Structures of Shikimate Dehydrogenase AroE and Its Paralog YdiB

Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Absent in metazoans, this pathway is an attractive target for nontoxic herbicides and drugs. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. Here we characterize YdiB as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. Structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 Å resolution, respectively. Both enzymes display a similar architecture with two α/β domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility suggesting that a switch between open and closed conformations occurs upon substrate binding. Sequence analysis and structural comparison led us to propose the catalytic machinery and a model for 3-dehydroshikimate recognition. Furthermore, we discuss the evolutionary and metabolic implications of the presence of two shikimate dehydrogenases in E. coli and other organisms. Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Absent in metazoans, this pathway is an attractive target for nontoxic herbicides and drugs. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. Here we characterize YdiB as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. Structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 Å resolution, respectively. Both enzymes display a similar architecture with two α/β domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility suggesting that a switch between open and closed conformations occurs upon substrate binding. Sequence analysis and structural comparison led us to propose the catalytic machinery and a model for 3-dehydroshikimate recognition. Furthermore, we discuss the evolutionary and metabolic implications of the presence of two shikimate dehydrogenases in E. coli and other organisms. The shikimate pathway, which links metabolism of carbohydrates to biosynthesis of aromatic compounds, is essential to plants, bacteria, and fungi (1Herrmann K.M. Weaver L.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 473-503Crossref PubMed Scopus (929) Google Scholar) as well as apicomplexan parasites (2Roberts F. Roberts C.W. Johnson J.J. Kyle D.E. Krell T. Coggins J.R. Coombs G.H. Milhous W.K. Tzipori S. Ferguson D.J. Chakrabarti D. McLeod R. Nature. 1998; 393: 801-805Crossref PubMed Scopus (172) Google Scholar). This seven-step metabolic route leads from phosphoenolpyruvate and erythrose 4-phosphate to chorismate, the common precursor for the synthesis of folic acid, ubiquinone, vitamins E and K, and aromatic amino acids (1Herrmann K.M. Weaver L.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 473-503Crossref PubMed Scopus (929) Google Scholar). This pathway is absent in metazoans, which must obtain the essential amino acids phenylalanine and tryptophan from their diet. Therefore, enzymes of this pathway are important targets for the development of nontoxic herbicides (3Kishore G.M. Shah D.M. Annu. Rev. Biochem. 1988; 57: 627-663Crossref PubMed Scopus (357) Google Scholar), as well as antimicrobial (4Davies G.M. Barrett-Bee K.J. Jude D.A. Lehan M. Nichols W.W. Pinder P.E. Thain J.L. Watkins W.J. Wilson R.G. Antimicrob. Agents Chemother. 1994; 38: 403-406Crossref PubMed Scopus (108) Google Scholar) and antiparasite (2Roberts F. Roberts C.W. Johnson J.J. Kyle D.E. Krell T. Coggins J.R. Coombs G.H. Milhous W.K. Tzipori S. Ferguson D.J. Chakrabarti D. McLeod R. Nature. 1998; 393: 801-805Crossref PubMed Scopus (172) Google Scholar) agents. The sixth step in the pathway, catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase, has already been successfully targeted, with the development of glyphosate, a broad spectrum herbicide (5Steinrücken H.C. Armhein N. Biochem. Biophys. Res. Commun. 1980; 94: 1207-1212Crossref PubMed Scopus (786) Google Scholar). However, after 20 years of extensive use, glyphosate-resistant weeds have recently emerged (6Baerson S.R. Rodriguez D.J. Tran M. Feng Y. Biest N.A. Dill G.M. Plant Physiol. 2002; 129: 1265-1275Crossref PubMed Scopus (285) Google Scholar), emphasizing the importance of maintaining target diversity. In order to design new inhibitors, crystal structures of several enzymes of the shikimate pathway have been elucidated recently: 3-dehydroquinate synthase (7Carpenter E.P. Hawkins A.R. Frost J.W. Brown K.A. Nature. 1998; 394: 299-302Crossref PubMed Scopus (118) Google Scholar), type I and II dehydroquinases (8Gourley D.G. Shrive A.K. Polikarpov I. Krell T. Coggins J.R. Hawkins A.R. Isaacs N.W. Sawyer L. Nat. Struct. Biol. 1999; 6: 521-525Crossref PubMed Scopus (128) Google Scholar), type I and II shikimate kinases (9Romanowski M.J. Burley S.K. Proteins. 2002; 47: 558-562Crossref PubMed Scopus (35) Google Scholar, 10Krell T. Coggins J.R. Lapthorn A.J. J. Mol. Biol. 1998; 278: 983-997Crossref PubMed Scopus (73) Google Scholar), and 5-enolpyruvylshikimate-3-phosphate synthase (11Schönbrunn E. Eschenburg S. Shuttleworth W.A. Schloss J.V. Amrhein N. Evans J.N. Kabsch W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1376-1380Crossref PubMed Scopus (395) Google Scholar), catalyzing the second, third, fifth, and sixth steps of the pathway, respectively. Shikimate dehydrogenase (EC 1.1.1.25) catalyzes the fourth reaction in the shikimate pathway, the NADP-dependent reduction of 3-dehydroshikimate to shikimate (Fig. 1A). Whereas dehydrogenases usually form oligomers, shikimate dehydrogenase, coded by the gene aroE in Escherichia coli, is present as a monomer in most bacteria (12Chaudhuri S. Coggins J.R. Biochem. J. 1985; 226: 217-223Crossref PubMed Scopus (45) Google Scholar, 13Anton I.A. Coggins J.R. Biochem. J. 1988; 249: 319-326Crossref PubMed Scopus (49) Google Scholar). In higher organisms this activity is part of a multifunctional enzyme. In plants shikimate dehydrogenase is associated with type I dehydroquinase to form a bifunctional enzyme (14Deka R.K. Anton I.A. Dunbar B. Coggins J.R. FEBS Lett. 1994; 349: 397-402Crossref PubMed Scopus (26) Google Scholar), whereas in fungi, such as Neurospora crassa, this enzyme forms the fifth domain of the pentafunctional AROM polypeptide, which catalyzes five of seven steps of the shikimate pathway (15Lambert J.M. Boocock M.R. Coggins J.R. Biochem. J. 1985; 226: 817-829Crossref PubMed Scopus (41) Google Scholar). However, the molecular basis of 3-dehydroshikimate recognition and enzymatic reduction is not known. Although in E. coli AroE is strictly specific for shikimate, some fungal shikimate dehydrogenases can also utilize quinic acid as a substrate. This compound, which differs from shikimic acid only by the addition of a hydroxyl group at C-1 (Fig. 1B), is the precursor to the ubiquitous plant secondary product chlorogenate (1Herrmann K.M. Weaver L.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 473-503Crossref PubMed Scopus (929) Google Scholar). To date, two independent families of quinate/shikimate dehydrogenases have been identified. The first consists of NAD-dependent dehydrogenases (16Hawkins A.R. Lamb H.K. Moore J.D. Charles I.G. Roberts C.F. J. Gen. Microbiol. 1993; 139: 2891-2899Crossref PubMed Scopus (79) Google Scholar), and the second consists of membrane-associated dehydrogenases that utilize pyrrolo-quinoline-quinone as a cofactor (17Onrston L.N. Neidle E.L. Tower K. Bergogne-Berezin E. Fewson C.A. The Biology of Scholar). Both of dehydrogenases are in the pathway, which of with as the by and metabolism by the pathway (16Hawkins A.R. Lamb H.K. Moore J.D. Charles I.G. Roberts C.F. J. Gen. Microbiol. 1993; 139: 2891-2899Crossref PubMed Scopus (79) Google Scholar, L.N. Neidle E.L. Tower K. Bergogne-Berezin E. Fewson C.A. The Biology of Scholar). J. W. D.J. Res. PubMed Scopus Google Scholar), as putative shikimate can as to AroE the of the the shikimate dehydrogenase shikimate also the NAD-dependent quinate/shikimate whereas the enzymes a This with other a dehydrogenase of the of E. coli and has the presence of a gene of which with AroE and YdiB are paralogs, the only two from the present in E. Here we the of YdiB and that is a quinate/shikimate dehydrogenase that can utilize either NAD or NADP as a cofactor. have determined crystal structures of AroE at 1.5 Å and YdiB at 2.5 Å structures are the first shikimate dehydrogenase structures to Comparison of their led us to propose the important amino acid and to at the molecular the structural to in cofactor specificity. Furthermore, we discuss the evolutionary and metabolic implications of the presence of two shikimate dehydrogenases in E. coli and other organisms. and and as J. K. S. Coggins J.R. Lapthorn A.J. Biol. PubMed Scopus Google Scholar). The to group with a and with molecules in the The gene a of from E. coli E. coli and were in W.A. J.R. D.M. J. PubMed Scopus Google Scholar) with at to an of with and at 20 for were by and in of and of were by and the by The to a with and the This to a of and the with of The target by the bound to by addition of by with the by to and the to 20 were by by The at from of and of and their after The are group with a with two molecules in the and were from AroE to 1.5 Å at at the a were by a with with the only that to the This at a to the at the a in a were and with the W. PubMed Scopus Google Scholar), the and group in I. from the Biol. 1994; 50: PubMed Scopus Google Scholar). the to G.M. W. Evans and Scholar), which were in the to the and the with and of the in in the independent molecules in the the Biol. PubMed Scopus Google Scholar). of the were as for of The the A. R. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar) and the independent that were of the and model and addition of the of and with the addition of molecules of and with the of in the of in a model with the of and of and as the J.M. J. 1993; Google Scholar). The with the with the and and and and and in for Å Å in for Å Å the is in for Å Å of with and of of Å in most and and in for Å Å the is with and of in a new YdiB were for in a in a to the and at in a were a at the at in and were the W. PubMed Scopus Google Scholar). and are in I. were a YdiB crystal to 2.5 Å at and the of the 20 were the of G.M. J. M. L.M. T. Biol. 1998; PubMed Scopus Google Scholar). The with this in a of of Å of the the by molecular and with G.M. J. M. L.M. T. Biol. 1998; PubMed Scopus Google Scholar), a of of The model with the M. A. 47: PubMed Scopus Google Scholar) the with G.M. J. M. L.M. T. Biol. 1998; PubMed Scopus Google Scholar) with the target The were only in the of The as well as the the presence of bound to YdiB The model for the at 2.5 Å has an of and an of and consists of two five and The is by the presence of two conformations of in which the to In the of several were also not the of The a as the J.M. J. 1993; Google Scholar). The is with the with the the of were a of YdiB at and in the presence or in the of The were a To analysis also a and with the by The YdiB and at in the The enzymatic of AroE and YdiB were at 20 by the reduction of or at in the presence of either shikimic acid or quinic To by the enzymatic activity of AroE in the and 20 To the for the of shikimic or quinic acid, and for the cofactor or and and and to the for the of or and for shikimic or quinic acid and and and To the of enzyme to the enzyme were in order to the reaction The at for a of the enzyme. in and a The were by the were the were from of YdiB as a coli YdiB with AroE from the I.A. Coggins J.R. Biochem. J. 1988; 249: 319-326Crossref PubMed Scopus (49) Google Scholar) and with the quinate/shikimate dehydrogenases from N. L. J. Mol. Biol. PubMed Scopus Google Scholar) and from A.R. Lamb H.K. M. J.W. Roberts C.F. Mol. Gen. 1988; PubMed Scopus Google Scholar). this as a to substrate specificity by the reduction of or in the presence of either shikimic acid or quinic a AroE the AroE shikimic acid as cofactor activity in the presence of The are similar for the cofactor and the substrate shikimate and acid, at a of is not a substrate for either in the presence of or To as a with to the of shikimic acid with a of and 20 the of the activity of AroE not that is not a of and that AroE not In YdiB is to shikimic acid by either or as cofactor. of shikimate, YdiB similar for cofactors The for the shikimic acid, to the type of cofactor at shikimate shikimate 20 to YdiB also a activity quinic acid, with either or as a cofactor. of YdiB a five for for This is for the of quinic acid, which is at of at of YdiB is the first quinate/shikimate dehydrogenase in E. Although this enzyme has a catalytic with that of this is by a substrate and cofactor specificity. The specific activity of YdiB not AroE the of this activity from E. coli (12Chaudhuri S. Coggins J.R. Biochem. J. 1985; 226: 217-223Crossref PubMed Scopus (45) Google Scholar). Although is that YdiB is dehydrogenase, we the that substrate is shikimate catalytic AroE and YdiB display a for their as by the similar of their Furthermore, YdiB shikimic and quinic acid, their are in the presence of and In the of YdiB is to which cofactor is YdiB has a to in the presence of as by the between the for at the of either or This by a for as by the cofactor in the presence of or by a of in a of E. coli AroE and of AroE molecules with and a of and of bound in the of A. The molecules are by as J. K. S. Coggins J.R. Lapthorn A.J. Biol. PubMed Scopus Google Scholar). The YdiB two molecules by with and The and are and are not in the we to to AroE with of YdiB in the between AroE and the two enzymes have similar structures that the and The molecules have a and two The first domain is of two and whereas the second domain Both domains have α/β and are by the and a The of two domains the a in which the cofactor is (Fig. of the shikimate dehydrogenase and dehydrogenase from bacteria, and plants are with the secondary structures of E. coli AroE and YdiB. and are as and and are with This the from E. coli AroE AroE dehydrogenase E. N. L. YdiB and E. coli YdiB and with the E. F. 1999; PubMed Scopus Google The domain consists of a and The order is with the to the other The first a with the and to the of the The is with the at to the of the and the to the The domain is by a and which the the as to the L. J. Mol. Biol. 1993; PubMed Scopus Google Scholar), this domain and structural with the domain of which has order to the other of can AroE with of 2.5 In the between and two and the a sixth at the The in is several and from the The of is also similar to the part of the cofactor biosynthesis of Å Although the two in the order in with to the other to a switch in the of and is a of several The domain or domain not from amino acid this domain a a with the order and to the The fourth present in the is in whereas the and fourth are by in a the domains are some of the most structural with Å and II dehydrogenase Å The a new of a the with other this from the Structures of AroE and AroE has been to a (12Chaudhuri S. Coggins J.R. Biochem. J. 1985; 226: 217-223Crossref PubMed Scopus (45) Google Scholar, J. K. S. Coggins J.R. Lapthorn A.J. Biol. PubMed Scopus Google Scholar), YdiB and in the presence and of that YdiB has a with a of that this forms This by the as a of of the the crystal of YdiB that the is between the two molecules in the The two are by with the by from and the of the two This of the domains a with The by from and is in The of from which is at the of for J. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). an is not as a has been for the of from T. J.M. Mol. 2002; PubMed Scopus Google Scholar). the YdiB has been the this are by or amino acids in YdiB and (Fig. amino acid the the of the YdiB a to the domain of AroE and this is present as a monomer in The of AroE and AroE and YdiB molecules the for the in YdiB and for is well (Fig. In the we to of YdiB and of AroE as have the The cofactor is the of at a switch in the of the The of the domains of AroE and YdiB in of and of their and an occurs in the of the of and The of the and is similar in AroE and YdiB. The group of the is to the group of two and the (Fig. The forms only to the The the that and (Fig. and forms to the of and and to the is in the (Fig. This is of the in NAD-dependent and are and is a is the Proteins. PubMed Scopus Google Scholar). the the in the AroE are the of a at the usually the presence of a at and a in of at the the to the of the which have a specificity for either NAD or NADP Proteins. PubMed Scopus Google Scholar), of the a of cofactor specificity. E. coli in is strictly NADP-dependent (12Chaudhuri S. Coggins J.R. Biochem. J. 1985; 226: 217-223Crossref PubMed Scopus (45) Google Scholar), whereas N. and E. display a for NAD L. J. Mol. Biol. PubMed Scopus Google Scholar, A.R. Lamb H.K. M. J.W. Roberts C.F. Mol. Gen. 1988; PubMed Scopus Google Scholar), and E. coli YdiB is to Therefore, the comparison of the cofactor in AroE and YdiB is of as reveals the structural to between NADP and NAD in the The of the by enzymes is for NADP-dependent dehydrogenases as an that the and a by that the group of the in NAD Proteins. PubMed Scopus Google Scholar). In the the between and two strictly and and which are in the recognition of the in their with the cofactor in the two In the the hydroxyl group of the is to as well as to the of in the (Fig. In the of forms a to the of the forms two with the other of the whereas group the of the This is by with from and by a with of the of and (Fig. The and a in as form an that the In YdiB are several the with (Fig. which of to the of the a U. J. Mol. Biol. PubMed Scopus Google Scholar). The of is by a of which the of a This for of and of to as well as the to a of (Fig. The in YdiB is by the of and of AroE by and respectively. is to the hydroxyl group of the and also a The a which is the in the AroE (Fig. and The of YdiB to also a conformational of to with the of YdiB with NADP that this cofactor is in a similar to that of The which is in this in order to a and as a is The and is by the of the of the cofactor and is by from the The is in a by the of the the of the of the between and and the first from the of the in the are in this and a or a is also in the (Fig. or is present in this in AroE and YdiB In molecules and of this is at the of the and is to the hydroxyl of and (Fig. whereas in the AroE and YdiB molecules at the of the to the of and and In of a is also present in this bound and hydroxyl to the AroE and and (Fig. The comparison of independent molecules of AroE and YdiB in the of their conformations are for AroE and whereas the two molecules of YdiB display similar domains of the an in the of of the domains of AroE and YdiB in an of Å for of and Å for of for the and respectively. However, for the molecules are Å for the independent AroE molecules and Å for the comparison of AroE and YdiB molecules (Fig. of AroE the most whereas of YdiB the most form of the enzyme (Fig. and The between two conformations to a of an the of and the of the domain a of Å between the open and closed This conformational is with the of the in the between the and the five in this and and are in the whereas a fourth is in of The is in of the and is by and in the of the In this which we a of by a group that of in a of a group at this In the open are by between their This in the closed as is to the of and this a to the of whereas group is to the closed in the that we that the conformational which the occurs upon substrate and is for the of a The of in the as a the open at the of a catalytic and the of the the substrate is The of presence in the closed of a and a the between shikimate dehydrogenase and substrate (Fig. The of the and and structural led us to propose a model for the recognition of 3-dehydroshikimate (Fig. The enzyme catalyzes the reduction of 3-dehydroshikimate to shikimate as of the substrate. that occurs from the of R. PubMed Scopus Google Scholar), which is with the of the cofactor in the of the two to the of must to the from of the The of the and of in the of the of is with such (Fig. the we that the form specific the In the other enzymes in the shikimate pathway, the of the substrate is bound by either an I dehydroquinase (8Gourley D.G. Shrive A.K. Polikarpov I. Krell T. Coggins J.R. Hawkins A.R. Isaacs N.W. Sawyer L. Nat. Struct. Biol. 1999; 6: 521-525Crossref PubMed Scopus (128) Google Scholar), synthase (7Carpenter E.P. Hawkins A.R. Frost J.W. Brown K.A. Nature. 1998; 394: 299-302Crossref PubMed Scopus (118) Google Scholar), 5-enolpyruvylshikimate-3-phosphate synthase (11Schönbrunn E. Eschenburg S. Shuttleworth W.A. Schloss J.V. Amrhein N. Evans J.N. Kabsch W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1376-1380Crossref PubMed Scopus (395) Google or II dehydroquinase D.A. Krell T. M. Coggins J.R. Lapthorn A.J. 2002; PubMed Scopus Google the of the the the between and by two a of a in a similar to the type II The at and most to that form two to the The in the domain the substrate is also to an with the The of the in this of the and with this (Fig. The substrate in this form between hydroxyl and the of and whereas the hydroxyl the with are between AroE and the and of (Fig. of shikimate dehydrogenase have that of the enzyme a whereas either a hydroxyl or group is for D. Biochem. J. PubMed Scopus Google Scholar). a of of 3-dehydroshikimate that the and and Coggins J.R. Lett. 1988; Scopus Google Scholar) that the hydroxyl of the substrate has the specificity of E. coli whereas the hydroxyl is of the A.R. 1988; PubMed Scopus Google between the hydroxyl and the enzyme that this hydroxyl forms a to a group Coggins J.R. Lett. 1988; Scopus Google Scholar). the of has been that this group is either a or an group D. J. Biochem. Scopus Google Scholar). In this a for the the hydroxyl with dehydrogenase M.J. M. K. A. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), an catalytic group is to a to the of 3-dehydroshikimate reduction and to a of The and are the most to this their to the and the and of (Fig. is the of the of the and in a to that in dehydrogenase J. A. B. J. Biochem. PubMed Scopus Google Scholar). The of AroE at is with a in the of AroE by at has been to the enzyme in a which by T. S. A.R. A. Coggins J.R. J. Res. 1998; PubMed Scopus Google Scholar). of the in AroE from by the presence of of as S. the of Escherichia coli Shikimate of Scholar). However, is a from the of the cofactor in the closed of and is not strictly in the a for the catalytic The and from the of Shikimate in the E. coli presence of two shikimate dehydrogenase in E. coli their specific The of a second shikimate dehydrogenase also the design of YdiB for the of Although the substrate specificity of YdiB has been is not YdiB in the shikimate pathway or has analysis of the in the that at two shikimate dehydrogenase at to and and that this is not only to E. coli or of display a to AroE and YdiB a However, a are either and or and and S. and with between and The of the aroE and in the E. coli is also AroE is by of or putative and to the shikimate In is between the gene a putative amino acid and the gene type I to the A. S. D. E. F. E. J. Res. 2001; PubMed Scopus Google Scholar), AroE and YdiB are whereas the is the of the of and in is also in bacteria L. L. and S. enzymes the Therefore, YdiB have a to that of to the shikimate dehydrogenase and a bifunctional in some plants and a bifunctional enzyme have by the of an gene I dehydroquinases are associated with whereas type II dehydroquinases are to in and The of in in the shikimate In the substrate and cofactor of YdiB in of a NAD-dependent quinate/shikimate dehydrogenases are in the Therefore, YdiB essential for of E. coli with as a (16Hawkins A.R. Lamb H.K. Moore J.D. Charles I.G. Roberts C.F. J. Gen. Microbiol. 1993; 139: 2891-2899Crossref PubMed Scopus (79) Google Scholar), the presence of a pathway in this J. for in the of AroE and N. for of the are also to R. for with the S. for in and J. D. for the the

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