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Structure–function studies of the C3/C5 epimerases and C4 reductases of the Campylobacter jejuni capsular heptose modification pathways

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100352

ISSN

1083-351X

Autores

Heba Barnawi, Laura Woodward, Natalie Fava, Mikhail Roubakha, Steven D. Shaw, Chelsea Kubinec, James H. Naismith, Carole Creuzenet,

Tópico(s)

Cassava research and cyanide

Resumo

Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6-deoxy-D-altro-heptose and GDP-6-OMe-L-gluco-heptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes—C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC—are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools. Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6-deoxy-D-altro-heptose and GDP-6-OMe-L-gluco-heptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes—C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC—are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools. Bacteria produce sugars often found as part of or attached to the cell wall or in a capsule where they are often important for virulence and in some cases cell viability. We have previously characterized the dehydratases, epimerases, and reductases that modify GDP-manno-heptose into various heptose forms found in the capsule of Campylobacter jejuni (Fig. 1) and in the lipopolysaccharide of Yersinia pseudotuberculosis (1Butty F.D. Aucoin M. Morrison L. Ho N. Shaw G.S. Creuzenet C. Elucidating the formation of 6-deoxyheptose: Biochemical characterization of the GDP-D-glycero-D-manno-heptose C6 dehydratase, DmhA and its associated C4 reductase, DmhB.Biochemistry. 2009; 48: 7764-7775Crossref PubMed Scopus (17) Google Scholar, 2McCallum M. Shaw G.S. Creuzenet C. Characterization of the dehydratase WcbK and the reductase WcaG involved in GDP-6-deoxy-manno-heptose biosynthesis in Campylobacter jejuni.Biochem. J. 2011; 439: 235-248Crossref PubMed Scopus (12) Google Scholar, 3McCallum M. Shaw G.S. Creuzenet C. Comparison of predicted epimerases and reductases of the Campylobacter jejuni D-altro- and L-gluco-heptose synthesis pathways.J. Biol. Chem. 2013; 288: 19569-19580Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 4McCallum M. Shaw S.D. Shaw G.S. Creuzenet C. Complete 6-deoxy-D-altro-heptose biosynthesis pathway from Campylobacter jejuni: More complex than anticipated.J. Biol. Chem. 2012; 287: 29776-29788Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) (not shown). Specifically, the Y. pseudotuberculosis pathway leading to 6-deoxy-D-manno-heptose was a simple two-step pathway consisting of C4, C6 dehydration, and C4 reduction of GDP-manno-heptose (1Butty F.D. Aucoin M. Morrison L. Ho N. Shaw G.S. Creuzenet C. Elucidating the formation of 6-deoxyheptose: Biochemical characterization of the GDP-D-glycero-D-manno-heptose C6 dehydratase, DmhA and its associated C4 reductase, DmhB.Biochemistry. 2009; 48: 7764-7775Crossref PubMed Scopus (17) Google Scholar). In contrast, the C. jejuni Ddah and Mlgh modification pathways leading to 6-deoxy-D-altro-heptose and 3,6-OMe-L-gluco heptose, respectively, have been found to be more complex (Fig. 1). Both pathways operate via a 6-deoxy-4-keto intermediate (P1 formed by DdahA (aka WcbK) (2McCallum M. Shaw G.S. Creuzenet C. Characterization of the dehydratase WcbK and the reductase WcaG involved in GDP-6-deoxy-manno-heptose biosynthesis in Campylobacter jejuni.Biochem. J. 2011; 439: 235-248Crossref PubMed Scopus (12) Google Scholar)), which is then processed by a pair of enzymes (DdahB/C and MlghB/C). Based on sequence similarity, all enzymes were predicted to be C3/C5 epimerases with DdahC and MlghC possessing additional C4 reductase activity (3McCallum M. Shaw G.S. Creuzenet C. Comparison of predicted epimerases and reductases of the Campylobacter jejuni D-altro- and L-gluco-heptose synthesis pathways.J. Biol. Chem. 2013; 288: 19569-19580Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 4McCallum M. Shaw S.D. Shaw G.S. Creuzenet C. Complete 6-deoxy-D-altro-heptose biosynthesis pathway from Campylobacter jejuni: More complex than anticipated.J. Biol. Chem. 2012; 287: 29776-29788Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). We showed experimentally that DdahB is a C3 epimerase leading to the formation of a single C3 epimerized product (denoted as P4α), whereas MlghB is a C3/C5 epimerase leading to the formation of three products encompassing the same C3 only epimer (P4α) as DdahB but also the C5 only epimer (denoted as P4β) and the double C3, C5 epimer (denoted as P4γ) (Fig. 1). DdahB and MlghB were devoid of reductase activity, but both DdahC and MlghC served as C4 reductases, reducing epimerized products made by DdahB and MlghB to generate products denoted as P5α and P5γ, respectively (3McCallum M. Shaw G.S. Creuzenet C. Comparison of predicted epimerases and reductases of the Campylobacter jejuni D-altro- and L-gluco-heptose synthesis pathways.J. Biol. Chem. 2013; 288: 19569-19580Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 4McCallum M. Shaw S.D. Shaw G.S. Creuzenet C. Complete 6-deoxy-D-altro-heptose biosynthesis pathway from Campylobacter jejuni: More complex than anticipated.J. Biol. Chem. 2012; 287: 29776-29788Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In addition, in vitro and in vivo studies demonstrated that all these enzymes are important for the function of the polysaccharide that incorporates their reaction products, namely contributing to capsule- or lipopolysaccharide-based resistance to serum, bile salts, and/or antibiotics, allowing epithelial cell invasion and playing an essential role in gut colonization and/or dissemination to deeper organs (5Ho N. Kondakova A.N. Knirel Y.A. Creuzenet C. The biosynthesis and biological role of 6-deoxyheptose in the lipopolysaccharide O-antigen of Yersinia pseudotuberculosis.Mol. Microbiol. 2008; 68: 424-447Crossref PubMed Scopus (19) Google Scholar, 6Kondakova A.N. Ho N. Bystrova O.V. Shashkov A.S. Lindner B. Creuzenet C. Knirel Y.A. Structural studies of the O-antigens of Yersinia pseudotuberculosis O:2a and mutants thereof with impaired 6-deoxy-D-manno-heptose biosynthesis pathway.Carbohydr. Res. 2008; 343: 1383-1389Crossref PubMed Scopus (27) Google Scholar, 7Wong A. Lange D. Houle S. Arbatsky N.P. Valvano M.A. Knirel Y.A. Dozois C.M. Creuzenet C. Role of capsular modified heptose in the virulence of Campylobacter jejuni.Mol. Microbiol. 2015; 96: 1136-1158Crossref PubMed Scopus (22) Google Scholar). Thus, these enzymes could be novel targets, allowing inhibition of colonization by Y. pseudotuberculosis or C. jejuni or by other mucosal pathogens that also produce similar modified heptoses, such as Burkholderia species (8Reckseidler S.L. DeShazer D. Sokol P.A. Woods D.E. Detection of bacterial virulence genes by subtractive hybridization: Identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant.Infect. Immun. 2001; 69: 34-44Crossref PubMed Scopus (209) Google Scholar, 9Perry M.B. MacLean L.L. Schollaardt T. Bryan L.E. Ho M. Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei.Infect. Immun. 1995; 63: 3348-3352Crossref PubMed Google Scholar). This would present alternatives to antibiotics of interest both in human and veterinary medicine (10Lin J. Yan M. Sahin O. Pereira S. Chang Y.J. Zhang Q. Effect of macrolide usage on emergence of erythromycin-resistant Campylobacter isolates in chickens.Antimicrob. Agents Chemother. 2007; 51: 1678-1686Crossref PubMed Scopus (80) Google Scholar, 11Marshall B.M. Levy S.B. Food animals and antimicrobials: Impacts on human health.Clin. Microbiol. Rev. 2011; 24: 718-733Crossref PubMed Scopus (1189) Google Scholar). C. jejuni is of particular concern because it causes severe gastrointestinal disease in humans and its high level of resistance to fluoroquinolones has warranted its classification as a high priority pathogen by the WHO in 2017. With ∼9500 reported cases yearly in Canada (12Public Health Agency of CanadaCanadian integrated surveillance report: Salmonella, Campylobacter, verotoxigenic E. coli and Shigella, from 2000 to 2004.Can. Commun. Dis. Rep. 2009; 35S3: 1-50Google Scholar) (http://publications.gc.ca/pub?id=9.507317&sl=0, https://diseases.canada.ca/notifiable/), campylobacteriosis is often contracted via consumption of contaminated undercooked chicken meat. It may be possible to exploit heptose-modifying enzymes as targets to reduce chicken colonization by C. jejuni to curtail transmission to humans. The DdahB/C and MlghB/C pairs of C. jejuni enzymes are present in most Campylobacters and unique to Campylobacters (13Parkhill J. Wren B.W. Mungall K. Ketley J.M. Churcher C. Basham D. Chillingworth T. Davies R.M. Feltwell T. Holroyd S. Jagels K. Karlyshev A.V. Moule S. Pallen M.J. Penn C.W. et al.The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences.Nature. 2000; 403: 665-668Crossref PubMed Scopus (1519) Google Scholar, 14Karlyshev A.V. Champion O.L. Churcher C. Brisson J.R. Jarrell H.C. Gilbert M. Brochu D. St Michael F. Li J. Wakarchuk W.W. Goodhead I. Sanders M. Stevens K. White B. Parkhill J. et al.Analysis of Campylobacter jejuni capsular loci reveals multiple mechanisms for the generation of structural diversity and the ability to form complex heptoses.Mol. Microbiol. 2005; 55: 90-103Crossref PubMed Scopus (133) Google Scholar), making them attractive targets. Understanding the molecular basis for their specificity would assist in the design of highly selective inhibitors, which would avoid problems of bacterial antibiotic resistance linked to the usage of broad-spectrum antibiotics in chicken farming (10Lin J. Yan M. Sahin O. Pereira S. Chang Y.J. Zhang Q. Effect of macrolide usage on emergence of erythromycin-resistant Campylobacter isolates in chickens.Antimicrob. Agents Chemother. 2007; 51: 1678-1686Crossref PubMed Scopus (80) Google Scholar, 11Marshall B.M. Levy S.B. Food animals and antimicrobials: Impacts on human health.Clin. Microbiol. Rev. 2011; 24: 718-733Crossref PubMed Scopus (1189) Google Scholar). The mechanisms involved in C3/C5 epimerization are fairly well understood. There are two main mechanisms extant, the first typified by the dTDP-6-deoxy-D-xylo-4-hexulose C3/C5 epimerase RmlC (15Giraud M.F. Leonard G.A. Field R.A. Berlind C. Naismith J.H. RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase.Nat. Struct. Biol. 2000; 7: 398-402Crossref PubMed Scopus (91) Google Scholar, 16Dong C. Major L.L. Allen A. Blankenfeldt W. Maskell D. Naismith J.H. High-resolution structures of RmlC from Streptococcus suis in complex with substrate analogs locate the active site of this class of enzyme.Structure. 2003; 11: 715-723Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 17Dong C. Major L.L. Srikannathasan V. Errey J.C. Giraud M.F. Lam J.S. Graninger M. Messner P. McNeil M.R. Field R.A. Whitfield C. Naismith J.H. RmlC, a C3' and C5' carbohydrate epimerase, appears to operate via an intermediate with an unusual twist boat conformation.J. Mol. Biol. 2007; 365: 146-159Crossref PubMed Scopus (51) Google Scholar). DdahB and MlghB are 38 and 37% identical to Salmonella enterica RmlC, respectively. The residues involved in RmlC activity are summarized in Table S1. They include the His 63-Asp 170 dyad, Tyr 133 and Lys 73, which are highly conserved in other bacteria, including Streptococcus suis (Table S1 and (17Dong C. Major L.L. Srikannathasan V. Errey J.C. Giraud M.F. Lam J.S. Graninger M. Messner P. McNeil M.R. Field R.A. Whitfield C. Naismith J.H. RmlC, a C3' and C5' carbohydrate epimerase, appears to operate via an intermediate with an unusual twist boat conformation.J. Mol. Biol. 2007; 365: 146-159Crossref PubMed Scopus (51) Google Scholar)). The His-Asp dyad is important for both C3 and C5 epimerization of the substrate because RmlC has no cofactor: His 63 acts as a base to deprotonate the sugar from the lower face of the ring at C3 and C5 positions (16Dong C. Major L.L. Allen A. Blankenfeldt W. Maskell D. Naismith J.H. High-resolution structures of RmlC from Streptococcus suis in complex with substrate analogs locate the active site of this class of enzyme.Structure. 2003; 11: 715-723Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and Asp 170 increases the basicity of His 63, enhancing its ability to deprotonate the sugar. The enolate anion is stabilized by Lys 73 (16Dong C. Major L.L. Allen A. Blankenfeldt W. Maskell D. Naismith J.H. High-resolution structures of RmlC from Streptococcus suis in complex with substrate analogs locate the active site of this class of enzyme.Structure. 2003; 11: 715-723Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 17Dong C. Major L.L. Srikannathasan V. Errey J.C. Giraud M.F. Lam J.S. Graninger M. Messner P. McNeil M.R. Field R.A. Whitfield C. Naismith J.H. RmlC, a C3' and C5' carbohydrate epimerase, appears to operate via an intermediate with an unusual twist boat conformation.J. Mol. Biol. 2007; 365: 146-159Crossref PubMed Scopus (51) Google Scholar). On the opposite face of the sugar ring, Tyr 133 donates a proton at C5 position from its hydroxyl group, thus acting as an acid. A conserved water molecule found close to C3 was proposed to play a role in C3 epimerization (17Dong C. Major L.L. Srikannathasan V. Errey J.C. Giraud M.F. Lam J.S. Graninger M. Messner P. McNeil M.R. Field R.A. Whitfield C. Naismith J.H. RmlC, a C3' and C5' carbohydrate epimerase, appears to operate via an intermediate with an unusual twist boat conformation.J. Mol. Biol. 2007; 365: 146-159Crossref PubMed Scopus (51) Google Scholar). The second mechanism is exhibited by GDP-L-fucose synthase (GFS) (also known as GMER), a GDP-4-keto-6-deoxy-D-mannose C3/C5 epimerase, C4 reductase (18Somoza J.R. Menon S. Schmidt H. Joseph-McCarthy D. Dessen A. Stahl M.L. Somers W.S. Sullivan F.X. Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose.Structure. 2000; 8: 123-135Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 19Lau S.T. Tanner M.E. Mechanism and active site residues of GDP-fucose synthase.J. Am. Chem. Soc. 2008; 130: 17593-17602Crossref PubMed Scopus (25) Google Scholar, 20Rizzi M. Tonetti M. Vigevani P. Sturla L. Bisso A. Flora A.D. Bordo D. Bolognesi M. GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from Escherichia coli, a key enzyme in the biosynthesis of GDP-L-fucose, displays the structural characteristics of the RED protein homology superfamily.Structure. 1998; 6: 1453-1465Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 21Somers W.S. Stahl M.L. Sullivan F.X. GDP-fucose synthetase from Escherichia coli: Structure of a unique member of the short-chain dehydrogenase/reductase family that catalyzes two distinct reactions at the same active site.Structure. 1998; 6: 1601-1612Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), or GME, a GDP-mannose C3/C5 epimerase from Arabidopsis thaliana. These enzymes have an NADP cofactor and carry out four reactions at a single active site: an oxidation, two epimerizations, and a reduction (21Somers W.S. Stahl M.L. Sullivan F.X. GDP-fucose synthetase from Escherichia coli: Structure of a unique member of the short-chain dehydrogenase/reductase family that catalyzes two distinct reactions at the same active site.Structure. 1998; 6: 1601-1612Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 22Major L.L. Wolucka B.A. Naismith J.H. Structure and function of GDP-mannose-3',5'-epimerase: An enzyme which performs three chemical reactions at the same active site.J. Am. Chem. Soc. 2005; 127: 18309-18320Crossref PubMed Scopus (66) Google Scholar). DdahC and MlghC are 44 and 38% identical to Escherichia coli GFS, respectively. Residues important for these activities are summarized in Table S2. The reduction reaction is catalyzed by Ser 107, Tyr 136, and Lys 140, which form the typical SYK catalytic triad of short-chain dehydrogenase reductase enzymes (23Jornvall H. Persson B. Krook M. Atrian S. Gonzalez-Duarte R. Jeffery J. Ghosh D. Short-chain dehydrogenases/reductases (SDR).Biochemistry. 1995; 34: 6003-6013Crossref PubMed Scopus (1140) Google Scholar). Ser 107 and Lys 140 lower the pKa of Tyr 136, which allows it to function as a general acid or base during catalysis through its hydroxyl side chain (19Lau S.T. Tanner M.E. Mechanism and active site residues of GDP-fucose synthase.J. Am. Chem. Soc. 2008; 130: 17593-17602Crossref PubMed Scopus (25) Google Scholar, 21Somers W.S. Stahl M.L. Sullivan F.X. GDP-fucose synthetase from Escherichia coli: Structure of a unique member of the short-chain dehydrogenase/reductase family that catalyzes two distinct reactions at the same active site.Structure. 1998; 6: 1601-1612Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). This residue is important for final reduction of the 4-keto intermediate once epimerization is complete. In GFS, Cys 109 and His 179 form a catalytic dyad where Cys 109 is the base and His 179 is the acid for epimerization at both C3 and C5 positions (19Lau S.T. Tanner M.E. Mechanism and active site residues of GDP-fucose synthase.J. Am. Chem. Soc. 2008; 130: 17593-17602Crossref PubMed Scopus (25) Google Scholar). Functionally equivalent residues are Cys 145/Lys 217 in GME. Because all known C3/C5 epimerases, C4 reductases, described above use hexoses as substrates while the Campylobacter enzymes function in heptose modification pathways, we undertook the investigation of the Campylobacter enzymes to decipher the extent of their heptose versus mannose specificity and also to understand why all enzymes predicted to be C3/C5 epimerases with C4 reductase activity performed different reactions on different heptose intermediates. The studies involve structural studies of both enzymes, modeling, site-directed mutagenesis, and functional analysis on GDP-manno-heptose and GDP-mannose–derived substrates. These studies will support the future applications mentioned above but are also of fundamental importance to provide a better understanding of complex glycan synthesis. MlghB and DdahB are the only C3/C5 epimerases demonstrated to have activity on heptose-based substrates to date. To assess their specificity, GDP-manno-heptose and GDP-mannose were converted into the 4-keto, 6-deoxy-derivatives P1 (7 carbon) and P1' (6 carbon) by DdahA and equimolar amounts were used as substrates for MlghB and DdahB. As established before (3McCallum M. Shaw G.S. Creuzenet C. Comparison of predicted epimerases and reductases of the Campylobacter jejuni D-altro- and L-gluco-heptose synthesis pathways.J. Biol. Chem. 2013; 288: 19569-19580Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 4McCallum M. Shaw S.D. Shaw G.S. Creuzenet C. Complete 6-deoxy-D-altro-heptose biosynthesis pathway from Campylobacter jejuni: More complex than anticipated.J. Biol. Chem. 2012; 287: 29776-29788Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), MlghB converts heptose-based P1 into C3, C5, and C3/C5 epimers (P4α, P4β, and P4γ, respectively) (Figs. 1 and 2, B and D). In contrast, DdahB mostly converts the P1 substrate to P4α (C3 epimer) (Fig. 2, B and F), but prolonged incubation (3 h) with DdahB led to appearance of P4β (C5 only) and P4γ (C3 and C5) (Fig. 2B) and concomitant decrease in the P4α peak. Because there was no further conversion of P1 in that time frame, this suggests that C5 epimerization also occurred, generating P4β and further converting P4α into P4γ. Both enzymes were able to epimerize a mannose (six carbon) derived substrate denoted P1' into product P4' (the nomenclature is chosen to mirror the heptose conversion with the addition of the prime denoting the six-carbon substrate). P1' was processed much less efficiently than P1, especially by DdahB where catalysis was limited even after a 3-h incubation and was accompanied by significant substrate degradation (Fig. 2, A, C and E). DdahB thus possesses clear "heptose preference." Definitely establishing the nature of P4' has proven impossible because of its instability and of the very small amounts of material produced. Because DdahB primarily catalyzes C3 epimerization activity of heptose (3McCallum M. Shaw G.S. Creuzenet C. Comparison of predicted epimerases and reductases of the Campylobacter jejuni D-altro- and L-gluco-heptose synthesis pathways.J. Biol. Chem. 2013; 288: 19569-19580Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 4McCallum M. Shaw S.D. Shaw G.S. Creuzenet C. Complete 6-deoxy-D-altro-heptose biosynthesis pathway from Campylobacter jejuni: More complex than anticipated.J. Biol. Chem. 2012; 287: 29776-29788Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), we reasoned that P4' would be the C3 epimer. This would imply MlghB was incapable of C5 epimerization of the smaller mannose-based substrate. The additional carbon of the P1 substrate is critical for enzyme turnover, appearing essential for the C5 position. DdahB and MlghB were purified as homodimers (Fig S1, A and B, Table S3), crystallized, and their structures solved to 1.30 Å and 2.14 Å resolution, respectively. DdahB crystallized in space group P1 21 1, with two chains in the asymmetric unit and MlghB crystallized in space group P21 21 21, with four chains in the asymmetric unit. Statistics on the final structures are reported in Table 1. The final structure of DdahB has two monomers in the asymmetric unit that are expected to form a stable dimer (24Krissinel E. Henrick K. Inference of macromolecular assemblies from crystalline state.J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (6289) Google Scholar), consistent with size-exclusion chromatography coupled with multiangle light scattering (SEC-MALS, Fig. 3A and Fig. S1A). Residues 1 to 139 and 145 to 174 of monomer A and residues 3 to 181 of monomer B are experimentally located. The monomer comprises 14 β-strands and two short α-helices, with nine β-strands, arranged in two β-sheets, forming a small antiparallel β-barrel, known as a jellyroll. The fold of the protein identifies it as a member of the cupin family (25Dunwell J.M. Culham A. Carter C.E. Sosa-Aguirre C.R. Goodenough P.W. Evolution of functional diversity in the cupin superfamily.Trends Biochem. Sci. 2001; 26: 740-746Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). MlghB has the same cupin fold as DdahB, and the monomers superimpose with an RMSD of 0.7 Å for the 163 overlapping Cα atoms and share 81% sequence identity (Table S4). Like DdahB, MlghB has two dimers (24Krissinel E. Henrick K. Inference of macromolecular assemblies from crystalline state.J. Mol. Biol. 2007; 372: 774-797Crossref PubMed Scopus (6289) Google Scholar) in the asymmetric unit (Fig. 3B); residues 2 to 178, 3 to 142 and 148 to 178; 3 to 138 and 147 to 173, and 2 to 140 are located in subunits A, B, C, and D, respectively. In the dimer, a β-strand from one monomer adds, in an antiparallel manner, to the β-sheet of the other monomer. The arrangement buries around 20% of the surface area of each monomer. No cofactor was detected in either structure.Table 1Additional crystallographic parameters for DdahB and MlghBParameter measuredDdahBMlghBDdahBGDP-mannoseMlghBGDP-mannoseData collection Space groupP21P212121P21P212121 Wavelength0.9160.9161.541.54 Unit cell dimensionsa, b, c (Å)47.8, 68.3, 53.747.3, 121.7, 153.848.0, 67.8, 53.142.4, 121.9, 154.1α, β, γ (°)90, 91.4, 9090, 90, 9090, 91.8, 9090, 90, 90 Resolution (Å)aValues in parenthesis are for the highest shell.34–1.3 (1.32–1.30)95–2.14 (2.20–2.14)53–2.35 (2.43–2.35)56.7–2.60 (2.67–2.60) RmergeaValues in parenthesis are for the highest shell.0.047 (0.57)0.06 (0.76)0.061 (0.169)0.162 (0.669) I/σIaValues in parenthesis are for the highest shell.13.9 (1.2)12.7 (1.1)16.3 (6.1)12.5 (3.5) CC1/2aValues in parenthesis are for the highest shell.1.0 (0.7)1.0 (0.5)1 (0.9)1.0 (0.8) CompletenessaValues in parenthesis are for the highest shell.95 (73)100 (100)98 (89)99 (92) RedundancyaValues in parenthesis are for the highest shell.2.7 (1.4)5.9 (4.2)3.6 (2.7)7.0 (6.8)Refinement Resolution (Å)aValues in parenthesis are for the highest shell.34–1.3 (1.32–1.30)95–2.1 (2.18–2.14)39–2.35 (2.43–2.35)57–2.60 (2.67–2.60) ReflectionsaValues in parenthesis are for the highest shell.76,397 (4400)41,762 (2944)13,466 (879)23,972 (1611) Rwork/Rfree %aValues in parenthesis are for the highest shell.14.8/17.5 (31.7/30.1)21.6/23.9 (42.1/42.4)18.5/24.3 (19.4/31.5)21.0/23.5 (29.4/34.3) No. of atomsProtein2742564827425858Water4667468Ligands--67112 Residual B factors (Å2)Protein2164222326Water48461116Ligand--4547 RMSDBond lengths (Å)0.0130.0100.0070.010Bond angles (°)1.61.41.31.6 RamachandranFavored (%)98999899Outliers (%)00007ANI7ANG7ANJ7AN4a Values in parenthesis are for the highest shell. Open table in a new tab The fold of the monomer and dimeric arrangement of both DdahB and MlghB closely resemble that of RmlC, the third enzyme in the dTDP-L-rhamnose pathway (Fig. 3C and Table S4 for RMSD values). RmlC was the first cupin C3/C5 epimerase enzyme structure to be described (15Giraud M.F. Leonard G.A. Field R.A. Berlind C. Naismith J.H. RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase.Nat. Struct. Biol. 2000; 7: 398-402Crossref PubMed Scopus (91) Google Scholar). The structural similarity is much higher than sequence similarity (38% identity). A search of the RCSB reveals many other cupin fold enzymes including multiple C3/C5 epimerases. As DdahB and MlghB can both epimerize GDP-mannose (in addition to their normal heptose-based substrates), we attempted to obtain cocomplexes with GDP-mannose. The structure of DdahB in the presence of GDP-mannose was solved to a resolution of 2.35 Å. Although the crystal was soaked in a solution of GDP-mannose prepared in mother liquor before freezing, the electron density was poor. We positioned GDP-mannose in one chain and GDP only in the other (Fig. 3A and Fig. S2). The presence of GDP-mannose in one chain suggested to us the problem was not degradation of GDP-mannose but rather the disorder. We attributed this to the fact GDP-mannose is not the true natural substrate and therefore its binding may be suboptimal. There was little change in the protein structure upon substrate binding (Table S4). MlghB in the presence of GDP-mannose crystallized in the same unit cell as native, diffracted to 2.6 Å, showed little change in structure, but we were only able to locate the GDP moiety for each monomer (Fig. S2 and Table S4). In both proteins, the guanine, ribose, and the phosphate attached to ribose portions of the GDP were bound at the same location, at the dimer interface. In both structures, the guanine ring makes hydrogen bonds to Asn 22, Thr 33, and Lys 54∗ (∗ denotes this residue is located in the other monomer in the dimer), with Ile 3 and Ile 31 stacking on opposite sides of t

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