Functional characterization of dehydratase/aminotransferase pairs from Helicobacter and Campylobacter: enzymes distinguishing the pseudaminic acid and bacillosamine biosynthetic pathways

Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-alpha-D-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His(6)-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-alpha-D-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-D-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose.     

Molecular architecture of DesI: a key enzyme in the biosynthesis of desosamine

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found, for example, in such macrolide antibiotics as erthyromycin, azithromycin, and clarithromycin. The efficacies of these macrolide antibiotics are markedly reduced in the absence of desosamine. In the bacterium Streptomyces venezuelae, six enzymes are required for the production of dTDP-desosamine. The focus of this X-ray crystallographic analysis is the third enzyme in the pathway, a PLP-dependent aminotransferase referred to as DesI. The structure of DesI was solved in complex with its product, dTDP-4-amino-4,6-dideoxyglucose, to a nominal resolution of 2.1 A. Each subunit of the dimeric enzyme contains 12 alpha-helices and 14 beta-strands. Three cis-peptides are observed in each subunit, Phe 330, Pro 332, and Pro 339. The two active sites of the enzyme are located in clefts at the subunit/subunit interface. Electron density corresponding to the bound product clearly demonstrates a covalent bond between the amino group of the product and C-4' of the PLP cofactor. Interestingly, there are no hydrogen-bonding interactions between the protein and the dideoxyglucosyl group of the product (within 3.2 A). The only other sugar-modifying aminotransferase whose structure is known in the presence of product is PseC from Helicobacter pylori. This enzyme, as opposed to DesI, catalyzes amino transfer to the axial position of the sugar. A superposition of the two active sites for these proteins reveals that the major differences in ligand binding occur in the orientations of the deoxyglucosyl and phosphoryl groups. Indeed, the nearly 180 degrees difference in hexose orientation explains the equatorial versus axial amino transfer exhibited by DesI and PseC, respectively.     

Structural studies of Salmonella typhimurium ArnB (PmrH) aminotransferase: a 4-amino-4-deoxy-L-arabinose lipopolysaccharide-modifying enzyme

Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as Salmonella, resistance to cationic antimicrobial peptides, including those derived from the innate immune system. ArnB catalysis of amino group transfer from glutamic acid to the 4"-position of a UDP-linked ketopyranose molecule to form UDP-4-amino-4-deoxy-L-arabinose represents a key step in the lipid A modification pathway. Structural and functional studies of the ArnB aminotransferase were undertaken by combining X-ray crystallography with biochemical analyses. High-resolution crystal structures were solved for two native forms and one covalently inhibited form of S. typhimurium ArnB. These structures permitted identification of key residues involved in substrate binding and catalysis, including a rarely observed nonprolyl cis peptide bond in the active site.     

Crystal Structure of Helicobacter pylori Pseudaminic Acid Biosynthesis N-Acetyltransferase PseH: Implications for Substrate Specificity and Catalysis

Helicobacter pylori infection is the common cause of gastroduodenal diseases linked to a higher risk of the development of gastric cancer. Persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. It belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. The crystal structure of the PseH complex with cofactor acetyl-CoA has been determined at 2.3 Å resolution. This is the first crystal structure of the GNAT superfamily member with specificity to UDP-4-amino-4,6-dideoxy-β-L-AltNAc. PseH is a homodimer in the crystal, each subunit of which has a central twisted β-sheet flanked by five α-helices and is structurally homologous to those of other GNAT superfamily enzymes. Interestingly, PseH is more similar to the GNAT enzymes that utilize amino acid sulfamoyl adenosine or protein as a substrate than a different GNAT-superfamily bacterial nucleotide-sugar N-acetyltransferase of the known structure, WecD. Analysis of the complex of PseH with acetyl-CoA revealed the location of the cofactor-binding site between the splayed strands β4 and β5. The structure of PseH, together with the conservation of the active-site general acid among GNAT superfamily transferases, are consistent with a common catalytic mechanism for this enzyme that involves direct acetyl transfer from AcCoA without an acetylated enzyme intermediate. Based on structural homology with microcin C7 acetyltransferase MccE and WecD, the Michaelis complex can be modeled. The model suggests that the nucleotide- and 4-amino-4,6-dideoxy-β-L-AltNAc-binding pockets form extensive interactions with the substrate and are thus the most significant determinants of substrate specificity. A hydrophobic pocket accommodating the 6’-methyl group of the altrose dictates preference to the methyl over the hydroxyl group and thus to contributes to substrate specificity of PseH.     

Molecular architecture of DesV from Streptomyces venezuelae: a PLP-dependent transaminase involved in the biosynthesis of the unusual sugar desosamine

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. Six enzymes are required for its biosynthesis in Streptomyces venezuelae. The focus of this article is DesV, which catalyzes the PLP-dependent replacement of a 3-keto group with an amino functionality in the fifth step of the pathway. For this study the three-dimensional structures of both the internal aldimine and the ketimine intermediate with glutamate were determined to 2.05 A resolution. DesV is a homodimer with each subunit containing 12 alpha-helical regions and 12 beta-strands that together form three layers of sheet. The structure of the internal aldimine demonstrates that the PLP-cofactor is held in place by residues contributed from both subunits (Asp 164 and Gln 167 from Subunit I and Tyr 221 and Asn 235 from Subunit II). When the ketimine intermediate is present in the active site, the loop defined by Gln 225 to Ser 228 from Subunit II closes down upon the active site. The structure of DesV is similar to another sugar-modifying enzyme referred to as PseC. This enzyme is involved in the biosynthesis of pseudaminic acid, which is a sialic acid-like nonulosonate found in the flagellin of Helicobacter pylori. In the case of PseC, however, the amino group is transferred to the C-4 rather than the C-3 position. Details concerning the structural analysis of DesV and a comparison of its molecular architecture to that of PseC are presented.     

Structural,genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori

Mass spectrometry analyses of the complex polar flagella from Helicobacter pylori demonstrated that both FlaA and FlaB proteins are post-translationally modified with pseudaminic acid (Pse5Ac7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno -n o n-ulosonic acid). Unlike Campylobacter, flagellar glycosylation in Helicobacter displays little heterogeneity in isoform or glycoform distribution, although all glycosylation sites are located in the central core region of the protein monomer in a manner similar to that found in Campylobacter. Bioinformatic analysis revealed five genes (HP0840, HP0178, HP0326A, HP0326B, HP0114) homologous to other prokaryote genes previously reported to be involved in motility, flagellar glycosylation or polysaccharide biosynthesis. Insertional mutagenesis of four of these homologues in Helicobacter (HP0178, HP0326A, HP0326B, HP0114) resulted in a non-motile phenotype, no structural flagella filament and only minor amounts of flagellin protein detectable by Western immunoblot. However, mRNA levels for the flagellin structural genes remained unaffected by each mutation. In view of the combined bioinformatic and structural evidence indicating a role for these gene products in glycan biosynthesis, subsequent investigations focused on the functional characterization of the respective gene products. A novel approach was devised to identify biosynthetic sugar nucleotide precursors from intracellular metabolic pools of parent and isogenic mutants using capillary electrophoresis-electrospray mass spectrometry (CE-ESMS) and precursor ion scanning. HP0326A, HP0326B and the HP0178 gene products are directly involved in the biosynthesis of the nucleotide-activated form of Pse, CMP-Pse. Mass spectral analyses of the cytosolic extract from the HP0326A and HP0326B isogenic mutants revealed the accumulation of a mono- and a diacetamido trideoxyhexose UDP sugar nucleotide precursor.     

Helicobacter pylori HP0518 affects flagellin glycosylation to alter bacterial motility

Helicobacter pylori is a human gastric pathogen associated with gastric and duodenal ulcers as well as gastric cancer. Mounting evidence suggests this pathogen's motility is prerequisite for successful colonization of human gastric tissues. Here, we isolated an H. pylori G27 HP0518 mutant exhibiting altered motility in comparison to its parental strain. We show that the mutant's modulated motility is linked to increased levels of O-linked glycosylation on flagellin A (FlaA) protein. Recombinant HP0518 protein decreased glycosylation levels of H. pylori flagellin in vitro, indicating that HP0518 functions in deglycosylation of FlaA protein. Furthermore, mass spectrometric analysis revealed increased glycosylation of HP0518 FlaA was due to a change in pseudaminic acid (Pse) levels on FlaA; HP0518 mutant-derived flagellin contained approximately threefold more Pse than the parental strain. Further phenotypic and molecular characterization demonstrated that the hyper-motile HP0518 mutant exhibits superior colonization capabilities and subsequently triggers enhanced CagA phosphorylation and NF-κB activation in AGS cells. Our study shows that HP0518 is involved in the deglycosylation of flagellin, thereby regulating pathogen motility. These findings corroborate the prominent function of H. pylori flagella in pathogen-host cell interactions and modulation of host cell responses, likely influencing the pathogenesis process.     

Cj1121c, a novel UDP-4-keto-6-deoxy-GlcNAc C-4 aminotransferase essential for protein glycosylation and virulence in Campylobacter jejuni

Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins carry diacetamidobacillosamine (DAB), a sugar that is not found in humans. Hence, the enzymes responsible for DAB synthesis represent potential therapeutic targets. We describe the biochemical characterization of Cj1121c, a putative aminotransferase encoded by the general protein glycosylation locus, to assess its role in DAB biosynthesis. By using overexpressed and affinity-purified enzyme, we demonstrate that Cj1121c has pyridoxal phosphate- and glutamate-dependent UDP-4-keto-6-deoxy-GlcNAc C-4 transaminase activity and produces UDP-4-amino-4,6-dideoxy-GlcNAc. This is consistent with a role in DAB biosynthesis and distinguishes Cj1121c from Cj1294, a homologous UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-4-hexulose C-4 aminotransferase that we characterized previously. We show that Cj1121c can also use this 4-keto-arabino sugar indirectly as a substrate, that Cj1121c and Cj1294 are active simultaneously in C. jejuni, and that the activity of Cj1121c is preponderant under standard growth conditions. Kinetic data indicate that Cj1121c has a slightly higher catalytic efficiency than Cj1294 with regard to the 4-keto-arabino substrate. By site-directed mutagenesis, we show that residues Glu-158 and Leu-131 are not essential for catalysis or for substrate specificity contrary to expectations. We further demonstrate that a cj1121c knock-out mutant is impaired for flagella-mediated motility, for invasion of intestinal epithelial cells, and for persistence in the chicken intestine, clearly demonstrating that Cj1121c is essential for host colonization and virulence. Finally, we show that cj1121c is necessary for protein glycosylation by lectin Western blotting. Collectively, these results validate Cj1121c as a promising drug target and provide the means to assay for inhibitors.     

Cloning and characterization of spermidine synthase and its implication in polyamine biosynthesis in Helicobacter pylori strain 26695

The HP0832 (speE) gene of Helicobacter pylori strain 26695 codes for a putative spermidine synthase, which belongs to the polyamine biosynthetic pathway. Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), which serves as an aminopropyl donor. The deduced amino acid sequence of the HP0832 gene shares less than 20% sequence identity with most spermidine synthases from mammalian cells, plants and other bacteria. In this study, the HP0832 open reading frame (786 bp) was cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0832 protein (31.9 kDa) was purified by Ni-NTA affinity chromatography at a yield of 15 mg/L of bacteria culture. Spermidine synthase activity of the recombinant protein was confirmed by the appearance of spermidine after incubating the enzyme with putrescine and dcSAM. Substrate specificity studies have shown that spermidine could not replace putrescine as the aminopropyl acceptor. Endogenous spermidine synthase of H. pylori was detected with an antiserum raised against the recombinant HP0832 protein. H. pylori strain 26695 contains putrescine and spermidine at a molar ratio of 1:3, but no detectable spermine or norspermidine was observed, suggesting that the spermidine biosynthetic pathway may provide the main polyamines in H. pylori strain 26695.     

Structural studies of the L-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica: the apo,substrate, and product-aldimine complexes

The evolution of biosynthetic pathways is difficult to reconstruct in hindsight; however, the structures of the enzymes that are involved may provide insight into their development. One enzyme in the cobalamin biosynthetic pathway that appears to have evolved from a protein with different function is L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica, which is structurally similar to histidinol phosphate aminotransferase [Cheong, C. G., Bauer, C. B., Brushaber, K. R., Escalante-Semerena, J. C., and Rayment, I. (2002) Biochemistry 41, 4798-4808]. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. To understand the relationship between this decarboxylase and the aspartate aminotransferase family to which it belongs, the structures of CobD in its apo state, the apo state complexed with the substrate, and its product external aldimine complex have been determined at 1.46, 1.8, and 1.8 A resolution, respectively. These structures show that the enzyme steers the breakdown of the external aldimine toward decarboxylation instead of amino transfer by positioning the carboxylate moiety of the substrate out of the plane of the pyridoxal ring and by placing the alpha-hydrogen out of reach of the catalytic base provided by the lysine that forms the internal aldimine. It would appear that CobD evolved from a primordial PLP-dependent aminotransferase, where the selection was based on similarities between the stereochemical properties of the substrates rather than preservation of the fate of the external aldimine. These structures provide a sequence signature for distinguishing between L-threonine-O-3-phosphate decarboxylase and histidinol phosphate aminotransferases, many of which appear to have been misannotated.     

Crystal structure and catalytic mechanism of PglD from Campylobacter jejuni

The carbohydrate 2, 4-diacetamido-2, 4, 6-trideoxy-alpha-D-glucopyranose (BacAc(2)) is found in a variety of eubacterial pathogens. In Campylobacter jejuni, PglD acetylates the C4 amino group on UDP-2-acetamido-4-amino-2, 4, 6-trideoxy-alpha-D-glucopyranose (UDP-4-amino-sugar) to form UDP-BacAc(2). Sequence analysis predicts PglD to be a member of the left-handed beta helix family of enzymes. However, poor sequence homology between PglD and left-handed beta helix enzymes with existing structural data precludes unambiguous identification of the active site. The co-crystal structures of PglD in the presence of citrate, acetyl coenzyme A, or the UDP-4-amino-sugar were solved. The biological assembly is a trimer with one active site formed between two protomers. Residues lining the active site were identified, and results from functional assays on alanine mutants suggest His-125 is critical for catalysis, whereas His-15 and His-134 are involved in substrate binding. These results are discussed in the context of implications for proteins homologous to PglD in other pathogens.     

Giant DNA virus mimivirus encodes pathway for biosynthesis of unusual sugar 4-amino-4,6-dideoxy-D-glucose (Viosamine)

Mimivirus is one the largest DNA virus identified so far, infecting several Acanthamoeba species. Analysis of its genome revealed the presence of a nine-gene cluster containing genes potentially involved in glycan formation. All of these genes are co-expressed at late stages of infection, suggesting their role in the formation of the long fibers covering the viral surface. Among them, we identified the L136 gene as a pyridoxal phosphate-dependent sugar aminotransferase. This enzyme was shown to catalyze the formation of UDP-4-amino-4,6-dideoxy-D-glucose (UDP-viosamine) from UDP-4-keto-6-deoxy-D-glucose, a key compound involved also in the biosynthesis of L-rhamnose. This finding further supports the hypothesis that Mimivirus encodes a glycosylation system that is completely independent of the amoebal host. Viosamine, together with rhamnose, (N-acetyl)glucosamine, and glucose, was found as a major component of the viral glycans. Most of the sugars were associated with the fibers, confirming a capsular-like nature of the viral surface. Phylogenetic analysis clearly indicated that L136 was not a recent acquisition from bacteria through horizontal gene transfer, but it was acquired very early during evolution. Implications for the origin of the glycosylation machinery in giant DNA virus are also discussed.     

Biochemical characterization of the Campylobacter jejuni Cj1294, a novel UDP-4-keto-6-deoxy-GlcNAc aminotransferase that generates UDP-4-amino-4,6-dideoxy-GalNAc

Campylobacter jejuni produces multiple glycoproteins whose glycans contain 4-amino 6-deoxy sugars or their derivatives, such as diacetamidobacillosamine or pseudaminic acid. Because the proteoglycans contribute to bacterial virulence and their constitutive sugars are not commonly found in humans, inhibitors developed against the enzymes that are responsible for their biosynthesis could be novel therapeutic targets to fight this important food-borne pathogen. The biosynthesis of diacetamidobacillosamine is anticipated to involve a sugar nucleotide C6 dehydratase, a C4 aminotransferase and an acetyltransferase. We have identified a set of genes (cj1293, cj1294, and cj1298) potentially encoding one of each enzymatic activity, and demonstrated earlier that Cj1293 was a UDP-GlcNAc-specific C6 dehydratase. Others have shown that Cj1293 was involved in protein glycosylation. Here, we report on our investigation of the potential activity of Cj1294 as a sugar nucleotide C4 aminotransferase. Our biochemical characterization of overexpressed and purified protein shows that Cj1294 is a pyridoxal phosphate-dependent aminotransferase specific for UDP-4-keto-6-deoxy-GlcNAc that uses preferentially glutamic acid as an amino donor. A detailed physicokinetic study of Cj1294 was performed to determine the K(m) of 1.28 +/- 0.2 mm and k(cat) of 11.5 +/- 1.3 min(-1). Also, two residues essential for protein stability and activity, Arg(228) and Lys(181), respectively, were identified by site-directed mutagenesis. Finally, we demonstrated by NMR analysis of purified reaction product that Cj1294 produces UDP-4-amino-4,6-dideoxy-GalNAc. These results indicate that Cj1294 is involved in the biosynthesis of diacetamidofucosamine, a C4 epimer of diacetamidobacillosamine not yet described in C. jejuni proteoglycans, suggesting that the composition of C. jejuni proteoglycans is more variable than anticipated.     

Crystal structure of Escherichia coli ArnA (PmrI) decarboxylase domain. A key enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance

Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa can modify the structure of lipid A in their outer membrane with 4-amino-4-deoxy-l-arabinose (Ara4N). Such modification results in resistance to cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin. ArnA is a key enzyme in the lipid A modification pathway, and its deletion abolishes both the Ara4N-lipid A modification and polymyxin resistance. ArnA is a bifunctional enzyme. It can catalyze (i) the NAD(+)-dependent decarboxylation of UDP-glucuronic acid to UDP-4-keto-arabinose and (ii) the N-10-formyltetrahydrofolate-dependent formylation of UDP-4-amino-4-deoxy-l-arabinose. We show that the NAD(+)-dependent decarboxylating activity is contained in the 360 amino acid C-terminal domain of ArnA. This domain is separable from the N-terminal fragment, and its activity is identical to that of the full-length enzyme. The crystal structure of the ArnA decarboxylase domain from E. coli is presented here. The structure confirms that the enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family. On the basis of sequence and structure comparisons of the ArnA decarboxylase domain with other members of the short-chain dehydrogenase/reductase (SDR) family, we propose a binding model for NAD(+) and UDP-glucuronic acid and the involvement of residues T(432), Y(463), K(467), R(619), and S(433) in the mechanism of NAD(+)-dependent oxidation of the 4'-OH of the UDP-glucuronic acid and decarboxylation of the UDP-4-keto-glucuronic acid intermediate.     

Crystal structure of 4-methyl-5-beta-hydroxyethylthiazole kinase from Bacillus subtilis at 1.5 A resolution

4-Methyl-5-beta-hydroxyethylthiazole kinase (ThiK) catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole (Thz). This enzyme is a salvage enzyme in the thiamin biosynthetic pathway and enables the cell to use recycled Thz as an alternative to its synthesis from 1-deoxy-D-xylulose-5-phosphate, cysteine, and tyrosine. The structure of ThiK in the rhombohedral crystal form has been determined to 1.5 A resolution and refined to a final R-factor of 21. 6% (R-free 25.1%). The structures of the enzyme/Thz complex and the enzyme/Thz-phosphate/ATP complex have also been determined. ThiK is a trimer of identical subunits. Each subunit contains a large nine-stranded central beta-sheet flanked by helices. The overall fold is similar to that of ribokinase and adenosine kinase, although sequence similarity is not immediately apparent. The area of greatest similarity occurs in the ATP-binding site where several key residues are highly conserved. Unlike adenosine kinase and ribokinase, in which the active site is located between two domains within a single subunit, the ThiK active site it formed at the interface between two subunits within the trimer. The structure of the enzyme/ATP/Thz-phosphate complex suggests that phosphate transfer occurs by an inline mechanism. Although this mechanism is similar to that proposed for both ribokinase and adenosine kinase, ThiK lacks an absolutely conserved Asp thought to be important for catalysis in the other two enzymes. Instead, ThiK has a conserved cysteine (Cys198) in this position. When this Cys is mutated to Asp, the enzymatic activity increases 10-fold. Further sequence analysis suggests that another thiamin biosynthetic enzyme (ThiD), which catalyzes the formation of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by two sequential phosphorylation reactions, belongs to the same family of small molecule kinases.     

Acyl group specificity at the active site of tetrahydridipicolinate N-succinyltransferase

Tetrahydrodipicolinate N-succinyltransferase (DapD) catalyzes the succinyl-CoA-dependent acylation of L-2-amino-6-oxopimelate to 2-N-succinyl-6-oxopimelate as part of the succinylase branch of the meso-diaminopimelate/lysine biosynthetic pathway of bacteria, blue-green algae, and plants. This pathway provides meso-diaminopimelate as a building block for cell wall peptidoglycan in most bacteria, and is regarded as a target pathway for antibacterial agents. We have solved the X-ray crystal structures of DapD in ternary complexes with pimelate/succinyl-CoA and L-2-aminopimelate with the nonreactive cofactor analog, succinamide-CoA. These structures define the binding conformation of the cofactor succinyl group and its interactions with the enzyme and place its thioester carbonyl carbon in close proximity to the nucleophilic 2-amino group of the acceptor, in support of a direct attack ternary complex mechanism. The acyl group specificity differences between homologous tetrahydrodipicolinate N-acetyl- and N-succinyltransferases can be rationalized with reference to at least three amino acids that interact with or give accessible active site volume to the cofactor succinyl group. These residues account at least in part for the substrate specificity that commits metabolic intermediates to either the succinylase or acetylase branches of the meso-diaminopimelate/lysine biosynthetic pathway.     

Accommodation of GDP-linked sugars in the active site of GDP-perosamine synthase

Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.     

Structure and mutagenic conversion of E1 dehydrase: at the crossroads of dehydration, amino transfer, and epimerization

Pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) are highly versatile coenzymes whose importance is well recognized. The capability of PLP/PMP-dependent enzymes to catalyze a diverse array of chemical reactions is attributed to fine-tuning of the cofactor-substrate interactions in the active site. CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E1), along with its reductase (E3), catalyzes the C-3 deoxygenation of CDP-4-keto-6-deoxy-D-glucose to form the dehydrated product, CDP-4-keto-3,6-dideoxy- d-glucose, in the ascarylose biosynthetic pathway. This product is the progenitor to most 3,6-dideoxyhexoses, which are the major antigenic determinants of many Gram-negative pathogens. The dimeric [2Fe-2S] protein, E 1, cloned from Yersinia pseudotuberculosis, is the only known enzyme whose catalysis involves the direct participation of PMP in one-electron redox chemistry. E1 also contains an unusual [2Fe-2S] cluster with a previously unknown binding motif (C-X 57-C-X 1-C-X 7-C). Herein we report the first X-ray crystal structure of E1, which exhibits an aspartate aminotransferase (AAT) fold. A comparison of the E1 active site architecture with homologous structures uncovers residues critical for the dehydration versus transamination activity. Site-directed mutagenesis of four E1 residues, D194H, Y217H, H220K, and F345H, converted E 1 from a PMP-dependent dehydrase to a PLP/glutamate-dependent aminotransferase. The E1 quadruple mutant, having been conferred this altered enzyme activity, can transaminate the natural substrate to CDP-4,6-dideoxy-4-amino-D-galactose without E3. Taken together, these results provide the molecular basis of the functional switch of E1 toward dehydration, epimerization, and transamination. The insights gained from these studies can be used for the development of inhibitors of disease-relevant PLP/PMP-dependent enzymes.     

5'-methylthioadenosine nucleosidase is implicated in playing a key role in a modified futalosine pathway for menaquinone biosynthesis in Campylobacter jejuni

Menaquinone (vitamin K(2)) serves as an electron carrier in the electron transport chain required for respiration in many pathogenic bacteria. Most bacteria utilize a common menaquinone biosynthetic pathway as exemplified by Escherichia coli. Recently, a novel biosynthetic pathway, the futalosine pathway, was discovered in Streptomyces. Bioinformatic analysis strongly suggests that this pathway is also operative in the human pathogens Campylobacter jejuni and Helicobacter pylori. Here, we provide compelling evidence that a modified futalosine pathway is operative in C. jejuni and that it utilizes 6-amino-6-deoxyfutalosine instead of futalosine. A key step in the Streptomyces pathway involves a nucleosidase called futalosine hydrolase. The closest homolog in C. jejuni has been annotated as a 5'-methylthioadenosine nucleosidase (MTAN). We have shown that this C. jejuni enzyme has MTAN activity but negligible futalosine hydrolase activity. However, the C. jejuni MTAN is able to hydrolyze 6-amino-6-deoxyfutalosine at a rate comparable with that of its known substrates. This suggests that the adenine-containing version of futalosine is the true biosynthetic intermediate in this organism. To demonstrate this in vivo, we constructed a C. jejuni mutant strain deleted for mqnA2, which is predicted to encode for the enzyme required to synthesize 6-amino-6-deoxyfutalosine. Growth of this mutant was readily rescued by the addition of 6-amino-6-deoxyfutalosine, but not futalosine. This provides the first direct evidence that a modified futalosine pathway is operative in C. jejuni. It also highlights the tremendous versatility of the C. jejuni MTAN, which plays key roles in S-adenosylmethionine recycling, the biosynthesis of autoinducer molecules, and the biosynthesis of menaquinone.     

Synthesis of mono- and di-fucosylated type I Lewis blood group antigens by Helicobacter pylori.

The identification of Helicobacter pylori isolates that expresses exclusively type I Lewis antigens is necessary to determine the biosynthetic pathway of these antigens. Fast-atom bombardment MS provides evidence that the H. pylori isolate UA1111 expresses predominantly Leb, with H type I and Lea in lesser amounts. Cloning and expression of the H. pylori fucosyltransferases (FucTs) allow comparisons with previously identified H. pylori enzymes and determination of the enzyme specificities. Although all FucTs, one alpha(1,2) FucT and two alpha(1,3/4) FucTs, appear to be functional in this isolate, their activities are lower and enzyme specificities are different to other H. pylori FucTs previously characterized. Studies of the cloned enzyme activities and mutational analysis indicate that Lea acts as the substrate for the synthesis of Leb. This is different from the human Leb biosynthetic pathway, but analogous to the biosynthetic pathway utilized by H. pylori for the production of Ley.