Crystal Structure of a Novel Shikimate Dehydrogenase from Haemophilus influenzae

To date two classes of shikimate dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroshikimate to shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroshikimate to shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of shikimate dehydrogenase annotated as shikimate dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of shikimate but not quinate. The turnover rate for the oxidation of shikimate is ∼1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of shikimate dehydrogenases, namely AroE, YdiB, and this newly characterized shikimate dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the shikimate dehydrogenase reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of shikimate dehydrogenase. To date two classes of shikimate dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroshikimate to shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroshikimate to shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of shikimate dehydrogenase annotated as shikimate dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of shikimate but not quinate. The turnover rate for the oxidation of shikimate is ∼1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of shikimate dehydrogenases, namely AroE, YdiB, and this newly characterized shikimate dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the shikimate dehydrogenase reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of shikimate dehydrogenase. The shikimate pathway occupies a central position for aromatic biosynthesis in microbes and plants but is not present in humans and other higher animals. The absence of the shikimate pathway in animals makes it an ideal target for herbicide and anti-microbial drug design. Recently the shikimate pathway was identified in apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, which has renewed interest in better understanding the enzymes in the pathway (1McConkey G.A. Exp. Parasitol. 2000; 94: 23-32Crossref PubMed Scopus (21) Google Scholar, 2Campbell S.A. Richards T.A. Mui E.J. Samuel B.U. Coggins J.R. McLeod R. Roberts C.W. Int. J. Parasitol. 2004; 34: 5-13Crossref PubMed Scopus (69) Google Scholar). The importance of the shikimate pathway is exemplified by the common herbicide glyphosate, which inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Recent studies have also shown that glyphosate blocks the shikimate pathway in apicomplexan parasites and is effective in controlling their growth (3Roberts C.W. Roberts F. Lyons R.E. Kirisits M.J. Mui E.J. Finnerty J. Johnson J.J. Ferguson D.J. Coggins J.R. Krell T. Coombs G.H. Milhous W.K. Kyle D.E. Tzipori S. Barnwell J. Dame J.B. Carlton J. McLeod R. J. Infect. Dis. 2002; 185: S25-S36Crossref PubMed Scopus (136) Google Scholar). The shikimate pathway consists of seven enzymatic steps initiated by the condensation of phosphoenolpyruvate and erythrose-4-phosphate by 3-deoxy-d-arabino-heptolosonate 7-phosphate synthase. The last enzymatic step produces the branch point intermediate, chorismate, which serves in turn as the precursor for a number of pathways including those involved in aromatic amino acid, phytoalexin, flavanoid, and lignin biosynthesis. The fourth enzyme in the pathway, shikimate dehydrogenase (shikimate:NADP+ oxidoreductase; EC 1.1.1.25), is involved in the NADPH-dependent reduction of dehydroshikimate to shikimate. This enzymatic reaction proceeds in both the forward and reverse direction with similar rates and similar Michaelis constants for substrates in either direction. Kinetic studies with substrate analogues and isotope exchange demonstrated that the shikimate dehydrogenase-catalyzed reaction is consistent with an ordered sequential mechanism (4Balinsky D. Dennis A.W. Cleland W.W. Biochemistry. 1971; 10: 1947-1952Crossref PubMed Scopus (29) Google Scholar). Two types of shikimate dehydrogenases from bacteria have been characterized to date, AroE and YdiB (quinate/shikimate dehydrogenase, EC 1.1.1.282). Although AroE has been established as the enzyme responsible for flux through the main trunk of the shikimate pathway, YdiB has been implicated in a branch point involving the metabolism of quinate (5Herrmann K.M. Plant Cell. 1995; 7: 907-919Crossref PubMed Scopus (398) Google Scholar, 6Herrmann K.M. Weaver L.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 473-503Crossref PubMed Scopus (929) Google Scholar). YdiB was first characterized in fungi and was shown to play a significant role in the quinate utilization pathway rather than in the shikimate pathway itself (7Giles N.H. Case M.E. Baum J. Geever R. Huiet L. Patel V. Tyler B. Microbiol. Rev. 1985; 49: 338-358Crossref PubMed Google Scholar, 8Wheeler K.A. Lamb H.K. Hawkins A.R. Biochem. J. 1996; 315: 195-205Crossref PubMed Scopus (19) Google Scholar). This pathway consists of three enzymes (quinate/shikimate dehydrogenase, 3-dehydroquinase, and dehydroshikimate dehydratase) that catabolize quinate into protocatechuic acid. In addition, quinate/shikimate dehydrogenase can also catalyze the reduction of 3-dehydroshikimate to shikimate. The structures of both the AroE and YdiB proteins have been determined (9Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In this study we identified a novel class of shikimate dehydrogenases termed shikimate dehydrogenase-like (SDH-L). 1The abbreviations used are: SDH-L, shikimate dehydrogenase-like; HI, Haemophilus influenzae. Phylogenetic and kinetic analyses show that SDH-L is distinct from both AroE and YdiB, yet, all three classes have similar three-dimensional structures. This class of shikimate dehydrogenase was annotated by sequencing projects as shikimate dehydrogenase-like because of the lack of supporting biological and biochemical data. Because the shikimate pathway enzymes are ideal targets for the development of antimicrobial and herbicidal compounds, identification and characterization of novel enzyme(s) will enhance our understanding of the pathway and may reveal other avenues for drug and herbicide design. In this study we present the crystal structure and kinetic analysis of the shikimate dehydrogenase-like enzyme (HI0607) from Haemophilus influenzae. Chemicals—Shikimic acid was a generous gift from Professor John Frost at Michigan State University. All other reagents are of molecular biology grade and were purchased from Sigma, Bioshop, or BDH. Cloning, Expression, and Purification—The gene (gene identifier 38233601) encoding the hypothetical shikimate 3-dehydrogenase-like protein HI0607 was amplified by polymerase chain reaction from H. influenzae genomic DNA and cloned into the MCSG7 vector as described elsewhere (10Stols L. Gu M. Dieckman L. Raffen R. Collart F.R. Donnelly M.I. Protein Expression Purif. 2002; 25: 8-15Crossref PubMed Scopus (429) Google Scholar). Expression and purification of HI0607 were conducted according to the published protocol by Christendat et al. (11Christendat D. Saridakis V. Dharamsi A. Bochkarev A. Pai E.F. Arrowsmith C.H. Edwards A.M. J. Biol. Chem. 2000; 275: 24608-24612Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In brief, HI0607 recombinants were expressed in the E. coli strain BL21 Gold in 1 liter of Luria-Bertani medium supplemented with 50 μg/ml kanamycin and 50 μg/ml ampicillin and incubated at 37 °C with shaking until the culture reached an A of 0.7 at 600 nm. The culture was then induced with 0.4 mm isopropyl-β-d-thiogalactopyranoside for 3 h at 37 °C and allowed to grow overnight with shaking at 24 °C. Cells were harvested by centrifugation and then disrupted by sonication, and the insoluble cellular material was removed by centrifugation. HI0607 was purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Purified HI0607 was concentrated and quantified at 280 nm using the extinction coefficient of 16640 m–1 cm–1 from a contribution of its aromatic amino acids. Selenomethionine-labeled protein was prepared by supplementing a methionine auxotroph BL21 Escherichia coli strain (B834 DE3 from Novagen) with selenomethionine in its growth media. The protein was purified and quantified as described above for native HI0607 with the addition of 5 mm β-mercaptoethanol in all buffers during the purification process. Site-directed Mutagenesis—Site-directed mutagenesis was carried out using the QuikChange™ protocol (Stratagene) in which the following complimentary oligonucleotides containing the required mutation (indicated by boldfaced codons) for the HI0607 active site were used: K67H, 5′-GCTGTTTCAATGCCATTCCACGAAACTTGTATGCC-3′; K67N, 5′-GCTGTTTCAATGCCATTCAACGAAACTTGTATGCC-3′; K67A, 5′-GCTGTTTCAATGCCATTCGCAGAAACTTGTATGCC-3′; D130N, 5′-GCGTGCATATAACACTAACTACATTGCCATCG-3′; and D103A, 5′-GCGTGCATATAACACTGCATACATTGCCATCG-3′. DNA encoding wild-type HI0607 was used as a template for the polymerase chain mutagenesis reaction. Briefly, 25 ng of template DNA were incubated with the appropriate mutagenic primers, dNTPs, and Pfu DNA polymerase using the cycling parameters recommended in the supplier's technical manual. Following completion of the amplification reaction, 10 units of DpnI were added to each reaction mixture and incubated at 37 °C for 6 h, and 1 μl of the resulting mix was then transformed into XL2-Blue cells as recommended by the supplier. Plasmid DNA was purified from the resulting colonies using the Mini Prep Kit (Qiagen), and all mutations were verified by DNA sequencing. Mutant proteins were expressed and purified using the identical protocol as that used for the wild-type protein described above. Enzyme Kinetics—The enzymatic activity of HI0607 was assayed by monitoring the reduction of NADP+ at 340 nm and 30 °C in the presence of either shikimate or quinate. The Km and Vmax values were determined by varying the concentrations of either shikimate or NADP+ while keeping the other substrate at saturation; 4 mm shikimate and 2 mm NADP+ were considered saturating. These substrate concentrations were at least 10 times higher than their respective Km values. The ability of HI0607 to utilize NAD+ was determined by assaying the enzyme at NAD+ concentrations up to 10 mm with shikimate at saturation or with varying the concentration of quinate up to 100 mm. The effect of divalent metals on the enzyme-catalyzed reaction rate was determined with either 5 mm MgCl2, ZnCl2, Zn(OAc)2, MnCl2, or CaCl2 in the reaction cuvette and saturating amounts of NADP+ and shikimate. The pH rate profile was prepared by conducting Michaelis-Menten saturation kinetics at each pH value, and both kcat and Km values were determined. To determine the effect of extreme pH on enzyme stability, the enzyme was preincubated at pH 7.5 and 9.5 and assayed for loss of activity after a specific time at pH 8.5. In addition, enzyme activity was also monitored at the two pH extremes over a 30-min period to determine whether a time-dependent inactivation was occurring. Data Analysis—The kinetic data were fitted to the following rate equations using the computer programs of Cleland or GraFit®. Initial velocity data were obtained by varying the concentration of either shikimate or NADP+ (A) and fitted to Equation 1, shown here, v=VAK+A (Eq. 1) to yield maximal velocity values (V) and the Michaelis constant (K). The variation of the values for V and V/K as a function of pH were fitted to the log form of Equation 2 or Equation 3, shown here, (Eq. (Eq. the of V or V/K at a pH value, the of the and are acid and is the Data and of HI0607 were using the J. J. Scopus Google Scholar). The 1 μl of protein and 1 μl of containing pH and The to with parameters of a and and protein The resulting protein crystal to and the data were from a crystal of native protein at a of 100 in a of selenomethionine protein to was and two data were at the point and the of the The data were and with the D. 2000; Full Text Full Text PDF Scopus Google Scholar). and were obtained with the by with the The protein was with and with as in the Biol. 2002; PubMed Scopus Google Scholar, J. Biol. 1999; PubMed Scopus Google Scholar). The resulting protein from the protein was with the data from the native protein crystal and used to of the protein structure with the A. R. Biol. 1999; PubMed Scopus Google of and HI0607 1 2 for for native and for are shown in for for native and for are shown in at protein for for native and for are shown in for for native and for are shown in from ideal for for native and for are shown in from ideal in a new The was and with the T.A. M. A. PubMed Scopus Google and with the Biol. 50: PubMed Scopus Google Scholar). for the data and structure are shown in A of all the amino in the that of have and were with allowed or as determined with the R. J. 1996; PubMed Scopus Google Scholar). All data were at of the at the acid analyses of proteins to the three classes of shikimate the first was annotated as a SDH-L the as shikimate dehydrogenase by the and the as quinate/shikimate dehydrogenase by the analysis three distinct YdiB, AroE, and SDH-L, a distinct The SDH-L protein was identified in a of including of and In contrast, AroE was identified in and YdiB was in a lower of analysis identified a number of in SDH-L as well as that are common to both AroE and two of residues, and were by consists of two both of which to the class of These two are by and and are important in keeping the and The first 100 the of HI0607 with a structure of a with the and by and The consists of a central with the and by and A central is in the of the which is by the and and an the of the The the of the from to and the it from to The the and from to which that at the two and of this from the of which of the of the A and studies on HI0607 indicate that the protein is a in which may also to its biological This is by the crystal are in a which is consistent with the protein a A and and and of the The of form a with the structure analysis of HI0607 was conducted by the Protein Data with the to of In addition to other shikimate dehydrogenases, we have identified a number of proteins with a of 6 or to that of proteins with to that of HI0607 dehydrogenase dehydrogenase and In each is in the as for The a specific to the shikimate dehydrogenase E. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of the and crystal structures of shikimate dehydrogenases were determined and are in the Protein Data These the structures of the AroE enzymes from E. coli H. influenzae and and the YdiB enzyme from E. coli and and HI0607, the enzyme from H. influenzae that is of the dehydrogenase structures with that of HI0607 that their substrate and are 3, A and The of E. coli and H. influenzae AroE have been identified by with either NADP+ coli or a influenzae of HI0607 is AroE and SDH-L proteins from different The crystal structure of the E. coli that this is with the of an is important for NAD+ and NADP+ A.M. Biol. 1995; PubMed Scopus Google Scholar, S. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, its chain with the and have been shown to the affinity for the B. R. M. M. L. E. A. Biochem. J. 2000; PubMed Scopus Google Scholar). This the of with the of is in with the enzymatic reaction, which that SDH-L NADP+ for but not Although crystal structures of shikimate dehydrogenases are analysis of the site is and important mechanistic that can obtained from structures has not been of the site of HI0607 is and The of this is by a number of groups including and into this at the are a number of groups including and the of this are including a of and to the on the of this are a number of groups including and of are the different shikimate dehydrogenases, that they are important Asp-103 and are invariant the three classes of shikimate dehydrogenases and are in the and Asp-103 and are involved in the catalytic mechanism of shikimate dehydrogenase, because they are the two groups in the site to the position of the structures of shikimate dehydrogenases with that of HI0607 and a the position of the the reaction of shikimate to from this catalytic pair and which is involved in acid and from the of which HI0607 the of analysis that HI0607 may to a new class of shikimate dehydrogenases we out to determine the kinetic properties of HI0607 for from HI0607, the AroE enzyme was also identified in H. a YdiB has not been compared the substrate and kinetic properties of HI0607 with those of the E. coli AroE and YdiB that HI0607 catalyzes the oxidation of shikimate in the presence of NADP+ with a kcat of and Michaelis constants of for shikimate and 37 6 for NADP+ the catalytic rate for shikimate was lower than that of the published rate for E. coli AroE but it was with the published rate for E. coli YdiB (9Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). This turnover rate can to substrate that shikimate may not the biological substrate for the SDH-L also determined that the of HI0607 were with those of AroE, because HI0607 was specific for NADP+ and not utilize NAD+ as a In addition, HI0607 not quinate as a substrate in the presence of either NADP+ or YdiB. These are consistent with our analysis that this shikimate dehydrogenase-like enzyme clusters from the AroE and YdiB shikimate the ability of HI0607 to catalyze the oxidation of shikimate to dehydroshikimate in the presence of the reverse reaction, is that this enzyme to the shikimate dehydrogenase class of of the structures of shikimate dehydrogenase reveals amino acid the active of the three classes of shikimate it is that a number of of active site may to the kinetic properties of These indicate that the SDH-L protein kinetic and properties with both AroE and YdiB shikimate dehydrogenases but may have an biological lower kcat for HI0607 as compared with that of the E. coli AroE protein can also indicate a for To this we at the of a number of divalent and to determine whether they the kinetic parameters of at concentrations up to 5 mm significant rate was also monitored the effect of from the protein by the addition of up to 10 mm in the reaction not the reaction that is divalent on the reaction pH pH rate profile of HI0607 was determined by the maximal enzyme rate the pH of The rate data from this analysis was fitted with the pH (3Roberts C.W. Roberts F. Lyons R.E. Kirisits M.J. Mui E.J. Finnerty J. Johnson J.J. Ferguson D.J. Coggins J.R. Krell T. Coombs G.H. Milhous W.K. Kyle D.E. Tzipori S. Barnwell J. Dame J.B. Carlton J. McLeod R. J. Infect. Dis. 2002; 185: S25-S36Crossref PubMed Scopus (136) Google described and The enzyme a pH rate with the lower activity at the pH and maximal activity at pH The rate profile that a with a of is involved in the catalytic reaction and to for kinetic and pH rate analysis for AroE shikimate dehydrogenase from the also a similar pH rate profile A.W. D. Int. J. Biochem. Scopus Google Scholar). site and are to in this pH to other active site as can also in this pH on their D. Saridakis Biochemistry. PubMed Scopus Google Scholar). This is in with the crystal structure of HI0607, which that and Asp-103 are at the of the the position for a catalytic in this protein the of the Asp-103 and with to each other that they may function as a catalytic in the reaction the role of two active site groups was by Although Asp-103 and are the two groups in the active other groups in the active site can at this pH on their mechanistic analysis in of pH and mutagenesis studies is on the AroE protein. Site-directed Asp-103 and function as a catalytic then the mutation of either will an two groups function we different kinetic varying of inactivation of HI0607 with each and a varying effect on the pH rate to and we also to a This is not in of the of its an mutation was also as a This mutation to for the of the chain and its ability to and Kinetic of Mutant The and purification properties of the enzymes were similar to those of the which that were to In addition, the profile to of each protein was similar to that of the wild-type protein. analysis that proteins with similar compared with the wild-type protein studies are for that all of the mutations at protein concentrations in our reaction from 10 to of protein in a reaction but not a reaction rate with of the is that mutations may have a effect on the active site may substrate To for this we the concentration of acid was to 10 mm and NADP+ to 10 which is at least times their respective Km and we monitored the reaction rate but were to enzyme mutations substrate we have been to enzyme activity at substrate the of the AroE the and in a reduction in shikimate dehydrogenase and D. data. we a similar reduction of HI0607 enzymatic activity for the a activity of for a HI0607 the rate units which is the of a using the extinction coefficient of m–1 cm–1 for NADPH. In the enzyme activity for active site for both AroE and HI0607, is consistent with our for catalytic the of two groups in the enzyme active the pH profile an of and the mutagenesis studies that HI0607 and the AroE we that and Asp-103 are catalytic groups and may function as a catalytic pair for HI0607 are this in addition to the of a number of other active site of the of HI0607 as a shikimate dehydrogenase-like protein. crystal structure is similar to those of AroE and YdiB, it catalyzes the oxidation of shikimate with and important active site are this protein and the two other classes of shikimate AroE and YdiB have distinct biological AroE in the main trunk of the shikimate pathway in both plants and the other YdiB is a branch point enzyme for quinate biosynthesis from the shikimate Because SDH-L catalyzes the oxidation of shikimate and is present in distinct classes of it is that this enzyme is involved in a novel branch from the shikimate all of the at for in conducting for the Saridakis for in the of the and John for with the

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