSRm) has been determined (19), the structure-function relationship of essential wild-type spSR (spSRw), the binding mode of activator Mg·ATP, the catalytic base to shuttle protons to the substrate d-serine, and the substrate recognition of the modified enzyme have not yet been uncovered. We now report the three-dimensional structures of unliganded spSRw in the open form, spSRw·AMP-PCP in the open form, and spSRm·serine in the closed form.     

Roles of Low-Molecular-Weight Penicillin-Binding Proteins in Bacillus subtilis Spore Peptidoglycan Synthesis and Spore Properties

The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by d,d-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparent d,d-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.Peptidoglycan (PG) is the structural element of the bacterial cell wall which determines cell shape and which resists the turgor pressure within the cell. The bacterial endospores produced by species of Bacillus, Clostridium, and several other bacterial genera are modified cells that are able to survive long periods and extreme conditions in a dormant, relatively dehydrated state. The PG wall within the endospore is required for maintenance of the dehydrated state (10, 11), which is the major determinant of spore heat resistance (2, 17, 22). Spore PG appears to be comprised of two distinct though contiguous layers. The thin inner layer, the germ cell wall, appears to have a structure similar to that of the vegetative wall and serves as the initial cell wall of the germinated spore (1, 20, 21, 31). The thicker outer layer, the spore cortex, has a modified structure which may determine its ability to carry out roles specific to the spore, and is rapidly degraded during spore germination (1, 20, 35, 37). The most dramatic of the cortex structural modifications results in partial cleavage or complete removal of ∼75% of the peptide side chains from the glycan strands. Loss of these peptides limits the cross-linking potential of the PG and results in the formation of only one peptide cross-link per 35 disaccharide units in the spore PG, compared to one peptide cross-link per 2.3 to 2.9 disaccharide units in the vegetative PG (1, 20, 36). This low degree of cross-linking has been predicted to give spore PG a flexibility that allows it to have a role in attainment of spore core dehydration (14, 34) in addition to its clear role in maintenance of dehydration. We are studying the structure and mechanism of synthesis of spore PG in an attempt to discern the roles of this structure and its individual components in determining spore properties.A family of proteins called the penicillin-binding proteins (PBPs) polymerizes PG on the external surface of the cell membrane (reviewed in reference 7). The high-molecular-weight (high-MW) members of this family (generally ≥60 kDa) carry the transglycosylase and transpeptidase activities involved in polymerization and cross-linking of the glycan strands. The low-MW PBPs have commonly been found to possess d,d-carboxypeptidase activity. This activity can remove the terminal d-alanine of the peptide side chains and thereby prevent the side chain from serving as a donor in the formation of a peptide cross-link. Analysis of the B. subtilis genome reveals six low-MW PBP-encoding genes: dacA (33), dacB (4), dacC (19), dacF (38), pbpE (23), and pbpX (accession no. {"type":"entrez-nucleotide","attrs":{"text":"Z99112","term_id":"32468745","term_text":"Z99112"}}Z99112). The four dac gene products exhibit very high sequence similarity to proven d,d-carboxypeptidases, and this activity has been demonstrated in vitro for the dacA and dacB products, PBP5 (12) and PBP5* (32), respectively. The sequences of the pbpE and pbpX products are more distantly related, and no activity has yet been established or ruled out for them.PBP5 is the major penicillin-binding and d,d-carboxypeptidase activity found in vegetative cells (12). Although dacA expression declines significantly during sporulation, a significant amount of PBP5 remains during the time of spore PG synthesis (29). A dacA-null mutation results in no obvious effects on vegetative growth, sporulation, spore characteristics, or spore germination (3, 33). However, loss of PBP5 does result in a reduction of cleavage of peptide side chains from the tetrapeptide to the tripeptide form in the spore PG (20). PBP5* is expressed only during sporulation and only in the mother cell compartment of the sporangium, under the control of the RNA polymerase ς^E subunit (4, 5, 28, 29). A dacB-null mutation leading to loss of this d,d-carboxypeptidase results in a fourfold increase in the effective cross-linking of the spore PG (1, 20, 22). This structural change is accompanied by only slight decreases in spore core dehydration and heat resistance (3, 22). The suspected d,d-carboxypeptidase activities of the products of the dacC and dacF genes have not been demonstrated. The latter two genes are expressed only during the postexponential growth phase: dacC is expressed during early stationary phase under the control of ς^H (19) and dacF is expressed only within the forespore under the control of ς^F (27, 38). Null mutations effecting either gene result in no obvious phenotype and no change in spore PG structure (19, 38).The multiplicity of these proteins in sporulating cells and the lack of effect of loss of some of them suggested redundancy of function among these proteins, a situation observed previously with PBPs of a high-MW class (25, 30, 39). In order to examine this possibility we have constructed mutants lacking multiple low-MW PBPs and have examined their sporulation efficiency, spore PG structure, spore heat resistance and wet density, and spore germination and outgrowth. The present study demonstrates a role for the dacF gene product in synthesis of spore PG, and we also present a model for the roles of the dacB and dacF gene products in spore PG formation.     

Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms and Escherichia coli

Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities of Bacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified as Aeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.Indicators of pollution (e.g., coliforms, fecal coliforms, and Escherichia coli) are traditionally used for monitoring the microbiological safety of water supplies and recreational water. Several techniques for detection of coliforms and E. coli are based on enzymatic hydrolysis of fluorogenic or chromogenic substrates for β-d-galactosidase and β-d-glucuronidase (9, 20). Current methods of recovery are usually culture based, and the analysis time is 18 to 24 h. In addition to enzymatic activity, these techniques use growth at appropriate temperatures in the presence of inhibitors, combined with demonstration of enzymatic activity, to selectively detect target bacteria.Rapid methods which require less than 6 h and are based on chromogenic, fluorogenic, or chemiluminogenic substrates for detection of coliforms, fecal coliforms, or E. coli have been described (1–3, 10, 27, 28). These rapid assays are based on the assumption that β-d-galactosidase and β-d-glucuronidase are markers for coliforms and E. coli, respectively. However, when the incubation time is 1 h or less, growth is not a selective step, and all β-d-galactosidase-positive or β-d-glucuronidase-positive microorganisms in a water sample contribute to the activity measured. At low initial concentrations of target bacteria (i.e., E. coli and total coliforms), increasing the preincubation time to 5 to 6 h did not result in a predominance of target bacteria compared to nontarget bacteria (28).The β-d-galactosidase or β-d-glucuronidase activity calculated per cultivable coliform or fecal coliform bacterium in environmental samples can be 1 to 2 log units higher than the activity per induced E. coli cell in pure culture (11, 26). The presence of active, noncultivable bacteria can be one reason for this. Studies of survival (7, 24, 25) and disinfection (26) of E. coli have shown that loss of cultivability does not necessarily result in a loss of β-d-galactosidase activity. The presence of false-positive bacteria can be another reason.β-d-Galactosidase has been found in numerous microorganisms, including gram-negative bacteria (e.g., strains belonging to the Enterobacteriaceae, Vibrionaceae, Pseudomonadaceae, and Neisseriaceae), several gram-positive bacteria, yeasts, protozoa, and fungi (17, 29). β-d-Glucuronidase is produced by most E. coli strains and also by other members of the Enterobacteriaceae, including some Shigella and Salmonella strains and a few Yersinia, Citrobacter, Edwardia, and Hafnia strains. Production of β-d-glucuronidase by Flavobacterium spp., Bacteroides spp., Staphylococcus spp., Streptococcus spp., anaerobic corynebacteria, and Clostridium has also been reported (12).High numbers of false-positive bacteria in sewage and contaminated water have been revealed by enumeration of β-d-galactosidase- and β-d-glucuronidase-positive CFU on nonselective agar supplemented with fluorogenic or chromogenic substrates (11, 28). Whether the activity from nontarget organisms can be neglected in a rapid assay depends on the number of nontarget organisms compared with the number of target bacteria and also on the level of their enzyme activity. Plant and algal biomass must be present at high concentrations to interfere in rapid bacterial β-d-galactosidase and β-d-glucuronidase assays (8).The main objective of this study was to investigate the enzyme characteristics of β-d-galactosidase- and β-d-glucuronidase-positive bacteria isolated from environmental water samples and to evaluate the potential influence of false-positive bacteria in rapid assays for coliform bacteria or E. coli in water. The effect of temperature on enzyme activity and on the interference of nontarget bacteria in the rapid assays was investigated as an important factor.(Some of the results were presented at the 97th General Meeting of the American Society for Microbiology 1997, Miami Beach, Fla., 4 to 8 May 1997.)     

Two d-2-Hydroxy-acid Dehydrogenases in Arabidopsis thaliana with Catalytic Capacities to Participate in the Last Reactions of the Methylglyoxal and ��-Oxidation Pathways

The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae d-lactate dehydrogenase (AtD-LDH). The recombinant protein is a homodimer of 59-kDa subunits with one FAD per monomer. A substrate screen indicated that AtD-LDH catalyzes the oxidation of d- and l-lactate, d-2-hydroxybutyrate, glycerate, and glycolate using cytochrome c as an electron acceptor. AtD-LDH shows a clear preference for d-lactate, with a catalytic efficiency 200- and 2000-fold higher than that for l-lactate and glycolate, respectively, and a K_m value for d-lactate of ∼160 μm. Knock-out mutants showed impaired growth in the presence of d-lactate or methylglyoxal. Collectively, the data indicated that the protein is a d-LDH that participates in planta in the methylglyoxal pathway. Web-based bioinformatic tools revealed the existence of a paralogous protein encoded by locus At4g36400. The recombinant protein is a homodimer of 61-kDa subunits with one FAD per monomer. A substrate screening revealed highly specific d-2-hydroxyglutarate (d-2HG) conversion in the presence of an organic cofactor with a K_m value of ∼580 μm. Thus, the enzyme was characterized as a d-2HG dehydrogenase (AtD-2HGDH). Analysis of knock-out mutants demonstrated that AtD-2HGDH is responsible for the total d-2HGDH activity present in A. thaliana. Gene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in β-oxidation and degradation of branched-chain amino acids and chlorophyll. It is proposed that AtD-2HGDH participates in the catabolism of d-2HG most probably during the mobilization of alternative substrates from proteolysis and/or lipid degradation.l- and d-lactate dehydrogenases belong to evolutionarily unrelated enzyme families (1). l-Lactate is oxidized by l-lactate:NAD oxidoreductase (EC 1.1.1.27), which catalyzes the reaction l-lactate + NAD → pyruvate + NADH, and by l-lactate cytochrome c oxidoreductase (l-lactate cytochrome c oxidoreductase, EC 1.1.2.3), which catalyzes the reaction l-lactate + 2 cytochrome c (oxidized) → pyruvate + 2 cytochrome c (reduced). Both groups are found in eubacteria, archebacteria, and eukaryotes. All known plant sequences belong to the EC 1.1.1.27 group (1). On the other hand, d-lactate is oxidized by d-lactate:NAD oxidoreductase (d-lactate:NAD oxidoreductase, EC 1.1.1.28), which catalyzes the reaction d-lactate + NAD → pyruvate + NADH, and by d-lactate cytochrome c oxidoreductase (d-lactate cytochrome c oxidoreductase, EC 1.1.2.4), which catalyzes the reaction d-lactate + 2 cytochrome c (oxidized) → pyruvate + 2 cytochrome c (reduced).Although l-lactate dehydrogenase belongs to the most intensely studied enzyme families (2, 3), our knowledge about the structure, kinetics, and biological function of d-LDH³ is limited. d-LDHs have mainly been identified in prokaryotes and fungi where they play an important role in anaerobic energy metabolism (4–10). In Saccharomyces cerevisiae and Kluyveromyces lactis, a mitochondrial flavoprotein d-lactate ferricytochrome c oxidoreductase (d-lactate cytochrome c oxidoreductase), catalyzing the oxidation of d-lactate to pyruvate, is required for the utilization of d-lactate (8, 11). In S. cerevisiae it was suggested that d-LDH is involved in the metabolism of methylglyoxal (MG) (12).In eukaryotic cells, d-lactate results from the glyoxalase system (13, 14). This system is the main MG catabolic pathway, comprising the enzymes glyoxalase I (lactoylglutathione lyase, EC 4.4.1.5) and glyoxalase II (hydroxyacylglutathione hydrolase, EC 3.1.2.6). MG (CH₃-CO-CHO; see structure in Fig. 4) is a cytotoxic compound formed primarily as a by-product of glycolysis through nonenzymatic phosphate elimination from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (15), and its production in various plants is enhanced under stress conditions such as salt, drought, cold, and heavy metal stress (16, 17). Moreover, the overexpression of glyoxalase I or II was shown to confer resistance to salt stress in tobacco and rice (17, 18). It is assumed that the role of the MG pathway, from MG synthase to d-lactate cytochrome c oxidoreductase in the extant metabolism, is to detoxify MG, whereas in the early state of metabolic development it might function as an anaplerotic route for the tricarboxylic acid cycle (15).Open in a separate windowFIGURE 4.Scheme showing the involvement of AtD-LDH in the methylglyoxal pathway and of AtD-2HGDH in the respiration of substrates from proteolysis and/or lipid degradation. d-Lactate resulting from the glyoxalase system is converted to pyruvate by AtD-LDH. The electrons originated may be transferred to the respiratory chain through cytochrome c in the intermembrane space. d-2-HG produced in the peroxisomes (as shown in supplemental Fig. S3) is transported to the mitochondria and converted to 2-ketoglutarate by AtD-2HGDH. Electrons are donated to the electron transport chain through the ETF/ETFQO system. Dotted files represent possible transport processes. 2-KG, 2-ketoglutarate. CIII, complex III. CIV, complex IV. e⁻, electron. ETF, electron transfer protein. ETFQO, ETF-ubiquinone oxidoreductase. GSH, glutathione. Pyr, pyruvate. TCA cycle, tricarboxylic acid cycle; UQ, ubiquinone.Glyoxalase I catalyzes the formation of S-d-lactoylglutathione from the hemithioacetal formed nonenzymatically from MG and glutathione, although glyoxalase II catalyzes the hydrolysis of S-d-lactoylglutathione to regenerate glutathione and liberate d-lactate. Glyoxalase I and II activities are present in all tissues of eukaryotic organisms. Glyoxalase I is found in the cytosol, whereas glyoxalase II localizes to the cytosol and mitochondria (13, 19, 20). Although glyoxalase I and II were extensively characterized, there are only few reports on the characterization of d-LDH. Recently, Atlante et al. (13) showed that externally added d-lactate caused oxygen consumption by mitochondria and that this metabolite was oxidized by a mitochondrial flavoprotein in Helianthus tuberosus.The complete sequence of Arabidopsis thaliana opened the way to search for genes encoding d-LDHs. Based on similarity with the d-LDH from S. cerevisiae (DLD1), an A. thaliana ortholog was identified. In this study, the isolation and structural and biochemical characterization of the recombinant mature d-LDH from A. thaliana (AtD-LDH) and its paralog, which was found to be a d-2-hydroxyglutarate dehydrogenase (AtD-2HGDH), is described. Whereas AtD-LDH has a narrow substrate specificity and the preferred substrates are d-lactate and d-2-hydroxybutyrate, AtD-2HGDH showed activity exclusively with d-2-hydroxyglutarate. Based on gene coexpression analysis and analysis of corresponding knock-out mutants, the participation of these previously unrecognized mitochondrial activities in plant metabolism is discussed.     

Biosynthesis and Structure of the Burkholderia cenocepacia K56-2 Lipopolysaccharide Core Oligosaccharide: TRUNCATION OF THE CORE OLIGOSACCHARIDE LEADS TO INCREASED BINDING AND SENSITIVITY TO POLYMYXIN B*

Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of genes for the biosynthesis of the core oligosaccharide (OS) moiety of the B. cenocepacia lipopolysaccharide. We constructed a panel of isogenic mutants with truncated core OS that facilitated functional gene assignments and the elucidation of the core OS structure in the prototypic strain K56-2. The core OS structure consists of three heptoses in the inner core region, 3-deoxy-d-manno-octulosonic acid, d-glycero-d-talo-octulosonic acid, and 4-amino-4-deoxy-l-arabinose linked to d-glycero-d-talo-octulosonic acid. Also, glucose is linked to heptose I, whereas heptose II carries a second glucose and a terminal heptose, which is the site of attachment of the O antigen. We established that the level of core truncation in the mutants was proportional to their increased in vitro sensitivity to polymyxin B (PmB). Binding assays using fluorescent 5-dimethylaminonaphthalene-1-sulfonyl-labeled PmB demonstrated a correlation between sensitivity and increased binding of PmB to intact cells. Also, the mutant producing a heptoseless core OS did not survive in macrophages as compared with the parental K56-2 strain. Together, our results demonstrate that a complete core OS is required for full PmB resistance in B. cenocepacia and that resistance is due, at least in part, to the ability of B. cenocepacia to prevent binding of the peptide to the bacterial cell envelope.Burkholderia cenocepacia is a Gram-negative opportunistic pathogen ubiquitously found in the environment (1, 2). Although generally harmless to healthy individuals, B. cenocepacia affects immunocompromised patients (1) such as those with cystic fibrosis and chronic granulomatous disease. Infected cystic fibrosis patients commonly develop chronic lung infections that are very difficult to treat because these bacteria are intrinsically resistant to virtually all clinically useful antibiotics as well as antimicrobial peptides (APs)⁵ (1, 3).Lipopolysaccharide (LPS) is the major surface component of Gram-negative bacteria and consists of lipid A, core oligosaccharide (OS), and in some bacteria O-specific polysaccharide or O antigen (4, 5). The O antigen acts as a protective barrier against desiccation, phagocytosis, and serum complement-mediated killing, whereas the core OS and the lipid A contribute to maintain the integrity of the outer membrane (4, 5). The lipid A also anchors the LPS molecule to the outer leaflet of the outer membrane and accounts for the endotoxic activity of LPS (4, 6). Lipid A is a bisphosphorylated β-1,6-linked glucosamine disaccharide substituted with fatty acids ester-linked at positions 3 and 3′ and amide-linked at positions 2 and 2′ (4). The core OS can be subdivided into the inner core and outer core. The inner core OS typically consists of one or two 3-deoxy-d-manno-octulosonic acid (Kdo) residues linked to the lipid A and three l-glycero-d-manno-heptose residues linked to the first Kdo (4). The outer core OS in enteric bacteria typically consists of 8–12 branched sugars linked to heptose II of the inner core. As a result of phosphate groups on the lipid A and core OS, the bacterial surface has a net negative charge. This plays an important role in the interaction of the bacterial surface with positively charged compounds such as cationic APs, which are cationic amphipathic molecules that kill bacteria by membrane permeabilization. In response to a series of environmental conditions such as low magnesium or high iron, bacteria can express modified LPS molecules that result in a less negative surface. This reduces the binding of APs and promotes resistance to these compounds. Previous studies have shown that Burkholderia LPS molecules possess unique properties. For example, Kdo cannot be detected by classic colorimetric methods in LPS from Burkholderia pseudomallei and Burkholderia cepacia, and the covalent linkage between Kdo and lipid A is more resistant to acid hydrolysis than in conventional LPS molecules (7). In B. cepacia, 4-amino-4-deoxy-l-arabinose (l-Ara4N) is bound to the lipid A by a phosphodiester linkage at position 4 of the nonreducing glucosamine (GlcN II) (8) and is also present as a component of the core OS. Also, instead of two Kdo molecules, the B. cepacia core OS has only one Kdo and the unusual Kdo analog, d-glycero-d-talo-octulosonic acid (Ko), which is nonstoichiometrically substituted with l-Ara4N forming a 1→8 linkage with α-Ko (7, 9). Although this is also the case for the inner core OS of B. cenocepacia J2315 (10), it is not a common feature for the core OS in all Burkholderia. For example, the inner core of Burkholderia caryophylli consists of two Kdo residues and does not possess l-Ara4N (11).Burkholderia species, including B. cenocepacia, are intrinsically resistant to human and non-human APs such as these produced by airway epithelial cells (12, 13), human β-defensin 3 (14), human neutrophil peptides (15), and polymyxin B (PmB) (15, 16). The minimum inhibitory concentration determined for some of these peptides in several Burkholderia species is greater than 500 μg/ml, which could aid these microorganisms during colonization of the respiratory epithelia (13). It has been proposed that the resistance of B. cepacia to cationic APs stems from ineffective binding to the outer membrane, as a consequence of the low number of phosphate and carboxylate groups in the lipopolysaccharide (17), but a systematic analysis of the molecular basis of AP resistance in B. cenocepacia and other Burkholderia is lacking. We have previously reported that a heptoseless B. cenocepacia mutant (SAL1) is significantly more sensitive than the parental clinical strain K56-2 to APs (15). This mutant has a truncated inner core and lacks the outer core, suggesting that a complete core OS is required for resistance of B. cenocepacia to APs.Apart from heptoses, the role of other sugar moieties of the B. cenocepacia core OS in AP resistance is not known. In this study, we report the structure of the core OS for B. cenocepacia strain K56-2 and its isogenic mutants XOA3, XOA7, and XOA8, which carry various core OS truncations. The structural analysis, combined with mutagenesis, allowed us to assign function to the majority of the genes involved in core OS biosynthesis and ligation of the O antigen and to establish that the degree of truncation of the core OS correlates with increased binding and bacterial sensitivity to PmB in vitro and reduced bacterial intracellular survival in macrophages.     

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala⁴-α-d-Lys³ cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala¹(α→α)d-Lys³ and d-Ala⁴(α→α)d-Lys³ types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)² cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala¹-γ-d-Glu²-X³-d-Ala⁴-d-Ala⁵, where X is most often meso-diaminopimelic acid (meso-A₂pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala⁴-d-Ala⁵ bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala⁴ and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X³-d-Ala⁴ bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A₂pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link.     

Biochemical Properties and Substrate Specificities of a Recombinantly Produced Azotobacter vinelandii Alginate Lyase

Alginate is a polysaccharide composed of β-d-mannuronic acid (M) and α-l-guluronic acid (G). An Azotobacter vinelandii alginate lyase gene, algL, was cloned, sequenced, and expressed in Escherichia coli. The deduced molecular mass of the corresponding protein is 41.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 39 kDa. Sixty-three percent of the amino acids in this mature protein are identical to those in AlgL from Pseudomonas aeruginosa. AlgL was partially purified, and the activity was found to be optimal at a pH of 8.1 to 8.4 and at 0.35 M NaCl. Divalent cations are not necessary for activity. The pI of the enzyme is 5.1. When an alginate rich in mannuronic acid was used as the substrate, the K_m was found to be 4.6 × 10⁻⁴ M (sugar residues). AlgL was found to cleave M-M and M-G bonds but not G-M or G-G bonds. Bonds involving acetylated residues were also cleaved, but this activity may be sensitive to the extent of acetylation.

Alginate is a family of 1-4-linked copolymers of β-d-mannuronic acid (M) and α-l-guluronic acid (G). It is produced by brown algae and by some bacteria belonging to the genera Azotobacter and Pseudomonas (8, 17, 18, 31). The polymer is widely used in industry and biotechnology (36, 44), and the genetics of its biosynthesis in Pseudomonas aeruginosa has been extensively studied due to its role in the disease cystic fibrosis (33). In bacterial alginates, some of the M residues may be O-2- and/or O-3-acetylated (42). The polymer is initially synthesized as mannuronan, and the G residues are introduced at the polymer level by mannuronan C-5-epimerases (13, 22, 23). The epimerized alginates contain a mixture of blocks of consecutive G residues (G blocks), consecutive M residues (M blocks), and alternating M and G residues (MG blocks). Alginates from Pseudomonas sp. do not contain G blocks (42).Alginate lyases catalyze the depolymerization of alginates by β-elimination, generating a molecule containing 4-deoxy-l-erythro-hex-4-enepyranosyluronate at the nonreducing end. Such lyases have been found in organisms using alginate as a carbon source, in bacteriophages specific for alginate-producing organisms, and in alginate-producing bacteria (45). An alginate molecule may contain four different glycosidic bonds, M-M, G-M, M-G, or G-G, and the relative rates at which each of these bonds are cleaved vary among different lyases (36a). The lyases also differ in the extent to which they are affected by acetylation (35, 43, 46).Davidson et al. (10) described an Azotobacter vinelandii lyase which preferred M blocks as a substrate. Kennedy et al. (28) later reported the purification of periplasmic alginate lyases from A. vinelandii and from Azotobacter chroococcum which also seemed to prefer deacetylated, M-rich alginate. The activities of these enzymes were found to be optimal at pH 6.8 and 7.2, respectively, while the enzyme reported by Davidson et al. (10) was found to display optimal activity at pH 7.8.A gene, algL, encoding an alginate lyase has been cloned from P. aeruginosa (2, 41). The gene was found to be located in a cluster containing most of the genes necessary for the biosynthesis of alginate. A homologous gene cluster has recently been identified in A. vinelandii (38) and shown to encode an alginate lyase (32). In our previous report, we showed that plasmid pHE102, which contains a part of this gene cluster, contains a DNA sequence sharing homology with algL from P. aeruginosa (38). We have now subcloned, sequenced, and expressed this gene in Escherichia coli. The lyase was shown to preferentially cleave deacetylated M-M and M-G bonds, but acetylated substrates were also cleaved.     

Identification of Two Genes from Streptomyces argillaceus Encoding Glycosyltransferases Involved in Transfer of a Disaccharide during Biosynthesis of the Antitumor Drug Mithramycin

Mithramycin is an antitumor polyketide drug produced by Streptomyces argillaceus that contains two deoxysugar chains, a disaccharide consisting of two d-olivoses and a trisaccharide consisting of a d-olivose, a d-oliose, and a d-mycarose. From a cosmid clone (cosAR3) which confers resistance to mithramycin in streptomycetes, a 3-kb PstI-XhoI fragment was sequenced, and two divergent genes (mtmGI and mtmGII) were identified. Comparison of the deduced products of both genes with proteins in databases showed similarities with glycosyltransferases and glucuronosyltransferases from different sources, including several glycosyltransferases involved in sugar transfer during antibiotic biosynthesis. Both genes were independently inactivated by gene replacement, and the mutants generated (M3G1 and M3G2) did not produce mithramycin. High-performance liquid chromatography analysis of ethyl acetate extracts of culture supernatants of both mutants showed the presence of several peaks with the characteristic spectra of mithramycin biosynthetic intermediates. Four compounds were isolated from both mutants by preparative high-performance liquid chromatography, and their structures were elucidated by physicochemical methods. The structures of these compounds were identical in both mutants, and the compounds are suggested to be glycosylated intermediates of mithramycin biosynthesis with different numbers of sugar moieties attached to C-12a-O of a tetracyclic mithramycin precursor and to C-2-O of mithramycinone: three tetracyclic intermediates containing one sugar (premithramycin A1), two sugars (premithramycin A2), or three sugars (premithramycin A3) and one tricyclic intermediate containing a trisaccharide chain (premithramycin A4). It is proposed that the glycosyltransferases encoded by mtmGI and mtmGII are responsible for forming and transferring the disaccharide during mithramycin biosynthesis. From the structures of the new metabolites, a new biosynthetic sequence regarding late steps of mithramycin biosynthesis can be suggested, a sequence which includes glycosyl transfer steps prior to the final shaping of the aglycone moiety of mithramycin.

Many bioactive drugs contain sugars attached to their aglycones which are usually important or, in some cases, essential for bioactivity. Most of these sugars belong to the family of the 6-deoxyhexoses (6-DOH) (18, 20, 27) and are transferred to the different aglycones as late steps in biosynthesis. Genes involved in the biosynthesis of different 6-DOH have been reported elsewhere and participate in the biosynthesis of erythromycin (9, 12, 31, 38, 39), daunorubicin (13, 26, 36), mithramycin (22), granaticin (2), streptomycin (10, 28), and tylosin (14, 23). However, information about the glycosyltransferases (GTFs) responsible for the transfer of the sugars to the respective aglycones is quite scarce. So far, only two GTFs from antibiotic producers have been biochemically characterized in detail, and they are involved in macrolide inactivation: Mgt, from Streptomyces lividans, a nonmacrolide producer (7, 17); and OleD, from the oleandomycin producer Streptomyces antibioticus (15, 29), which inactivates oleandomycin by addition of glucose to the 2′-OH group of the desosamine attached to the macrolactone ring (40). In the last several years, a few genes have been proposed to encode GTFs involved in the transfer of sugars to various aglycones during biosynthesis: dnrS and dnrH, from Streptomyces peucetius, involved in daunorubicin (26) and baumycin (36) biosynthesis, respectively; gra-orf5, involved in granaticin biosynthesis (2); eryCIII and eryBV, involved in the transfer of desosamine and mycarose, respectively, in erythromycin biosynthesis (12, 32, 38); and tylM2, from Streptomyces fradiae, involved in sugar transfer during tylosin biosynthesis (14).Mithramycin (Fig. ​(Fig.1)1) is an aromatic polyketide which shows antibacterial activity against gram-positive bacteria and also antitumor activity (30, 37). Together with the chromomycins and the olivomycins, mithramycin constitutes the so-called aureolic acid group of antitumor drugs. The polyketide moiety of mithramycin is derived from the condensation of 10 acetate building blocks in a series of reactions catalyzed by a type II polyketide synthase (5, 21). The mithramycin aglycone is glycosylated at positions 6 and 2 with disaccharide (d-olivose- d-olivose) and trisaccharide (d-olivose-d-oliose-d-mycarose) moieties, respectively. All of these sugars belong to the 6-DOH family. In the mithramycin pathway, two genes (mtmD and mtmE) encoding two enzymes (glucose-1-phosphate:TTP thymidylyl transferase and dTDP-4,6-dehydratase, respectively) involved in the biosynthesis of the mithramycin 6-DOH have been cloned, and their participation in mithramycin biosynthesis has been demonstrated by insertional inactivation (22). Here we report the characterization of two Streptomyces argillaceus genes (mtmGI and mtmGII) that encode two putative GTFs responsible for the formation and transfer of the disaccharide chain. Inactivation of these genes by gene replacement showed identical accumulated compounds and allowed the isolation of four glycosylated compounds which are likely to be intermediates in mithramycin biosynthesis. Open in a separate windowFIG. 1Structures of mithramycin, premithramycinone, and the new premithramycins.