Biosynthesis of Cytidine Diphosphate-6-d-Glucitol for the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative pathogenic bacterium commonly found in chickens and is the leading cause of human diarrheal disease worldwide. The various serotypes of C. jejuni produce structurally distinct capsular polysaccharides (CPSs) on the exterior surfaces of the cell wall. The capsular polysaccharide from C. jejuni serotype HS:5 is composed of a repeating sequence of d-glycero-d-manno-heptose and d-glucitol-6-phosphate. We previously defined the pathway for the production of d-glycero-d-manno-heptose in C. jejuni. Here, we elucidate the biosynthetic pathway for the assembly of cytidine diphosphate (CDP)-6-d-glucitol by the combined action of two previously uncharacterized enzymes. The first enzyme catalyzes the formation of CDP-6-d-fructose from cytidine triphosphate (CTP) and d-fructose-6-phosphate. The second enzyme reduces CDP-6-d-fructose with NADPH to generate CDP-6-d-glucitol. Using sequence similarity network (SSN) and genome neighborhood network (GNN) analyses, we predict that these pairs of proteins are responsible for the biosynthesis of CDP-6-d-glucitol and/or CDP-d-mannitol in the lipopolysaccharides (LPSs) and capsular polysaccharides in more than 200 other organisms. In addition, high resolution X-ray structures of the second enzyme are reported, which provide novel insight into the manner in which an open-chain nucleotide-linked sugar is harbored in an active site cleft.

Structural analysis of a bacterial UDP-sugar 2-epimerase reveals the active site architecture before and after catalysis.

The sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, was first identified ∼40 years ago in the O-antigen of Pseudomonas aeruginosa O:3,a,d. Since then, it has been observed on the O-antigens of various pathogenic Gram-negative bacteria including Bordetella pertussis, Escherichia albertii, and Pseudomonas mediterranea. Previous studies have established that five enzymes are required for its biosynthesis beginning with uridine dinucleotide (UDP)-N-acetyl-d-glucosamine (UDP-GlcNAc). The final step in the pathway is catalyzed by a 2-epimerase, which utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate. Curious as to whether this biochemical pathway is found in extreme thermophiles, we examined the published genome sequence for Thermus thermophilus HB27 and identified five ORFs that could possibly encode for the required enzymes. The focus of this investigation is on the ORF WP_011172736, which we demonstrate encodes for a 2-epimerase. For this investigation, ten high resolution X-ray crystallographic structures were determined to resolutions of 2.3 Å or higher. The models have revealed the manner in which the 2-epimerase anchors its UDP-sugar substrate as well as its UDP-sugar product into the active site. In addition, this study reveals for the first time the manner in which any sugar 2-epimerase can simultaneously bind UDP-sugars in both the active site and the allosteric binding region. We have also demonstrated that the T. thermophilus enzyme is allosterically regulated by UDP-GlcNAc. Whereas the sugar 2-epimerases that function on UDP-GlcNAc have been the focus of past biochemical and structural analyses, this is the first detailed investigation of a 2-epimerase that specifically utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate.

Characterization of a novel inhibitor for the New Delhi metallo-ß-lactamase-4: Implications for drug design and combating bacterial drug resistance.

The bacterial metallo-ß-lactamases (MBLs) catalyze the inactivation of ß-lactam antibiotics. Identifying novel pharmacophores remains crucial for the clinical development of additional MBL inhibitors. Previously, 1-hydroxypyridine-2(1H)-thione-6-carboxylic acid, hereafter referred to as 1,2-HPT-6-COOH, was reported as a low cytotoxic nanomolar ß-lactamase inhibitor of Verona-integron-encoded metallo-ß-lactamase 2, capable of rescuing ß-lactam antibiotic activity. In this study, we explore its exact mechanism of inhibition and the extent of its activity through structural characterization of its binding to New Delhi metallo-ß-lactamase 4 (NDM-4) and its inhibitory activity against both NDM-1 and NDM-4. Of all the structure-validated MBL inhibitors available, 1,2-HPT-6-COOH is the first discovered compound capable of forming an octahedral coordination sphere with Zn2 of the binuclear metal center. This unexpected mechanism of action provides important insight for the further optimization of 1,2-HPT-6-COOH and the identification of additional pharmacophores for MBL inhibition.

Bifunctional Epimerase/Reductase Enzymes Facilitate the Modulation of 6-Deoxy-Heptoses Found in the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and the leading cause of food poisoning in the United States and Europe. Surrounding the exterior surface of this bacterium is a capsular polysaccharide (CPS) that consists of a repeating sequence of common and unusual carbohydrate segments. At least 10 different heptose sugars have thus far been identified in the various strains of C. jejuni. The accepted biosynthetic pathway for the construction of the 6-deoxy-heptoses begins with the 4,6-dehydration of GDP-d-glycero-d-manno-heptose by a dehydratase, followed by an epimerase that racemizes C3 and/or C5 of the product GDP-6-deoxy-4-keto-d-lyxo-heptose. In the final step, a C4-reductase catalyzes the NADPH reduction of the resulting 4-keto product. However, in some strains and serotypes of C. jejuni, there are two separate C4-reductases with different product specificities in the gene cluster for CPS formation. Five pairs of these tandem C4-reductases were isolated, and the catalytic properties were ascertained. In four out of five cases, one of the two C4-reductases is able to catalyze the isomerization of C3 and C5 of GDP-6-deoxy-4-keto-d-lyxo-heptose, in addition to the catalysis of the reduction of C4, thus bypassing the requirement for a separate C3/C5-isomerase. In each case, the 3'-end of the gene for the first C4-reductase contains a poly-G tract of 8-10 guanine residues that may be used to control the expression and/or catalytic activity of either C4-reductase. The three-dimensional structure of the C4-reductase from serotype HS:15, which only does a reduction of C4, was determined to 1.45 Å resolution in the presence of NADPH and GDP.

Investigation of the enzymes required for the biosynthesis of 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid in Psychrobacter cryohalolentis K5T.

Psychrobacter cryohalolentis K5T is a Gram-negative bacterium first isolated from Siberian permafrost in 2006. It has a complex O-antigen containing l-rhamnose, d-galactose, two diacetamido-sugars, and one triacetamido-sugar. The biosynthetic pathway for one of the diacetamido-sugars, namely 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid, is presently unknown. Utilizing the published genome sequence of P. cryohalolentis K5T , we hypothesized that the genes designated Pcryo_0613, Pcryo_0614, Pcryo_0616, and Pcryo_0615 encode for a uridine dinucleotide (UDP)-N-acetyl-d-glucosamine 6-dehydrogenase, an nicotinamide adenine dinucleotide (oxidized) (NAD+ )-dependent dehydrogenase, a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase, and an N-acetyltransferase, respectively, activities of which would be required for the biosynthesis of this unusual carbohydrate. Here we present the cloning, overexpression, and purification of these hypothetical proteins. Kinetic data on the enzymes encoded by Pcryo_0613, Pcryo_0614, and Pcryo_0615 confirmed their postulated biochemical activities. In addition, the high-resolution X-ray structures of both the internal and external aldimine forms of the aminotransferase were determined to 1.25 and 1.0 Å, respectively. Finally, the three-dimensional architecture of the N-acetyltransferase in complex with its substrate and coenzyme A was solved to 1.8 Å resolution. Strikingly, the N-acetyltransferase was shown to adopt a new motif for UDP-sugar binding. The data presented herein provide additional insight into sugar biosynthesis in Gram-negative bacteria.

C3- and C3/C5-Epimerases Required for the Biosynthesis of the Capsular Polysaccharides from Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and one of the leading causes of food poisoning in Europe and the United States. The outside of the bacterium is coated with a capsular polysaccharide that assists in the evasion of the host immune system. Many of the serotyped strains of C. jejuni contain a 6-deoxy-heptose moiety that is biosynthesized from GDP-d-glycero-d-manno-heptose by the successive actions of a 4,6-dehydratase, a C3/C5-epimerase, and a C4-reductase. We identified 18 different C3/C5-epimerases that could be clustered together into three groups at a sequence identity of >89%. Four of the enzymes from the largest cluster (from serotypes HS:3, HS:10, HS:23/36, and HS:41) were shown to only catalyze the epimerization at C3. Three enzymes from the second largest cluster (HS:2, HS:15, and HS:42) were shown to catalyze the epimerization at C3 and C5. Enzymes from the third cluster were not characterized. The three-dimensional structures of the epimerases from serotypes HS:3, HS:23/36, HS:15, and HS:41 were determined to resolutions of 1.5-1.9 Å. The overall subunit architecture places these enzymes into the diverse "cupin" superfamily. Within X-ray coordinate error, the immediate regions surrounding the active sites are identical, suggesting that factors extending farther out may influence product outcome. The X-ray crystal structures are consistent with His-67 and Tyr-134 acting as general acid/base catalysts for the epimerization of C3 and/or C5. Two amino acid changes (A76V/C136L) were enough to convert the C3-epimerase from serotype HS:3 to one that could now catalyze the epimerization at both C3 and C5.

Reaction Mechanism and Three-Dimensional Structure of GDP-d-glycero-α-d-manno-heptose 4,6-Dehydratase from Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in the United States and Europe. Surrounding the outside of the bacterium is a carbohydrate coat known as the capsular polysaccharide. Various strains of C. jejuni have different sequences of unusual sugars and an assortment of decorations. Many of the serotypes have heptoses with differing stereochemical arrangements at C2 through C6. One of the many common modifications is a 6-deoxy-heptose that is formed by dehydration of GDP-d-glycero-α-d-manno-heptose to GDP-6-deoxy-4-keto-d-lyxo-heptose via the action of the enzyme GDP-d-glycero-α-d-manno-heptose 4,6-dehydratase. Herein, we report the biochemical and structural characterization of this enzyme from C. jejuni 81-176 (serotype HS:23/36). The enzyme was purified to homogeneity, and its three-dimensional structure was determined to a resolution of 2.1 Å. Kinetic analyses suggest that the reaction mechanism proceeds through the formation of a 4-keto intermediate followed by the loss of water from C5/C6. Based on the three-dimensional structure, it is proposed that oxidation of C4 is assisted by proton transfer from the hydroxyl group to the phenolate of Tyr-159 and hydride transfer to the tightly bound NAD+ in the active site. Elimination of water at C5/C6 is most likely assisted by abstraction of the proton at C5 by Glu-136 and subsequent proton transfer to the hydroxyl at C6 via Ser-134 and Tyr-159. A bioinformatic analysis identified 19 additional 4,6-dehydratases from serotyped strains of C. jejuni that are 89-98% identical in the amino acid sequence, indicating that each of these strains should contain a 6-deoxy-heptose within their capsular polysaccharides.

Structure and function of an N-acetyltransferase from the human pathogen Acinetobacter baumannii isolate BAL_212.

Acinetobacter baumannii is a Gram-negative bacterium commonly found in soil and water that can cause human infections of the blood, lungs, and urinary tract. Of particular concern is its prevalence in health-care settings where it can survive on surfaces and shared equipment for extended periods of time. The capsular polysaccharide surrounding the organism is known to be the major contributor to virulence. The structure of the K57 capsular polysaccharide produced by A. baumannii isolate BAL_212 from Vietnam was recently shown to contain the rare sugar 4-acetamido-4,6-dideoxy-d-glucose. Three enzymes are required for its biosynthesis, one of which is encoded by the gene H6W49_RS17300 and referred to as VioB, a putative N-acetyltransferase. Here, we describe a combined structural and functional analysis of VioB. Kinetic analyses show that the enzyme does, indeed, function on dTDP-4-amino-4,6-dideoxy-d-glucose with a catalytic efficiency of 3.9 x 104  M-1  s-1 (±6000), albeit at a reduced value compared to similar enzymes. Three high-resolution X-ray structures of various enzyme/ligand complexes were determined to resolutions of 1.65 Å or better. One of these models represents an intermediate analogue of the tetrahedral transition state. Differences between the VioB structure and those determined for the N-acetyltransferases from Campylobacter jejuni (PglD), Caulobacter crescentus (PerB), and Psychrobacter cryohalolentis (Pcryo_0637) are highlighted. Taken together, this investigation sheds new insight into the Type I sugar N-acetyltransferases.

4-Deoxy-4-fluoro-GalNAz (4FGalNAz) Is a Metabolic Chemical Reporter of O-GlcNAc Modifications, Highlighting the Notable Substrate Flexibility of O-GlcNAc Transferase.

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

Biochemical investigation of an N-acetyltransferase from Helicobacter pullorum.

N-acetylated sugars are often found, for example, on the lipopolysaccharides of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on the capsular polysaccharides. Key enzymes involved in their biosynthesis are the sugar N-acetyltransferases. Here, we describe a structural and functional analysis of one such enzyme from Helicobacter pullorum, an emerging pathogen that may be associated with gastroenteritis and gallbladder and liver diseases. For this analysis, the gene BA919-RS02330 putatively encoding an N-acetyltransferase was cloned, and the corresponding protein was expressed and purified. A kinetic analysis demonstrated that the enzyme utilizes dTDP-3-amino-3,6-dideoxy-d-glucose as a substrate as well as dTDP-3-amino-3,6-dideoxy-d-galactose, albeit at a reduced rate. In addition to this kinetic analysis, a similar enzyme from Helicobacter bilis was cloned and expressed, and its kinetic parameters were determined. Seven X-ray crystallographic structures of various complexes of the H. pullorum wild-type enzyme (or the C80T variant) were determined to resolutions of 1.7 Å or higher. The overall molecular architecture of the H. pullorum N-acetyltransferase places it into the Class II left-handed-ß-helix superfamily (LßH). Taken together, the data presented herein suggest that 3-acetamido-3,6-dideoxy-d-glucose (or the galactose derivative) is found on either the H. pullorum O-antigen or in another of its complex glycoconjugates. A BLAST search suggests that more than 50 non-pylori Helicobacter spp. have genes encoding N-acetyltransferases. Given that there is little information concerning the complex glycans in non-pylori Helicobacter spp. and considering their zoonotic potential, our results provide new biochemical insight into these pathogens.

Investigation of the enzymes required for the biosynthesis of an unusual formylated sugar in the emerging human pathogen Helicobacter canadensis.

It is now well established that the Gram-negative bacterium, Helicobacter pylori, causes gastritis in humans. In recent years, it has become apparent that the so-called non-pylori Helicobacters, normally infecting pigs, cats, and dogs, may also be involved in human pathology via zoonotic transmission. Indeed, more than 30 species of non-pylori Helicobacters have been identified thus far. One such organism is Helicobacter canadensis, an emerging pathogen whose genome sequence was published in 2009. Given our long-standing interest in the biosynthesis of N-formylated sugars found in the O-antigens of some Gram-negative bacteria, we were curious as to whether H. canadensis produces such unusual carbohydrates. Here, we demonstrate using both biochemical and structural techniques that the proteins encoded by the HCAN_0198, HCAN_0204, and HCAN_0200 genes in H. canadensis, correspond to a 3,4-ketoisomerase, a pyridoxal 5'-phosphate aminotransferase, and an N-formyltransferase, respectively. For this investigation, five high-resolution X-ray structures were determined and the kinetic parameters for the isomerase and the N-formyltransferase were measured. Based on these data, we suggest that the unusual sugar, 3-formamido-3,6-dideoxy-d-glucose, will most likely be found in the O-antigen of H. canadensis. Whether N-formylated sugars found in the O-antigen contribute to virulence is presently unclear, but it is intriguing that they have been observed in such pathogens as Francisella tularensis, Mycobacterium tuberculosis, and Brucella melitensis.

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus.

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.

Biosynthesis of d-glycero-l-gluco-Heptose in the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. The exterior cell surface of C. jejuni is coated with a capsular polysaccharide (CPS) that is essential for the maintenance and integrity of the bacterial cell wall and evasion of the host immune response. The identity and sequences of the monosaccharide components of the CPS are quite variable and dependent on the specific strain of C. jejuni. It is currently thought that the immediate precursor for the multiple variations found in the heptose moieties of the C. jejuni CPS is GDP-d-glycero-α-d-manno-heptose. In C. jejuni NCTC 11168, the heptose moiety is d-glycero-l-gluco-heptose. It has previously been shown that Cj1427 catalyzes the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose using α-ketoglutarate as a cosubstrate. Cj1430 was now demonstrated to catalyze the double epimerization of this product at C3 and C5 to form GDP-d-glycero-4-keto-ß-l-xylo-heptose. Cj1428 subsequently catalyzes the stereospecific reduction of this GDP-linked heptose by NADPH to form GDP-d-glycero-ß-l-gluco-heptose. The three-dimensional crystal structure of Cj1430 was determined to a resolution of 1.85 Å in the presence of bound GDP-d-glycero-ß-l-gluco-heptose, a product analogue. The structure shows that it belongs to the cupin superfamily. The three-dimensional crystal structure of Cj1428 was solved in the presence of NADPH to a resolution of 1.50 Å. Its fold places it into the short-chain dehydrogenase/reductase superfamily. Typically, members in this family display a characteristic signature sequence of YXXXK, with the conserved tyrosine serving a key role in catalysis. In Cj1428, this residue is a phenylalanine.

Characterization of two enzymes from Psychrobacter cryohalolentis that are required for the biosynthesis of an unusual diacetamido-d-sugar.

Psychrobacter cryohalolentis strain K5T is a Gram-negative organism first isolated in 2006. It has a complex O-antigen that contains, in addition to l-rhamnose and d-galactose, two diacetamido- and a triacetamido-sugar. The biochemical pathways for the production of these unusual sugars are presently unknown. Utilizing the published genome sequence of the organism, we hypothesized that the genes 0612, 0638, and 0637 encode for a 4,6-dehydratase, an aminotransferase, and an N-acetyltransferase, respectively, which would be required for the biosynthesis of one of the diacetamido-sugars, 2,4-diacetamido-2,4,6-trideoxy-d-glucose, starting from UDP-N-acetylglucosamine. Here we present functional and structural data on the proteins encoded by the 0638 and 0637 genes. The kinetic properties of these enzymes were investigated by a discontinuous HPLC assay. An X-ray crystallographic structure of 0638, determined in its external aldimine form to 1.3 Å resolution, demonstrated the manner in which the UDP ligand is positioned into the active site. It is strikingly different from that previously observed for PglE from Campylobacter jejuni, which functions on the same substrate. Four X-ray crystallographic structures were also determined for 0637 in various complexed states at resolutions between 1.3 and 1.55 Å. Remarkably, a tetrahedral intermediate mimicking the presumed transition state was trapped in one of the complexes. The data presented herein confirm the hypothesized functions of these enzymes and provide new insight into an unusual sugar biosynthetic pathway in Gram-negative bacteria. We also describe an efficient method for acetyl-CoA synthesis that allowed us to overcome its prohibitive cost for this analysis.

The high-resolution structure of a UDP-L-rhamnose synthase from Acanthamoeba polyphaga Mimivirus.

For the field of virology, perhaps one of the most paradigm-shifting events so far in the 21st century was the identification of the giant double-stranded DNA virus that infects amoebae. Remarkably, this virus, known as Mimivirus, has a genome that encodes for nearly 1,000 proteins, some of which are involved in the biosynthesis of unusual sugars. Indeed, the virus is coated by a layer of glycosylated fibers that contain d-glucose, N-acetyl-d-glucosamine, l-rhamnose, and 4-amino-4,6-dideoxy-d-glucose. Here we describe a combined structural and enzymological investigation of the protein encoded by the open-reading frame L780, which corresponds to an l-rhamnose synthase. The structure of the L780/NADP+ /UDP-l-rhamnose ternary complex was determined to 1.45 Å resolution and refined to an overall R-factor of 19.9%. Each subunit of the dimeric protein adopts a bilobal-shaped appearance with the N-terminal domain harboring the dinucleotide-binding site and the C-terminal domain positioning the UDP-sugar into the active site. The overall molecular architecture of L780 places it into the short-chain dehydrogenase/reductase superfamily. Kinetic analyses indicate that the enzyme can function on either UDP- and dTDP-sugars but displays a higher catalytic efficiency with the UDP-linked substrate. Site-directed mutagenesis experiments suggest that both Cys 108 and Lys 175 play key roles in catalysis. This structure represents the first model of a viral UDP-l-rhamnose synthase and provides new details into these fascinating enzymes.

Structural Analysis of Cj1427, an Essential NAD-Dependent Dehydrogenase for the Biosynthesis of the Heptose Residues in the Capsular Polysaccharides of Campylobacter jejuni.

Many strains of Campylobacter jejuni display modified heptose residues in their capsular polysaccharides (CPS). The precursor heptose was previously shown to be GDP-d-glycero-α-d-manno-heptose, from which a variety of modifications of the sugar moiety have been observed. These modifications include the generation of 6-deoxy derivatives and alterations of the stereochemistry at C3-C6. Previous work has focused on the enzymes responsible for the generation of the 6-deoxy derivatives and those involved in altering the stereochemistry at C3 and C5. However, the generation of the 6-hydroxyl heptose residues remains uncertain due to the lack of a specific enzyme to catalyze the initial oxidation at C4 of GDP-d-glycero-α-d-manno-heptose. Here we reexamine the previously reported role of Cj1427, a dehydrogenase found in C. jejuni NTCC 11168 (HS:2). We show that Cj1427 is co-purified with bound NADH, thus hindering catalysis of oxidation reactions. However, addition of a co-substrate, α-ketoglutarate, converts the bound NADH to NAD+. In this form, Cj1427 catalyzes the oxidation of l-2-hydroxyglutarate back to α-ketoglutarate. The crystal structure of Cj1427 with bound GDP-d-glycero-α-d-manno-heptose shows that the NAD(H) cofactor is ideally positioned to catalyze the oxidation at C4 of the sugar substrate. Additionally, the overall fold of the Cj1427 subunit places it into the well-defined short-chain dehydrogenase/reductase superfamily. The observed quaternary structure of the tetrameric enzyme, however, is highly unusual for members of this superfamily.

Biochemical analysis of a sugar 4,6-dehydratase from Acanthamoeba polyphaga Mimivirus.

The exciting discovery of the giant DNA Mimivirus in 2003 challenged the conventional description of viruses in a radical way, and since then, dozens of additional giant viruses have been identified. It has now been demonstrated that the Mimivirus genome encodes for the two enzymes required for the production of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, namely a 4,6-dehydratase and an aminotransferase. In light of our long-standing interest in the bacterial 4,6-dehydratases and in unusual sugars in general, we conducted a combined structural and functional analysis of the Mimivirus 4,6-dehydratase referred to as R141. For this investigation, the three-dimensional X-ray structure of R141 was determined to 2.05 Å resolution and refined to an R-factor of 18.3%. The overall fold of R141 places it into the short-chain dehydrogenase/reductase (SDR) superfamily of proteins. Whereas its molecular architecture is similar to that observed for the bacterial 4,6-dehydratases, there are two key regions where the polypeptide chain adopts different conformations. In particular, the conserved tyrosine that has been implicated as a catalytic acid or base in SDR superfamily members is splayed away from the active site by nearly 12 Å, thereby suggesting that a major conformational change must occur upon substrate binding. In addition to the structural analysis, the kinetic parameters for R141 using either dTDP-d-glucose or UDP-d-glucose as substrates were determined. Contrary to a previous report, R141 demonstrates nearly identical catalytic efficiency with either nucleotide-linked sugar. The data presented herein represent the first three-dimensional model for a viral 4,6-dehydratase and thus expands our understanding of these fascinating enzymes.

Misannotations of the genes encoding sugar N-formyltransferases.

Tens of thousands of bacterial genome sequences are now known due to the development of rapid and inexpensive sequencing technologies. An important key in utilizing these vast amounts of data in a biologically meaningful way is to infer the function of the proteins encoded in the genomes via bioinformatics techniques. Whereas these approaches are absolutely critical to the annotation of gene function, there are still issues of misidentifications, which must be experimentally corrected. For example, many of the bacterial DNA sequences encoding sugar N-formyltransferases have been annotated as l-methionyl-tRNA transferases in the databases. These mistakes may be due in part to the fact that until recently the structures and functions of these enzymes were not well known. Herein we describe the misannotation of two genes, WP_088211966.1 and WP_096244125.1, from Shewanella spp. and Pseudomonas congelans, respectively. Although the proteins encoded by these genes were originally suggested to function as l-methionyl-tRNA transferases, we demonstrate that they actually catalyze the conversion of dTDP-4-amino-4,6-dideoxy-d-glucose to dTDP-4-formamido-4,6-dideoxy-d-glucose utilizing N10 -formyltetrahydrofolate as the carbon source. For this analysis, the genes encoding these enzymes were cloned and the corresponding proteins purified. X-ray structures of the two proteins were determined to high resolution and kinetic analyses were conducted. Both enzymes display classical Michaelis-Menten kinetics and adopt the characteristic three-dimensional structural fold previously observed for other sugar N-formyltransferases. The results presented herein will aid in the future annotation of these fascinating enzymes.

Structural and Functional Characterization of YdjI, an Aldolase of Unknown Specificity in Escherichia coli K12.

The ydj gene cluster is found in 80% of sequenced Escherichia coli genomes and other closely related species in the human microbiome. On the basis of the annotations of the enzymes located in this cluster, it is expected that together they catalyze the catabolism of an unknown carbohydrate. The focus of this investigation is on YdjI, which is in the ydj gene cluster of E. coli K-12. It is predicted to be a class II aldolase of unknown function. Here we describe a structural and functional characterization of this enzyme. YdjI catalyzes the hydrogen/deuterium exchange of the pro-S hydrogen at C3 of dihydroxyacetone phosphate (DHAP). In the presence of DHAP, YdjI catalyzes an aldol condensation with a variety of aldo sugars. YdjI shows a strong preference for higher-order (seven-, eight-, and nine-carbon) monosaccharides with specific hydroxyl stereochemistries and a negatively charged terminus (carboxylate or phosphate). The best substrate is l-arabinuronic acid with an apparent kcat of 3.0 s-1. The product, l-glycero-l-galacto-octuluronate-1-phosphate, has a kcat/Km value of 2.1 × 103 M-1 s-1 in the retro-aldol reaction with YdjI. This is the first recorded synthesis of l-glycero-l-galacto-octuluronate-1-phosphate and six similar carbohydrates. The crystal structure of YdjI, determined to a nominal resolution of 1.75 Å (Protein Data Bank entry 6OFU ), reveals unusual positions for two arginine residues located near the active site. Computational docking was utilized to distinguish preferable binding orientations for l-glycero-l-galacto-octuluronate-1-phosphate. These results indicate a possible alternative binding orientation for l-glycero-l-galacto-octuluronate-1-phosphate compared to that observed in other class II aldolases, which utilize shorter carbohydrate molecules.