GalE and Gne are UDP-Glc and UDP-GlcNAc 4-epimerases

The human UDP-Glc 4-epimerase can also use UDP-GlcNAc as a substrate, but this is not the case for the E. coli GalE enzyme [3]. However Bengoechea et al. [4] showed that a galE-like gene in the Yersinia enterocolitica O:8 OPS gene cluster coded for a UDP-GlcNAc 4-epimerase that could also use UDP-Glc as substrate. This is in addition to the galE gene in the gal operon. The new gene was named gne, a name first used for a UDP-GlcNAc epimerase gene in Bacillus subtilis [5].

Bengoechea et al. [4] also showed that for both the Y. enterocolitica Gne and the E. coli GalE epimerases, the ability to handle UDP-GlcNAc is determined by the size of the active site. Amino-acid substitutions that reduce its size in the Y. enterocolitica O8 Gne protein prevented use of UDP-GlcNAc, and substitutions in GalE of E. coli that expand its size allowed UDP-GlcNAc to become a substrate. Clearly in both cases we have what are effectively a Gne epimerase and a GalE epimerase that differ in a single amino acid residue.

Initial transferases in the Enterobacteriaceae

The Enterobacteriaceae are unusual in that most OPS gene clusters lack an initial transferase (IT) gene. The Enterobacterial Common Antigen (ECA), which is generally present in this family, has GlcNAc as its first sugar, and it has become clear that WecA, the IT for ECA, also acts as the IT for most OPS in the family. The role of WecA in initiation of OPS synthesis by formation of UndPP-GlcNAc was shown directly for several E. coli serogroups [6]. The extrapolation to most Enterobacterial OPS is based on the observation that those Enterobacterial OPS that lack an IT gene in their gene cluster have either GlcNAc, GalNAc or both in their structure. Where GlcNAc is first sugar, the role of WecA is assumed to be the same as for those studied by Alexander et al [6]. When, in the absence of a GlcNAc residue, GalNAc was implicated as first sugar, a galE-like gene was often observed in the gene cluster. This was originally assumed to be a gne gene, with the Gne product converting UDP-GlcNAc to UDP-GalNAc to provide an alternative substrate for WecA, while WecA was assumed to be able to use GalNAc as well as GlcNAc as the initial sugar. However two recent publications revealed fatal flaws in this hypothesis.

GlcNAc epimerase function in E. coli O157

The E. coli O157 repeat unit contains a single GalNAc but no GlcNAc residue and the gene cluster has no IT gene [7], so OPS synthesis was expected to be initiated by WecA acting on UDP-GalNAc. A galE homologue was observed just upstream of the OPS gene cluster and this was assigned as the then predicted gne gene [7], although the level of similarity to other gne genes was low. In 2010 Rush et al. [8] showed that the O157 epimerase had no activity on UDP-GlcNAc, but instead acted on UndPP-GlcNAc to give UndPP-GalNAc. The pathway was shown to be formation of UndPP-GlcNAc by WecA, followed by conversion to UndPP-GalNAc by the “Gne” epimerase previously assumed to convert UDP-GlcNAc to UDP-GalNAc. Clearly some of the enzymes presumed to be UDP-GlcNAc epimerases (Gne) have a different function.

Rush et al. [8] also compared the O157 epimerase with epimerases from other gene clusters for GalNAc-containing OPS, and showed that those where GalNAc was the first sugar were in a different homology group than those where GalNAc was not the first sugar.

E. coli O86 and Yersinia pseudotuberculosis O6 and O7 have separate UDP-GlcNAc and UndPP-GlcNAc epimerases for OPS synthesis

The second observation was with the well-studied E. coli O86 OPS. The gene cluster region contains two gne-like genes, referred to as gne1 and gne2, and knockouts of either resulted in an altered LPS phenotype, indicating that both are required for normal OPS synthesis [9], and hence must have different functions. This could not be understood at the time but with clarification of the situation in E. coli O157:H7 as discussed above, the explanation became clear. There are two GalNAc residues that are the first and second sugars in the biological repeat unit [10]. The gne1 gene is upstream of the repeat-unit gene cluster, at the same locus as the E. coli O157 gne gene, and the encoded proteins are identical. Gne1 of O86 must also be an UndPP-GlcNAc epimerase, converting the first sugar to UndPP-GalNAc. The gne2 gene is the first gene within the repeat-unit gene cluster (after the flanking gene galF), and Gne2 must of necessity be the UDP-GlcNAc epimerase producing the UDP-GalNAc substrate required as donor of the second GalNAc residue of the repeat unit, because it is required for OPS expression and it is the only conceivable function.

The Y. pseudotuberculosis O6 and O7 OPS also have two GalNAc residues, and share a pair of gne-like genes. In this case both genes are within the main gene clusters and are separated by a shared gene proposed to encode the GT linking the two GalNAc residues, which are the first and second sugars of the O unit. The genes are named gne1 and gne2 in map order, and on the basis of their sequence similarities, and knowing the role of the E. coli O157 gene, were proposed to encode UndPP-GlcNAc and UDP-GlcNAc epimerases respectively [11]. The three genes, present at the 3’ end in both gene clusters, constitute a module for assembly of the GalNAc-(1–3)-GalNAc-UndPP structure, starting with UndPP-GlcNAc formed by WecA [11].

Epimerisation in the formation of UDP-GalNAcA

There has also been a recent clarification on function of a “gne” gene in the UDP-GalNAcA pathway. Synthesis of UDP-GalNAcA from UDP-GlcNAc requires 2 steps, epimerisation at C4 and oxygenation at C6. In the case of the Pseudomonas aeruginosa O6 the two proteins are encoded in the OPS gene cluster and have been well studied. The pathway has recently been shown to be oxygenation of UDP-GlcNAc to UDP-GlcNAcA (WbpO) followed by epimerisation to UDP-GalNAcA (WbpP) [12]. This is the reverse of the originally proposed order [13]. WbpP, the epimerase, had previously been shown in vitro to convert UDP-GlcNAc to UDP-GalNAc as for the original Gne proteins, and WbpO to convert UDP-GalNAc to UDP-GalNAcA [13]. However it is now clear that while it is possible in vitro to carry out the oxygenation step before epimerisation [13,14], in vivo the epimerisation reaction is carried out on UDP-GlcNAcA after the oxygenation step [12,15].

Subdivision of Gne-like epimerases

In 2004 Ishiyama et al. [16] compiled a tree of UDP-hexose 4-epimerases using 56 sequences that had been annotated as UDP-Glc-4-epimerases, and divided them into three groups based on phylogeny and in vitro biochemical assays: those with preference for UDP-Glc (group 1), those with an equivalent activity on both UDP-Glc and UDP-GlcNAc (group 2), and those with a preference for UDP-GlcNAc (group 3) [16]. However, those studies predated the experiments discussed above and we undertook a reappraisal of the relationships.

Selection of Protien sequences and analysis

We assembled a set of protein sequences for analysis using BLASTP with the amino-acid sequences of 1/ Gne2 from E. coli O86, 2/ the Gne protein from E. coli O157, 3/ WbpP from P. aeruginosa O6, and 4/ Gne from Y. enterocolitica O8, that represent the diversity of UDP-GlcNAc and UndPP-GlcNAc 4-epimerases that have been characterised in recent years. These four proteins belong to the NAD-dependent epimerase/dehydratase family, PF01370. We searched only genome sequences and confined the BLASTP search to the Gammaproteobacteria to keep the output to a manageable level. Additional Gne protein sequences were extracted from known OPS gene cluster sequences of several species of Enterobacteriaceae, and the original 56 sequences from the Ishiyama study [16] were also included (Table S1). These 56 proteins added taxonomic depth to this study by including several firmicute, archae and eucaryote proteins as well as the characterised human, Bacillus subtilus and E. coli GalE proteins. After excluding duplicates there were 295 proteins that were aligned using the CLUSTAL program, and the alignment was used to construct a neighbour-joining tree by MEGA (Figure 1 Figure S1). Bootstrap values above 50% are shown in Figure S1 adjacent to nodes (percentage of 1000 bootstrap trees that contain the node), and values for major nodes are shown in Figure 1.

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Figure 1. Tree of Gne and related proteins in theGammaproteobacteriaceae and other selected species as found by genome BLAST or taken from the literature as described in the text and listed in Table S1.

The tree is based on that shown in Figure S1 in which all of the strains are named. The four major clades are shown with branches coloured where additional information is available as indicated to the right of the Figure and in the text. The proteins indicated by an asterisk were from the Ishiyama et al (16) study.

https://doi.org/10.1371/journal.pone.0067646.g001

Distribution of gnu and gne genes in E. coli serotype strains

We took a set of E. coli strains to represent serogroups for which an O antigen structure has been determined, and for which we have the O-antigen gene cluster from sequence data, which is to be published separately. We excluded O antigens with the genes of the ABC transporter pathway and one with an initial sugar transferase gene. The 115 that remained had wzx and wzy genes in the gene cluster, so are assumed to use GlcNAc or GalNAc as the first sugar. We undertook two PCR reactions on each to test for the presence of a clade D gnu gene, using primers either based on sequence of the gnu gene of the E. coli O157 strain [8] (5-ACAGATTGGTGATGTTCG-3 and 5-ATCAAAGCAATATCCACC-3), or sequences between gnu and upstream gene galF (one primer in gnu (5-ATCGAACTCTGCAGTATGAATT-3), and the other in galF(5-ATGTACTGAATCATTGGCTTGT-3)).

Clade A - galE and gne genes - UDP-Glc epimerases and UDP-GlcNAc epimerases

Clade A epimerases include those that act on UDP-GlcNAc in E. coli O86, Y. enterocolitica O8, and Y. pseudotuberculosis O6 and O7, and are the Rush et al. [8] homology group of epimerases for OPS with GalNAc residues that were not the first sugar in the repeat unit. Clade A proteins also include the original UDP-Glc/UDP-Gal 4-epimerases known as GalE, including the human and E. coli forms and others from Trypanosoma, Streptococcus thermophilus, B. subtilus, Pasteurella multicoida, Erwinia amylovora, and Avibacterium paragallinarum that are spread throughout this clade. Some of the UDP-Glc/UDP-Gal 4-epimerase functions is supported by their genes being associated with other genes of the gal operon. Thus for clade A, there are two known functions.

The clade A Gne proteins are the original UDP-GlcNAc epimerases, and we propose that they retain that name, but those in the other clades are now shown to have different functions and receive new names. In a few cases it has been shown that the substrate specificity of clade A proteins is due to the size of the substrate-binding pocket as discussed in the introduction. In such cases the pocket size determines if UDP-GlcNAc and UDP-GalNAc can enter the site, but discrimination against UDP-Glc or UDP-Gal has not been tested when the active site was made larger by amino acid substitution.

We are not aware of any protein in the other clades being primarily a UDP-Glc or UDP-GlcNAc epimerase although, as discussed above, some are known to have one or both functions in vitro. We recommend that the use of “galE” and “gne” names be confined to those in clade A unless there is experimental evidence to the contrary, with the default name being “galE” unless there is reason to use “gne”.

Clade D -gnu genes - UndPP-GlcNAc epimerases

Clade D proteins include those for the known UndPP-GlcNAc epimerases. Clade D corresponds to the homology group identified by Rush et al. [8] for Gne proteins associated with repeat units having GalNAc as the first sugar. The substrate is quite different to that of the traditional Gne epimerase substrate, UDP-GlcNAc, and in addition, the O157 enzyme, when tested, did not show any activity towards UDP-GlcNAc. We propose the name gnu for these genes. There is support for many of these having the UndPP-GlcNAc epimerase function. For the 15 proteins from E. coli (including Shigella), S. enterica, Citrobacter, Cronobacter, Edwardsiella or Serratia, the corresponding gene is located upstream of the OPS gene cluster at the same locus as for the characterised E. coli O157 UndPP-GlcNAc epimerase.

There is good statistical support for this gene being associated with GalNAc residues. Wang et al [7] showed by PCR that 14 of 22 E. coli with GalNAc in the structure had the clade D gene, but only 4 of the 40 strains without GalNAc were positive. There are now many more available O-antigen structures so we extended this analysis to cover 115 E. coli serogroups, and also included information on presence of a gne gene in the gene cluster from sequence data to be published separately. The 115 E. coli O antigen gene clusters include a wzx and wzy gene but lack an initial transferase gene. All of them have either or both of GlcNAc and GalNAc residues, and these are assumed to include the first sugar. They are divided into 6 groups based on the patterns of GlcNAc and GalNAc in the structures (Table S2). Each pattern gives a specific expectation in terms of presence of the gna and gne genes as shown. In group 1 there is a main-chain GalNAc and a side-chain GalNAc but no GlcNAc, so one GalNAc has to be the first sugar and the other is not, so both gnu and gne are needed. In group 2 there is a main-chain GalNAc and a main-chain GlcNAc. Our O-unit structures do not tell us which sugar is the first, so either gnu or gne is needed depending on which is the first sugar. The other patterns give different predictions as shown in Table S2, together with our expectations for gnu and gne. The data is also shown in Table S2. For 101 of the O-antigen structures (groups 3-6) we can determine the needs for gnu and gne genes independently and for groups 2 and 3 we can infer the need for one or other of the gnu and gne genes depending on whether a single main-chain GalNAc residue is the first or a later sugar. We find a remarkably close fit for both genes to expectation providing very strong support for the functions proposed for clade B and D genes in E. coli. In 104 serogroups the gnu gene is present or absent as expected on the basis of chemical structure, and likewise in 105 serogroups for the gne gene. There are only 7 cases of a gnu or gne gene missing when it is expected and only 10 cases where a gene is present when not predicted (Table S2). We propose the name gnu for the clade D genes. There are additional proteins from Shigella, S. enterica, Citrobacter, Cronobacter, Edwardsiella and Serratia, for which the corresponding gene is located upstream of the OPS gene cluster at the same locus as for the characterised E. coli O157 UndPP-GlcNAc epimerase.

We discussed above that for Y. pseudotuberculosis O6 and O7, with both clade A and D proteins, a functional assignment can be made based on the OPS sequence and structure. This clade also includes the Y. pseudotuberculosis O3, O10 and O15 Gnu proteins that are close to the O6 and O7 proteins in the tree and 5 additional Y. pseudotuberculosis epimerases not included in the tree (O1c, O2b, O2c, O4a, O5a and O5b) [17–20] that are very similar to the others, a total of 11 Y. pseudotuberculosis serotypes with a gnu gene [17–20], all of which have GalNAc as first sugar. Serotypes O1b, O11 and O12, with GlcNAc as first sugar, have generally similar gene clusters to those of O1c, O2b, O2c, O3, O4a, O5a, O5b and O15, but lack the gnu gene (see alignment in [18]). The distribution of gnu in Y. pseudotuberculosis correlates absolutely with the presence of GalNAc as first sugar, and this again gives very strong support for these members of clade D having Gnu function. We have 35 proteins in clade D if we include all of the Y. pseudotuberculosis serotypes listed above, with good support for the gnu function for 26 of them.

Clade D is the most divergent of the four clades, and is itself quite diverse in sequence with E. coli and Y. pseudotuberculosis strains the most divergent with differences in 50% of the amino-acid residues for Y. pseudotuberculosis O10 and E. coli O157. It is important that the best evidence for Gnu function is for these same species. For E. coli we have good biochemical evidence for the Gnu function and statistical evidence for association with presence of GalNAc in the structure, and for Y. pseudotuberculosis we have 11 sequences with very strong support for the Gnu function.

Clade B - gnaB genes - UDP-GlcNAcA epimerases

Clade B proteins, where function is known, are involved in the synthesis of UDP-GalNAcA or its derivatives. The clade B proteins are ALL associated with a homologue of WbpO, with the genes either adjacent or, in Marinobacter sp. ELB17, separated by two ORFs. It is normal practice and reasonable to generalise from one well-documented example, and we assume that this pair of proteins generally act in the same way as documented in P. aeruginosa. We propose that the clade B epimerase genes be named gnaB, with the genes for the oxygenation step becoming gnaA (previously gna) genes. This proposal fits the standard pattern for nomenclature of OPS genes [21,22] by using gna for the UDP-GalNAcA pathway, with gnaA and gnaB named in function order [21,22]. The biological function proposed for these genes is supported by the presence of a GalNAcA residue (or derivative) in the OPS where the structure is known, including a group of P. aeruginosa OPS, several E. coli OPS and others in V. cholerae, and also the Vi antigen of S. enterica.

There is also a Giardia enzyme near the boundary of clades A and B, which is a UDP-GlcNAc epimerase that lacks UDP-Glc/UDP-Gal 4-epimerase activity [23]. There are several uncharacterised enzymes in this area, all on long terminal branches.

There are now structures of WbpP [16] from P. aeruginosa and WbgU from Plesiomonas shigelloides [24]. Both papers report substantial differences at the active site, when compared to previously characterised UDP-GlcNAc epimerases, in agreement with the finding that they have different substrates. However although they are now shown to function primarily as UDP-GlcNAcA epimerases, at the time of publication they were treated as UDP-GlcNAc epimerases [15], and UDP-GlcNAcA was not tested as a substrate or in complex with the enzymes.

Clade C - a clade of proteins of unknown function. To our knowledge clade C includes only one protein that has been characterised. WbpV from P. aeruginosa O6 is thought to be the final C-4 reductase in formation of UDP-D-QuiNAc from UDP-4-keto-D-QuiNAc [15]. Nucleotide 4-keto-sugar reductases are in the same family as the epimerases that we are discussing, and indeed in GalE the epimerisation has been shown to involve a 4-keto intermediate equivalent to the product of the 4-keto-sugar reductases [25]. It is thus not surprising to find a nucleotide 4-keto-sugar reductase grouping with 4-epimerases and it will be interesting to know the functions of other members of this clade.

Relationship of earlier groupings to clades A, B and D

With regard to earlier classifications, the group 3 “GlcNAc-specific” epimerases of Ishiyama et al [16] are in our clade B, which contains proteins now known to be epimerases for UDP-GlcNAcA [12] with no in vivo activity on UDP-GlcNAc, that we have renamed gnaB. The Ishiyama group 1 and 2 enzymes fall into clade A, which includes all of those with UDP-Glc and/or UDP-GlcNAc as in vivo substrates.

Recommendations for Annotation

We suggest that in annotation of genomes, any gene that falls into clades B or D be named gnaB or gnu respectively. In both cases there are well-documented cases for assignment of function that make this the best choice for annotation. In the case of clade B well-documented genes are the P. aeruginosa O6 gene currently annotated wbpP gene [12], and the S. enterica Vi antigen gene previously named viaC [26] and also tviC [27].

In the case of clade D we have the E. coli O157 UndPP-GlcNAc epimerase gene [8] that was originally named gne by us [7]. The Yersinia clade D genes also clearly have gnu function.

In the case of Clade A we have well documented gne genes from E coli O86 [10], Aeromonas hydrophila O34 [28] and Y. enterocolitica O8 [4], and galE genes from E. coli [29], Aeromonas hydrophila O34 [28], Erwina amylovora [30], A. paragallinarum [31], Pasturella multocida [32], Y. enterocolitica O8 [4], B. subtilis [5] and Human [33].

However there remains a difficulty in annotation of clade A genes, as GalE and Gne proteins cannot as yet be distinguished from each other by sequence alone, and for genome sequences there is often no information on possible OPS involvement. However many galE genes are in an operon for utilisation of exogenous galactose (see Figure 1) and association of a clade A gene sequence with galK or galT genes is a good indication that it is a galE gene with its primary role being galactose utilisation. In contrast the presence of a clade A gene in an OPS gene cluster is a good indication that it is a gne gene, particularly if there is a galE gene elsewhere in the genome. Comparison with other genomes in the same species group may add further clarification.

While the number of detailed studies is low for clades B and D in particular, this is not unusual in annotation, and the extension of the functions across the respective clades is strongly supported by the data discussed in the clade descriptions. However more data is highly desirable and we hope our analysis will encourage further work in the area and provide a framework for selection of strains for further study. If type strains are needed for comparison, then Y. enterocollitica O8 (gne), E. coli O157 (gnu) and P. aeruginosa O6 (gnaB/wbpP) are the best characterized. E. coli O86 and Y. pseudotuberculosis O6 and O7 have both gne and gnu and may also be useful in this regard.