Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4′-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′-O-β-d-galactoside, resveratrol 4′-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.     

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.     

The neurotoxicity of β-N-oxalyl-l-αβ-diaminopropionic acid, the neurotoxin from the pulse Lathyrus sativus

Intraperitoneal administration of β-N-oxalyl-l-αβ-diaminopropionic acid, the neurotoxin from Lathyrus sativus, to 12-day-old rats causes typical convulsions within 10min. There is a striking accumulation of glutamine in the brain, and chronic ammonia toxicity is indicated. There are no changes in the amounts of urea, aspartic acid and glutamic acid in the brain. Adult rats, even when injected with a dose of excess of β-N-oxalyl-l-αβ-diaminopropionic acid, do not develop symptoms, and there are no changes in the amounts of glutamine or ammonia in the brain. A significant concentration of β-N-oxalyl-l-αβ-diaminopropionic acid can be detected in the brain of the young rat but not in that of the adult animal. It is concluded that β-N-oxalyl-l-αβ-diaminopropionic acid interferes with the ammonia-generating or -fixing mechanisms in the brain and leads to chronic ammonia toxicity.     

Kinetics and mechanism of catalysis by proteolytic enzymes. A comparison of the kinetics of hydrolysis of synthetic substrates by bovine α- and β-trypsin

Several esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of S-(3-aminopropyl)-l-cysteine and the methyl ester of S-(4-aminobutyl)-N-toluene-p-sulphonyl-l-cysteine were synthesized. The kinetics of hydrolysis of these and esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of l-arginine, l-lysine, S-(2-aminoethyl)-l-cysteine and esters of γ-guanidino-l-α-toluene-p-sulphonamidobutyric acid and α-N-toluene-p-sulphonyl-l-homoarginine by α- and β-trypsin were compared. On the basis of values of the specificity constants (k_cat./K_m), the two enzymes display similar catalytic efficiency towards some substrates. In other cases α-trypsin is less efficient than β-trypsin. It is possible that α-trypsin possesses greater molecular flexibility than β-trypsin.     

Commentary on Urinary l-erythro-β-hydroxyasparagine: a novel serine racemase inhibitor and substrate of the Zn2+-dependent d-serine dehydratase

The analysis of the urine contents can be informative of physiological homoeostasis, and it has been speculated that the levels of urinary d-serine (d-ser) could inform about neurological and renal disorders. By analysing the levels of urinary d-ser using a d-ser dehydratase (DSD) enzyme, Ito et al. (Biosci. Rep.(2021) 41, BSR20210260) have described abundant levels of l-erythro-β-hydroxyasparagine (l-β-EHAsn), a non-proteogenic amino acid which is also a newly described substrate for DSD. The data presented support the endogenous production l-β-EHAsn, with its concentration significantly correlating with the concentration of creatinine in urine. Taken together, these results could raise speculations that l-β-EHAsn might have unexplored important biological roles. It has been demonstrated that l-β-EHAsn also inhibits serine racemase with K_i values (40 μM) similar to its concentration in urine (50 μM). Given that serine racemase is the enzyme involved in the synthesis of d-ser, and l-β-EHAsn is also a substrate for DSD, further investigations could verify if this amino acid would be involved in the metabolic regulation of pathways involving d-ser.     

Cytokinins in tRNA Obtained from Spinacia oleracea L. Leaves and Isolated Chloroplasts

Cytokinin-active ribonucleosides have been isolated from tRNA of whole spinach (Spinacia oleracea L.) leaves and isolated spinach chloroplasts. The tRNA from spinach leaf blades contained: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine (cis and trans isomers), 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (cis and trans isomers). A method for isolation of large amounts of intact chloroplasts was developed and subsequently used for the isolation of chloroplast tRNA. The chloroplast tRNA contained 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (the cis isomer only). The structures of these compounds were assigned on the basis of their chromatographic properties and mass spectra of trimethylsilyl derivatives which were identical with those of the corresponding synthetic compounds. The results of this study indicate that ribosylzeatin was present in spinach leaf tRNA, but absent from the purified chloroplast tRNA preparation.     

Identification and Characterization of a Novel Terrabacter ginsenosidimutans sp. nov. β-Glucosidase That Transforms Ginsenoside Rb1 into the Rare Gypenosides XVII and LXXV

A new β-glucosidase from a novel strain of Terrabacter ginsenosidimutans (Gsoil 3082^T) obtained from the soil of a ginseng farm was characterized, and the gene, bgpA (1,947 bp), was cloned in Escherichia coli. The enzyme catalyzed the conversion of ginsenoside Rb1 {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to the more pharmacologically active rare ginsenosides gypenoside XVII {3-O-β-d-glucopyranosyl-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, gypenoside LXXV {20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K [20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol]. A BLAST search of the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. Expressed in E. coli, β-glucosidase had apparent K_m values of 4.2 ± 0.8 and 0.14 ± 0.05 mM and V_max values of 100.6 ± 17.1 and 329 ± 31 μmol·min⁻¹·mg of protein⁻¹ against p-nitrophenyl-β-d-glucopyranoside and Rb1, respectively. The enzyme catalyzed the hydrolysis of the two glucose moieties attached to the C-3 position of ginsenoside Rb1, and the outer glucose attached to the C-20 position at pH 7.0 and 37°C. These cleavages occurred in a defined order, with the outer glucose of C-3 cleaved first, followed by the inner glucose of C-3, and finally the outer glucose of C-20. These results indicated that BgpA selectively and sequentially converts ginsenoside Rb1 to the rare ginsenosides gypenoside XVII, gypenoside LXXV, and then C-K. Herein is the first report of the cloning and characterization of a novel ginsenoside-transforming β-glucosidase of the glycoside hydrolase family 3.Ginseng refers to the roots of members of the plant genus Panax, which have been used as a traditional medicine in Asian countries for over 2,000 years due to their observed beneficial effects on human health. Ginseng saponins, also referred to as ginsenosides, are the major active components of ginseng (27). Various biological activities have been ascribed to ginseng saponins, including anti-inflammatory activity (43), antitumor effects (23, 39), and neuroprotective and immunoprotective (15, 31) effects.Ginsenosides can be categorized as protopanaxadiol (PPD), protopanaxatriol, and oleanane saponins, based on the structure of the aglycon, with a dammarane skeleton (29). The PPD-type ginsenosides are further classified into subgroups based on the position and number of sugar moieties attached to the aglycon at positions C-3 and C-20. For example, one of the largest PPD-type ginsenosides, Rb1 {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, contains 4 glucose moieties, two each attached via glycosidic linkages to the C-3 and C-20 positions of the aglycon (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Chemical structures of protopanaxadiol and protopanaxatriol ginsenosides (5). The ginsenosides represented here are all (S)-type ginsenosides. glc, β-d-glucopyranosyl; arap, α-l-arabinopyranosyl; araf, α-l-arabinofuranosyl; rha, α-l-rhamnopyranosyl; Gyp, gypenoside; C, compound.Because of their size, low solubility, and poor permeability across the cell membrane, it is difficult for human body to directly absorb large ginsenosides (44), although these components constitute the major portion of the total ginsenoside in raw ginseng (30). Moreover, the lack of the availability of the rare ginsensoides limits the research on their biological and medicinal properties. Therefore, transformation of these major ginsenosides into smaller deglycosylated ginsenosides, which are more effective in in vivo physiological action, is required (1, 37).The production of large amounts of rare ginsenosides from the major ginsenosides can be accomplished through a number of physiochemical methods such as heating (17), acid treatment (2), and alkali treatment (48). However, these approaches produce nonspecific racemic mixtures of rare ginsenosides. As an alternative, enzymatic methods have been explored as a way to convert the major ginsenosides into more pharmacologically active rare ginsenosides in a more specific manner (14, 20).To date, three types of glycoside hydrolases, β-d-glucosidase, α-l-arabinopyranosidase, and α-l-arabinofuranosidase, have been found to be involved in the biotransformation of PPD-type ginsenosides. For example, a β-glucosidase isolated from a fungus converts Rb1 to C-K [20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol] (45), and an α-l-arabinopyranosidase and α-l-arabinofuranosidase have been isolated from an intestinal bacterium that hydrolyze, respectively, Rb2 {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[α-l-arabinopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to Rd {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} and Rc {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O- [α-l-arabinofuranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to Rd (34). Two recombinant enzymes that convert major ginsenosides into rare ginsenosides have been cloned and expressed in Escherichia coli: Solfolobus solfataricus β-glycosidase, which transforms Rb1 or Rc to C-K (28), and β-glucosidase from a soil metagenome, which transforms Rb1 to Rd (16). Both of these glycoside hydrolases are family 1 glycoside hydrolases.Here, we report the cloning and expression in E. coli of a gene (bgpA) encoding a new ginsenoside-hydrolyzing β-glucosidase from a novel bacterial strain, Terrabacter ginsenosidimutans sp. nov. Gsoil 3082, isolated from a ginseng farm in Korea. BgpA is a family 3 glycoside hydrolase, and the recombinant enzyme employs a different enzymatic pathway from ginsenoside-hydrolyzing family 1 glycoside hydrolases. BgpA preferentially and sequentially hydrolyzed the terminal and inner glucoses at the C-3 position of ginsenoside Rb1 and then the outer glucose at the C-20 position. Thus, BgpA could be effective in the biotransformation of ginsenoside Rb1 to gypenoside (Gyp) XVII {3-O-β-d-glucopyranosyl-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, Gyp LXXV {20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K.     

Cytokinins in pisum transfer ribonucleic Acid

Five cytokinin-active ribonucleosides have been isolated from the transfer RNA of 7-day-old green pea shoots (Pisum sativum L. var. Alaska). Ultraviolet spectroscopy and mass spectrometry have been used to identify 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β- d-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine. The latter was separated into the cis- and trans-isomers by thin layer chromatography. The fifth cytokinin is indicated to be 6-(3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine on the basis of its chromatographic properties.     

Inhibition of glycosidases by aldonolactones of corresponding configuration. The specificity of α-l-arabinosidase

1. The previous study (Conchie, Gelman & Levvy, 1967b) of the specificity of β-glucosidase, β-galactosidase and β-d-fucosidase in barley, limpet, almond emulsin and rat epididymis was extended to α-l-arabinosidase. 2. The inhibitory action of l-arabinono-(1→5)-lactone was tested against all four types of enzyme, and α-l-arabinosidase was examined for inhibition by glucono-, galactono- and d-fucono-lactone. 3. In emulsin, the enzyme that hydrolyses β-glucosides, β-galactosides and β-d-fucosides also hydrolyses α-l-arabinosides. Rat epididymis resembles emulsin except that, as already noted, it lacks β-glucosidase activity. 4. In the limpet, α-l-arabinosidase activity is associated with the enzyme that hydrolyses β-glucosides and β-d-fucosides, and not with the separate β-galactosidase. 5. The effects of the different lactones on the barley preparation suggest that α-l-arabinosidase activity is associated with the β-galactosidase rather than with the enzyme that hydrolyses β-glucosides and β-d-fucosides. Fractionation and heat-inactivation experiments indicate that there is also a separate α-l-arabinosidase in the preparation.     

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.     

Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove α-l-Araf residues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket.     

alpha-l-Arabinofuranosidase from Radish (Raphanus sativus L.) Seeds

An α-l-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl α-l-arabinofuranoside and p-nitrophenyl β-d-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding α-l-arabinopyranoside and β-d-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg²⁺, Ag⁺, l-arabino-(1→4)-lactone, and d-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off α-l-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of α-l-arabinofuranosidase and β-d-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety.     

Promotion of seed germination by cyanide

Potassium cyanide at 3 μm to 10 mm promotes germination of Amaranthus albus, Lactuca sativa, and Lepidium virginicum seeds. l-Cysteine hydrogen sulfide lyase, which catalyzes the reaction of HCN with l-cysteine to form β-l cyanoalanine, is active in the seeds. β-l-Cyanoalanine is the most effective of the 23 α-amino acids tested for promoting germination of A. albus seeds. Aspartate, which is produced by enzymatic hydrolysis of asparagine formed by hydrolysis from β-cyanoalanine, is the second most effective of the 23 amino acids. Uptake of aspartate-4-¹⁴C is much lower than of cyanide.     

Purification and characterization of dihydrodipicolinate synthase from pea

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a combination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicolinate synthase from a plant source. The pea dihydrodipicolinate synthase has an apparent molecular weight of 127,000 and is composed of three identical subunits of 43,000 as determined by gel filtration and cross-linking experiments. The trimeric quaternary structure resembles the trimeric structure of other aldolases, such as 2-keto-3-deoxy-6-phosphogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference concerns the methionine content: dihydrodipicolinate synthase from pea contains 22 moles of methionine residue per mole of native protein, contrary to the E. coli enzyme, which does not contain this amino acid at all. Dihydrodipicolinate synthase from pea is highly specific for the substrates pyruvate and l-aspartate-β-semialdehyde; it follows Michaelis-Menten kinetics for both substrates. The pyruvate and l-aspartate-β-semialdehyde have Michaelis constant values of 1.70 and 0.40 millimolar, respectively. l-Lysine, S-(2-aminoethyl)-l-cysteine, and l-α-(2-aminoethoxyvinyl)glycine are strong allosteric inhibitors of the enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, respectively. The inhibition by l-lysine and l-α-(2-aminoethoxyvinyl)glycine is noncompetitive towards l-aspartate-β-semialdehyde, whereas S-(2-aminoethyl)-l-cysteine inhibits dihydrodipicolinate synthase competitively with respect to l-aspartate-β-semialdehyde. Furthermore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy-heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid (1.2 millimolar) activates dihydrodipicolinate synthase from pea by a factor of 1.4 and 1.2, respectively. This is the first reported activation process found for dihydrodipicolinate synthase.     

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2′:2,1′-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2′:2,1′-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).     

The isolation of oligosaccharides from the cell-wall polysaccharide of Lactobacillus casei, serological group C

1. A number of disaccharides and oligosaccharides have been isolated from the products of mild acid hydrolysis of the specific substance from Lactobacillus casei, serological group C. 2. The major disaccharide is O-β-d-glucopyranosyl-(1→3)-N-acetyl- d-galactosamine (B4) and evidence is presented for the structure of a tetrasaccharide composed of O-β-d-glucopyranosyl-(1→6)-d-galactose (B1) joined through its reducing end group to B4. 3. Disaccharide B1 is also a component of a trisaccharide O-β-d-glucopyranosyl-(1→6)-O-β- d-galactopyranosyl-(1→6)-N-acetyl-d-glucosamine (A7). 4. A number of other oligosaccharides have been shown to be related structurally. 5. The ability of certain of the oligosaccharides to inhibit the precipitin reaction has been studied. The disaccharide B1 is more effective as an inhibitor than gentiobiose and the trisaccharide A7 is considerably more effective than B1. 6. These results have been compared with those obtained previously for the composition of the cell wall.     

Sugar-nucleotide precursors of arabinopyranosyl, arabinofuranosyl, and xylopyranosyl residues in spinach polysaccharides

Cultured spinach (Spinacia oleracea L. cv Monstrous Viroflay) cells incorporated exogenous l-[³H]arabinose sequentially into β-l-arabinopyranose-1-phosphate, uridine diphospho-β-l-arabinopyranose, uridine diphospho-α-d-xylopyranose and (in some experiments) α-d-xylopyranose-1-phosphate. The amount of ³H in each of these compounds reached a plateau after a few minutes, and could be rapidly chased with nonradioactive l-arabinose, demonstrating rapid turnover. After a few minutes' lag, incorporation of ³H into the arabinofuranosyl, arabinopyranosyl, and xylopyranosyl residues of polysaccharides was linear with respect to time. The kinetics of labeling were compatible with UDP-β-l-arabinopyranose and UDP-α-d-xylopyranose being the immediate precursors of arabians (both the pyranose and the furanose residues) and xylans, respectively. No other radioactive nucleotides were formed; in particular, UDP-arabinofuranose was absent. There was no evidence for conversion of arabinopyranose to arabinofuranose within the polysaccharides, suggesting that this conversion occurs during polymer synthesis. The glycolipids detected showed too slow a turnover to be intermediates of pentosan synthesis.     

delta-Aminolevulinic Acid Transaminase in Chlorella vulgaris

An enzyme catalyzing the formation of δ-aminolevulinic acid by transamination of γ,δ-dioxovaleric acid with l-α-alanine, l-glutamic acid, or l-phenylalanine has been detected in extracts of Chlorella vulgaris. The activity of this enzyme does not appear to parallel changes in chlorophyll content in a Chlorella mutant which requires light for chlorophyll production. The role of this enzyme in δ-aminolevulinic acid metabolism in plants is not clearly understood.     

Crystal Structure of an Exo-1,5-α-l-arabinofuranosidase from Streptomyces avermitilis Provides Insights into the Mechanism of Substrate Discrimination between Exo- and Endo-type Enzymes in Glycoside Hydrolase Family 43

Exo-1,5-α-l-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-l-arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-l-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-l-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite −1, formed by the flexible loop region Tyr-281–Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.     

A Galacturonic Acid–Containing Xyloglucan Is Involved in Arabidopsis Root Hair Tip Growth

Root hairs provide a model system to study plant cell growth, yet little is known about the polysaccharide compositions of their walls or the role of these polysaccharides in wall expansion. We report that Arabidopsis thaliana root hair walls contain a previously unidentified xyloglucan that is composed of both neutral and galacturonic acid–containing subunits, the latter containing the β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→ and/or α-l-fucosyl-(1→2)-β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→) side chains. Arabidopsis mutants lacking root hairs have no acidic xyloglucan. A loss-of-function mutation in At1g63450, a root hair–specific gene encoding a family GT47 glycosyltransferase, results in the synthesis of xyloglucan that lacks galacturonic acid. The root hairs of this mutant are shorter than those of the wild type. This mutant phenotype and the absence of galacturonic acid in the root xyloglucan are complemented by At1g63450. The leaf and stem cell walls of wild-type Arabidopsis contain no acidic xyloglucan. However, overexpression of At1g63450 led to the synthesis of galacturonic acid–containing xyloglucan in these tissues. We propose that At1g63450 encodes XYLOGLUCAN-SPECIFIC GALACTURONOSYLTRANSFERASE1, which catalyzes the formation of the galactosyluronic acid-(1→2)-α-d-xylopyranosyl linkage and that the acidic xyloglucan is present only in root hair cell walls. The role of the acidic xyloglucan in root hair tip growth is discussed.