Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.

Iron-Sulfur Cluster Repair Contributes to Yersinia pseudotuberculosis Survival within Deep Tissues.

To successfully colonize host tissues, bacteria must respond to and detoxify many different host-derived antimicrobial compounds, such as nitric oxide (NO). NO has direct antimicrobial activity through attack on iron-sulfur (Fe-S) cluster-containing proteins. NO detoxification plays an important role in promoting bacterial survival, but it remains unclear if repair of Fe-S clusters is also important for bacterial survival within host tissues. Here we show that the Fe-S cluster repair protein YtfE contributes to the survival of Yersinia pseudotuberculosis within the spleen following nitrosative stress. Y. pseudotuberculosis forms clustered centers of replicating bacteria within deep tissues, where peripheral bacteria express the NO-detoxifying gene hmp. ytfE expression also occurred specifically within peripheral cells at the edges of microcolonies. In the absence of ytfE, the area of microcolonies was significantly smaller than that of the wild type (WT), consistent with ytfE contributing to the survival of peripheral cells. The loss of ytfE did not alter the ability of cells to detoxify NO, which occurred within peripheral cells in both WT and ΔytfE microcolonies. In the absence of NO-detoxifying activity by hmp, NO diffused across ΔytfE microcolonies, and there was a significant decrease in the area of microcolonies lacking ytfE, indicating that ytfE also contributes to bacterial survival in the absence of NO detoxification. These results indicate a role for Fe-S cluster repair in the survival of Y. pseudotuberculosis within the spleen and suggest that extracellular bacteria may rely on this pathway for survival within host tissues.

Wzx flippases exhibiting complex O-unit preferences require a new model for Wzx-substrate interactions.

The Wzx flippase is a critical component of the O-antigen biosynthesis pathway, being responsible for the translocation of oligosaccharide O units across the inner membrane in Gram-negative bacteria. Recent studies have shown that Wzx has a strong preference for its cognate O unit, but the types of O-unit structural variance that a given Wzx can accommodate are poorly understood. In this study, we identified two Yersinia pseudotuberculosis Wzx that can distinguish between different terminal dideoxyhexose sugars on a common O-unit main-chain, despite both being able to translocate several other structurally-divergent O units. We also identified other Y. pseudotuberculosis Wzx that can translocate a structurally divergent foreign O unit with high efficiency, and thus exhibit an apparently relaxed substrate preference. It now appears that Wzx substrate preference is more complex than previously suggested, and that not all O-unit residues are equally important determinants of translocation efficiency. We propose a new "Structure-Specific Triggering" model in which Wzx translocation proceeds at a low level for a wide variety of substrates, with high-frequency translocation only being triggered by Wzx interacting with one or more preferred O-unit structural elements found on its cognate O unit(s).

GTP Preference of d-Glycero-α-d-manno-Heptose-1-Phosphate Guanylyltransferase from Yersinia pseudotuberculosis.

d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) is the fourth enzyme synthesizing a building component of lipopolysaccharide (LPS) of Gram-negative bacteria. Since HddC is a potential new target to develop antibiotics, the analysis of the structural and functional relationship of the complex structure will lead to a better idea to design inhibitory compounds. X-ray crystallography and biochemical experiments to elucidate the guanine preference were performed based on the multiple sequence alignment. The crystal structure of HddC from Yersinia pseudotuberculosis (YPT) complexed with guanosine 5'-(ß-amino)-diphosphate (GMPPN) has been determined at 1.55 Å resolution. Meanwhile, the mutants revealed their reduced guanine affinity, instead of acquiring noticeable pyrimidine affinity. The complex crystal structure revealed that GMPPN is docked in the catalytic site with the aid of Glu80 positioning on the conserved motif EXXPLGTGGA. In the HddC family, this motif is expected to recruit nucleotides through interacting with bases. The crystal structure shows that oxygen atoms of Glu80 forming two hydrogen bonds play a critical role in interaction with two nitrogen atoms of the guanine base of GMPPN. Interestingly, the binding of GMPPN induced the formation of an oxyanion hole-like conformation on the L(S/A/G)X(S/G) motif and consequently influenced on inducing a conformational shift of the region around Ser55.

Crystal structure of d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase from Yersinia pseudotuberculosis.

The Gram-negative bacterium Yersinia pseudotuberculosis is the causative agent of yersiniosis. d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC) is the fourth enzyme of the GDP-d-glycero-α-d-manno-heptose biosynthesis pathway which is important for the virulence of the microorganism. Therefore, HddC is a potential target of antibiotics against yersiniosis. In this study, HddC from the synthesized HddC gene of Y. pseudotuberculosis has been expressed, purified, crystallized. Synchrotron X-ray data from a selenomethionine-substituted HddC crystal were also collected and its structure was determined at 2.0Å resolution. Structure analyses revealed that it belongs to the glycosyltransferase A type superfamily members with the signature motif GXGXR for nucleotide binding. Despite of remarkable structural similarity, HddC uses GTP for catalysis instead of CTP and UTP which are used for other major family members, cytidylyltransferase and uridylyltransferase, respectively. We suggest that EXXPLGTGGA and L(S/A/G)X(S/G) motifs are probably essential to bind with GTP and a FSFE motif with substrate.

HmuS from Yersinia pseudotuberculosis is a non-canonical heme-degrading enzyme to acquire iron from heme.

Some Gram-negative pathogens import host heme into the cytoplasm and utilize it as an iron source for their survival. We report here that HmuS, encoded by the heme utilizing system (hmu) locus, cleaves the protoporphyrin ring to release iron from heme. A liquid chromatography/mass spectrometry analysis revealed that the degradation products of this reaction are two biliverdin isomers that result from transformation of a verdoheme intermediate. This oxidative heme degradation by HmuS required molecular oxygen and electrons supplied by either ascorbate or NADPH. Electrons could not be directly transferred from NADPH to heme; instead, ferredoxin-NADP+ reductase (FNR) functioned as a mediator. Although HmuS does not share amino acid sequence homology with heme oxygenase (HO), a well-known heme-degrading enzyme, absorption and resonance Raman spectral analyses suggest that the heme iron is coordinated with an axial histidine residue and a water molecule in both enzymes. The substitution of axial His196 or distal Arg102 with an alanine residue in HmuS almost completely eliminated heme-degradation activity, suggesting that Fe-His coordination and interaction of a distal residue with water molecules in the heme pocket are important for this activity.

YPTB3816 of Yersinia pseudotuberculosis strain IP32953 is a virulence-related metallo-oligopeptidase.

BACKGROUND: Although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations. RESULTS: In this study we found that oligopeptidase OpdA of Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli. Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used. CONCLUSIONS: The results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-oligopeptidases in particular. This is the first study demonstrating that opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity.

Lipopolysaccharide Biosynthesis Genes of Yersinia pseudotuberculosis Promote Resistance to Antimicrobial Chemokines.

Antimicrobial chemokines (AMCs) are a recently described family of host defense peptides that play an important role in protecting a wide variety of organisms from bacterial infection. Very little is known about the bacterial targets of AMCs or factors that influence bacterial susceptibility to AMCs. In an effort to understand how bacterial pathogens resist killing by AMCs, we screened Yersinia pseudotuberculosis transposon mutants for those with increased binding to the AMCs CCL28 and CCL25. Mutants exhibiting increased binding to AMCs were subjected to AMC killing assays, which revealed their increased sensitivity to chemokine-mediated cell death. The majority of the mutants exhibiting increased binding to AMCs contained transposon insertions in genes related to lipopolysaccharide biosynthesis. A particularly strong effect on susceptibility to AMC mediated killing was observed by disruption of the hldD/waaF/waaC operon, necessary for ADP-L-glycero-D-manno-heptose synthesis and a complete lipopolysaccharide core oligosaccharide. Periodate oxidation of surface carbohydrates also enhanced AMC binding, whereas enzymatic removal of surface proteins significantly reduced binding. These results suggest that the structure of Y. pseudotuberculosis LPS greatly affects the antimicrobial activity of AMCs by shielding a protein ligand on the bacterial cell surface.

Substrate-Determined Diastereoselectivity in an Enzymatic Carboligation.

Thiamine diphosphate-dependent enzymes catalyze the formation of C-C bonds, thereby generating chiral secondary or tertiary alcohols. By the use of vibrational circular dichroism (VCD) spectroscopy we studied the stereoselectivity of carboligations catalyzed by YerE, a carbohydrate-modifying enzyme from Yersinia pseudotuberculosis. Conversion of the non-physiological substrate (R)-3-methylcyclohexanone led to an R,R-configured tertiary alcohol (diastereomeric ratio (dr) >99:1), whereas the corresponding reaction with the S enantiomer gave the S,S-configured product (dr>99:1). This suggests that YerE-catalyzed carboligations can undergo either an R- or an S-specific pathway. We show that, in this case, the high stereoselectivity of the YerE-catalyzed reaction depends on the substrate's preference to acquire a low-energy conformation.

A Newly Identified Glutaminase-Free ʟ-Asparaginase (ʟ-ASPG86) from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens.

ʟ-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the Lasparaginase gene (ʟ-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of ʟ-ASPG86 (ʟ-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The ʟ-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant ʟ-asparaginase (r-ʟ-ASPG86) showed optimum conditions at 37-40oC, pH 9. Moreover, r-ʟ-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-LASPG86 was 687.1 units/mg under optimum conditions (37°C, pH 9, and 5 mM MnSO4).

Recombinant Phospholipase A1 of the Outer Membrane of Psychrotrophic Yersinia pseudotuberculosis: Expression, Purification, and Characterization.

The pldA gene encoding membrane-bound phospholipase A1 of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A1 (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A1 was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A1 were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A1 from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.

Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague.

Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host.

[Bacterial recombinant L-asparaginases: properties, structure and anti-proliferative activity].

For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.

Identification of potential Tpx inhibitors against pathogen-host interactions.

Yersinia organisms cause many infectious diseases by invading human cells and delivering their virulence factors via the type three secretion system (T3SS). One alternative strategy in the fight against these pathogenic organisms is to interfere with their T3SS. Previous studies demonstrated that thiol peroxidase, Tpx is functional in the assembly of T3SS and its inhibition by salicylidene acylhydrazides prevents the secretion of pathogenic effectors. In this study, the aim was to identify potential inhibitors of Tpx using an integrated approach starting with high throughput virtual screening and ending with molecular dynamics simulations of selected ligands. Virtual screening of ZINC database of 500,000 compounds via ligand-based and structure-based pharmacophore models retrieved 10,000 hits. The structure-based pharmacophore model was validated using high-throughput virtual screening (HTVS). After multistep docking (SP and XP), common scaffolds were used to find common substructures and the ligand binding poses were optimized using induced fit docking. The stability of the protein-ligand complex was examined with molecular dynamics simulations and the binding free energy of the complex was calculated. As a final outcome eight compounds with different chemotypes were proposed as potential inhibitors for Tpx. The eight ligands identified by a detailed virtual screening protocol can serve as leads in future drug design efforts against the destructive actions of pathogenic bacteria.

Silencing urease: a key evolutionary step that facilitated the adaptation of Yersinia pestis to the flea-borne transmission route.

The arthropod-borne transmission route of Yersinia pestis, the bacterial agent of plague, is a recent evolutionary adaptation. Yersinia pseudotuberculosis, the closely related food-and water-borne enteric species from which Y. pestis diverged less than 6,400 y ago, exhibits significant oral toxicity to the flea vectors of plague, whereas Y. pestis does not. In this study, we identify the Yersinia urease enzyme as the responsible oral toxin. All Y. pestis strains, including those phylogenetically closest to the Y. pseudotuberculosis progenitor, contain a mutated ureD allele that eliminated urease activity. Restoration of a functional ureD was sufficient to make Y. pestis orally toxic to fleas. Conversely, deletion of the urease operon in Y. pseudotuberculosis rendered it nontoxic. Enzymatic activity was required for toxicity. Because urease-related mortality eliminates 30-40% of infective flea vectors, ureD mutation early in the evolution of Y. pestis was likely subject to strong positive selection because it significantly increased transmission potential.

A cis-encoded sRNA controls the expression of fabH2 in Yersinia.

YsrH is a novel cis-encoded sRNA located on the opposite strand to fabH2, which is essential for fatty acid biosynthesis in bacteria. In this study, YsrH-mediated regulation of fabH2 expression was investigated in Yersinia pseudotuberculosis. Constitutive and inducible over-expression of YsrH decreased the mRNA level of fabH2, while expression of downstream fabD and fabG remained unaffected. Polynucleotide phosphorylase (PNPase) also played an important role in this regulation process by mediating YsrH decay in the exponential phase. Thus, our data defines a cis-encoded sRNA that regulates fatty acid synthesis via a regulatory mechanism also involving PNPase.

Immunization with a DNA adenine methylase over-producing Yersinia pseudotuberculosis vaccine confers robust cross-protection against heterologous pathogenic serotypes.

Yersinia pseudotuberculosis is a foodborne pathogen that can cause serious human illness. Although the source and route of transmission often remain obscure, livestock have been implicated in some cases. The diversity of yersiniae present on farms and their widespread distribution in animal and environmental reservoirs necessitates the use of broad prophylactic strategies that are efficacious against many serotypes simultaneously. Herein, immunization of mice with a modified, live attenuated Y. pseudotuberculosis vaccine that overproduces the DNA adenine methylase (Dam(OP)) conferred robust protection against virulent challenge (150-fold LD50) with homologous and heterologous serotypes that have been associated with human disease (O:1, O:1a, O:3). Further, the dam gene was shown to be essential for cell viability in all (7 of 7) Y. pseudotuberculosis strains tested. Direct selection for the inheritance of dam mutant alleles in Y. pseudotuberculosis resulted in dam strain variants that contained compensatory (second-site suppressor) mutations in genes encoding methyl-directed mismatch repair proteins (mutHLS) that are involved in suppression of the non-viable cell phenotype in all (19/19) strains tested. Such dam mutH variants exhibited a significant increase in virulence and spontaneous mutation frequency relative to that of a Dam(OP) vaccine strain. These studies indicate that Y. pseudotuberculosis Dam(OP) strains conferred potent cross-protective efficacy as well as decreased virulence and spontaneous mutation frequency relative to those that lack Dam, which have compensatory mutations in mutHLS loci. These data suggest that development of yersiniae livestock vaccines based on Dam overproduction is a viable mitigation strategy to reduce these potential foodborne contaminants.

[Cross-immunogenicity of various bacterial L-asparaginases].

AIM: Evaluate immune response in mice against various L-asparaginases and determine their cross-immunogenicity. MATERIALS AND METHODS: The studies were carried out in C57Bl(6j) line mice. Immunogenicity of L-asparaginases was studied: Escherichia coli type II (recombinant) (Medak, Germany) (EcA); Erwinia carotovora type II (ErA); Yersinia pseudotuberculosis type II (YpA); Rhodospirillum rubrum type I (RrA); Wollinella succinogenes type II (WsA). Immune response against the administered antigens was determined in EIA. RESULTS: Y. pseudotuberculosis L-asparaginase was the most immunogenic, E. coli--the least immunogenic. E. carotovora, R. rubrum, W. succinogenes asparaginases displayed intermediate immunogenicity. The results of cross-immunogenicity evaluation have established, that blood sera of mice, that had received YpA, showed cross-immunogenicity against all the other L-asparaginase preparations except E. carotovora. During immunization with E. coli L-asparaginase the developed antibodies also bound preparation from E. carotovora. Sera from mice immunized with W. succinogenes, E. carotovora and R. rubrum L-asparaginases had cross-reaction only with EcA and did not react with other preparations. CONCLUSION: Cross-immunogenicity of the studied L-asparaginases was determined. A sequence of administration of the studied preparation is proposed that allows to minimize L-asparaginase neutralization by cross-reacting antibodies.

Evidence that the fosfomycin-producing epoxidase, HppE, is a non-heme-iron peroxidase.

The iron-dependent epoxidase HppE converts (S)-2-hydroxypropyl-1-phosphonate (S-HPP) to the antibiotic fosfomycin [(1R,2S)-epoxypropylphosphonate] in an unusual 1,3-dehydrogenation of a secondary alcohol to an epoxide. HppE has been classified as an oxidase, with proposed mechanisms differing primarily in the identity of the O2-derived iron complex that abstracts hydrogen (H•) from C1 of S-HPP to initiate epoxide ring closure. We show here that the preferred cosubstrate is actually H2O2 and that HppE therefore almost certainly uses an iron(IV)-oxo complex as the H• abstractor. Reaction with H2O2 is accelerated by bound substrate and produces fosfomycin catalytically with a stoichiometry of unity. The ability of catalase to suppress the HppE activity previously attributed to its direct utilization of O2 implies that reduction of O2 and utilization of the resultant H2O2 were actually operant.

Biosynthesis of UDP-GlcNAc, UndPP-GlcNAc and UDP-GlcNAcA involves three easily distinguished 4-epimerase enzymes, Gne, Gnu and GnaB.

We have undertaken an extensive survey of a group of epimerases originally named Gne, that were thought to be responsible for inter-conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc). The analysis builds on recent work clarifying the specificity of some of these epimerases. We find three well defined clades responsible for inter-conversion of the gluco- and galacto-configuration at C4 of different N-acetylhexosamines. Their major biological roles are the formation of UDP-GalNAc, UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) and undecaprenyl pyrophosphate-N-acetylgalactosamine (UndPP-GalNAc) from the corresponding glucose forms. We propose that the clade of UDP-GlcNAcA epimerase genes be named gnaB and the clade of UndPP-GlcNAc epimerase genes be named gnu, while the UDP-GlcNAc epimerase genes retain the name gne. The Gne epimerases, as now defined after exclusion of those to be named GnaB or Gnu, are in the same clade as the GalE 4-epimerases for inter-conversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). This work brings clarity to an area that had become quite confusing. The identification of distinct enzymes for epimerisation of UDP-GlcNAc, UDP-GlcNAcA and UndPP-GlcNAc will greatly facilitate allocation of gene function in polysaccharide gene clusters, including those found in bacterial genome sequences. A table of the accession numbers for the 295 proteins used in the analysis is provided to enable the major tree to be regenerated with the inclusion of additional proteins of interest. This and other suggestions for annotation of 4-epimerase genes will facilitate annotation.