Induced Fit Movements and Metal Cofactor Selectivity of Class II Aldolases

Fructose-1,6-bisphosphate (FBP) aldolase is an essential glycolytic enzyme that reversibly cleaves its ketohexose substrate into triose phosphates. Here we report the crystal structure of a metallo-dependent or class II FBP aldolase from an extreme thermophile, Thermus aquaticus (Taq). The quaternary structure reveals a tetramer composed of two dimers related by a 2-fold axis. Taq FBP aldolase subunits exhibit two distinct conformational states corresponding to loop regions that are in either open or closed position with respect to the active site. Loop closure remodels the disposition of chelating active site histidine residues. In subunits corresponding to the open conformation, the metal cofactor, Co2+, is sequestered in the active site, whereas for subunits in the closed conformation, the metal cation exchanges between two mutually exclusive binding loci, corresponding to a site at the active site surface and an interior site vicinal to the metal-binding site in the open conformation. Cofactor site exchange is mediated by rotations of the chelating histidine side chains that are coupled to the prior conformational change of loop closure. Sulfate anions are consistent with the location of the phosphate-binding sites of the FBP substrate and determine not only the previously unknown second phosphate-binding site but also provide a mechanism that regulates loop closure during catalysis. Modeling of FBP substrate into the active site is consistent with binding by the acyclic keto form, a minor solution species, and with the metal cofactor mediating keto bond polarization. The Taq FBP aldolase structure suggests a structural basis for different metal cofactor specificity than in Escherichia coli FBP aldolase structures, and we discuss its potential role during catalysis. Comparison with the E. coli structure also indicates a structural basis for thermostability by Taq FBP aldolase. Fructose-1,6-bisphosphate (FBP) aldolase is an essential glycolytic enzyme that reversibly cleaves its ketohexose substrate into triose phosphates. Here we report the crystal structure of a metallo-dependent or class II FBP aldolase from an extreme thermophile, Thermus aquaticus (Taq). The quaternary structure reveals a tetramer composed of two dimers related by a 2-fold axis. Taq FBP aldolase subunits exhibit two distinct conformational states corresponding to loop regions that are in either open or closed position with respect to the active site. Loop closure remodels the disposition of chelating active site histidine residues. In subunits corresponding to the open conformation, the metal cofactor, Co2+, is sequestered in the active site, whereas for subunits in the closed conformation, the metal cation exchanges between two mutually exclusive binding loci, corresponding to a site at the active site surface and an interior site vicinal to the metal-binding site in the open conformation. Cofactor site exchange is mediated by rotations of the chelating histidine side chains that are coupled to the prior conformational change of loop closure. Sulfate anions are consistent with the location of the phosphate-binding sites of the FBP substrate and determine not only the previously unknown second phosphate-binding site but also provide a mechanism that regulates loop closure during catalysis. Modeling of FBP substrate into the active site is consistent with binding by the acyclic keto form, a minor solution species, and with the metal cofactor mediating keto bond polarization. The Taq FBP aldolase structure suggests a structural basis for different metal cofactor specificity than in Escherichia coli FBP aldolase structures, and we discuss its potential role during catalysis. Comparison with the E. coli structure also indicates a structural basis for thermostability by Taq FBP aldolase. Aldolases are essential enzymes that catalyze carbon-carbon bond formation in living organisms. They are ubiquitous and highly abundant in pathways of intermediate cellular metabolism such as gluconeogenesis, the Calvin cycle, and glycolysis, where they reversibly cleave ketohexose sugars. In synthetic chemistry, the action of aldolases is precisely controlled by the stereochemistry of these reactions, and thus these enzymes are often used as an alternative to conventional chemical methods in biotransformations and synthetic organic chemistry (1Takayama S. McGarvey G.J. Wong C.H. Annu. Rev. Microbiol. 1997; 51: 285-310Crossref PubMed Scopus (83) Google Scholar, 2Wong C.-H. Whitesides G.M. Tetrahedron Organic Chemistry Series: Enzymes in Synthetic Organic Chemistry. 12. Pergamon Press, New York1994Google Scholar) and especially in the synthesis of novel antibiotics (3Wagner J. Lerner R.A. Barbas III, C.F. Science. 1997; 270: 1797-1800Crossref Scopus (418) Google Scholar, 4Barbas III, C.F. Heine A. Zhong G. Hoffmann T. Gramatikova S. Bjornestedt R. List B. Anderson J. Stura E.A. Wilson I.A. Lerner R.A. Science. 1997; 278: 2085-2092Crossref PubMed Scopus (366) Google Scholar). Aldolases that cleave ketohexose substrates are among the most studied enzymes and, depending on their reaction mechanism, fall into two distinct groups. The class I enzymes utilize a lysine in Schiff base formation during catalysis and are mainly found in higher order organisms. Determination of the crystal structures of several class I enzymes (5Izard T. Lawrence M.C. Malby R.L. Lilley G.G. Colman P.M. Structure. 1994; 15: 361-369Abstract Full Text Full Text PDF Scopus (106) Google Scholar, 6Sygusch J. Beaudry D. Allaire M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7846-7850Crossref PubMed Scopus (212) Google Scholar, 7Gamblin S.J. Cooper B. Millar J.R. Davies G.J. Littlechild J.A. Watson H.C. FEBS Lett. 1990; 262: 282-286Crossref PubMed Scopus (57) Google Scholar, 8Hester G. Brenner-Holzach O. Rossi F.A. Struck-Donatz M. Winterhalter K.H. Smith J.D.G. Piontek K. FEBS Lett. 1991; 292: 237-242Crossref PubMed Scopus (87) Google Scholar, 9Kim H. Certa U. Dobeli H. Jakob P. Hol W.G. Biochem. 1998; 37: 4388-4396Crossref PubMed Scopus (73) Google Scholar, 10Choi K.H. Mazurkie A.S. Morris A.J. Utheza D. Tolan D.R. Allen K.N. Biochemistry. 1999; 38: 12655-12664Crossref PubMed Scopus (51) Google Scholar, 11Chudzik D.M. Michels P.A. de Walque S. Hol W.G. J. Mol. Biol. 2000; 300: 697-707Crossref PubMed Scopus (61) Google Scholar, 12Dalby A.R. Tolan D.R. Littlechild J.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1526-1533Crossref PubMed Scopus (41) Google Scholar) together with biochemical studies (13Lai C.Y. Tchola O. Cheng T. Horecker B.L. J. Biol. Chem. 1965; 240: 1347-1350Abstract Full Text PDF PubMed Google Scholar, 14Anai M. Lai C.Y. Horecker B.L. Arch. Biochem. Biophys. 1973; 156: 712-719Crossref PubMed Scopus (27) Google Scholar, 15Hannappel E. MacGregor J.S. Davoust S. Horecker B.L. Arch. Biochem. Biosphys. 1974; 214: 293-298Crossref Scopus (22) Google Scholar, 16Berthiaume L. Loisel T. Sygusch J. J. Biol. Chem. 1991; 266: 17092-17105Abstract Full Text PDF PubMed Google Scholar, 17Dobeli H. Itin C. Meier B. Certa U. Acta Leidensia. 1991; 60: 135-140PubMed Google Scholar, 18Berthiaume L. Tolan D.R. Sygusch J. J. Biol. Chem. 1993; 268: 10826-10835Abstract Full Text PDF PubMed Google Scholar, 19Maurady A. Zdanov A. de Moissac D. Beaudry D. Sygusch J. J. Biol. Chem. 2002; 277: 9474-9483Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) have provided mechanistic details for ligand recognition and catalysis in class I aldolases. Structurally, these aldolases display an (α/β)8 barrel in a homotetrameric arrangement. In contrast, class II enzymes, found in yeast, bacteria, fungi, and blue-green algae, are most often homodimeric (α/β)8 barrels (20Blom N. Tetreault S. Coulombe R. Sygusch J. Nat. Struc. Biol. 1996; 3: 856-862Crossref PubMed Scopus (80) Google Scholar, 21Cooper S.J. Leonard G.A. McSweeney S.M. Thompson A.W. Naismith J.H. Qamar S. Plater A. Berry A. Hunter W.N. Structure. 1996; 4: 1303-1315Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and require for catalysis a divalent metal cation, typically a transition metal such as Zn2+. The divalent cation functions as a Lewis acid to polarize the carbonyl bond of the incoming ketoses, thereby promoting cleavage of the adjacent carbon-carbon bond as well as proton transfer during enamine formation. Class II aldolases are activated by monovalent cations, such as NH4+, are generally more stable than their class I counterparts, exhibit a wide range of substrate specificity, and are preferred for use in biotransformation chemistry (22von der Osten C.H. Sinskey A.J. Barbas C.F. Pederson R.L. Wang Y.-F. Wong C.-H. J. Am. Chem. Soc. 1989; 111: 3924-3927Crossref Scopus (197) Google Scholar, 23Schoevaart R. van Rantwijk F. Sheldon R.A. J. Org. Chem. 2001; 66: 4559-4562Crossref PubMed Scopus (38) Google Scholar). Their reaction mechanisms are diverse. For instance, in one class II enzyme, 2-dehydro-3-deoxy-galactarate aldolase, a than an acid side proton transfer during enamine formation T. N. J. 2000; PubMed Scopus (51) Google Scholar). among class II aldolases is and in the of the and recognition and on details of active site with substrate D.R. Leonard G.A. Berry A. Hunter W.N. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Class II aldolases also potential for the of and they to and order class II FBP used Thermus used Thermus aldolase of its role in intermediate The enzyme the cleavage of FBP to the triose and cleavage during glycolysis, and the is used during or the Calvin sites corresponding to the divalent metal as well as the site of the monovalent cation in the FBP aldolase crystal structure from Escherichia coli S.J. Leonard G.A. McSweeney S.M. Thompson A.W. Naismith J.H. Qamar S. Plater A. Berry A. Hunter W.N. Structure. 1996; 4: 1303-1315Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). the crystal structures of E. coli FBP aldolase crystal structure in with a triose transition structural in substrate recognition and D.R. Leonard G.A. Berry A. Hunter W.N. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). that that class II FBP aldolases during the and their with active site conformational during the crystal structure of class II FBP aldolase to from the extreme Thermus in the of a The crystal structure not only the role of the conformational during the but also provided an as to the metal cofactor by Taq FBP aldolase for Co2+, than is preferred in such as E. of Taq FBP aldolase as Sygusch J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: PubMed Scopus Google Scholar) by of of at of and of the solution and that of the solution at K. The in to in The of the of Taq FBP aldolase from Sygusch J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: PubMed Scopus Google Scholar) that of of solution of Taq FBP aldolase and of the solution of and that of the solution at K. The in with in for the of Taq FBP aldolase and the in with previously Sygusch J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: PubMed Scopus Google Scholar) and is in The structure solution of the structure of the in and to by the in together with the Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; PubMed Scopus Google Scholar) of the sites in the Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; Scopus Google Scholar). The not of with the Acta Crystallogr. Sect. A. 1991; PubMed Scopus Google Scholar) and by of and from to the Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; PubMed Scopus Google Scholar). The for the at The of the to the and of an by the Acta Crystallogr. Sect. A. 1991; PubMed Scopus Google Scholar). The and of the two loop regions and the active site, in a by the higher enzyme The used as a to determine the Taq FBP aldolase crystal structure by the Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; PubMed Scopus Google Scholar). solution two in the of the enzyme, and for the loop the subunits the related by 2-fold The in Taq aldolase loop regions to an open conformation, whereas only one loop in the Taq aldolase and to the loop a closed conformation. not with loop in the closed Taq FBP aldolase structures with the J. M. Science. Scopus Google Scholar, G.M. P. J. N. T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; PubMed Scopus Google Scholar) The PubMed Scopus Google Scholar) the II the for the In the for and in the closed and and in the open The subunits in their open for and in one and and in the The of these to only with the to The also for one of the two anions to the active site of in their open conformation, and their to in the and for and The the and the for the by the Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; PubMed Scopus Google Scholar) indicates that of in most and in The structure also that are than at the a in the of Taq of of acid of of range where the of and the of where the of and the of the for a from the prior to The of the and not used the for a from the prior to The of the and not used side from bond where the of and the of the for a from the prior to The of the and not used in a acyclic of FBP from into the active site of the structure by the the The substrate in the active site such that its with the in the closed and keto the metal In the the second site. of at the to by substrate into the active site. The of the and the substrate not with the of the Taq FBP structure of the Taq FBP aldolase of an (α/β)8 barrel The of the are in and in with a of The barrel is closed on its by an The of the structure of an and is by an In to the the structure also a a in the of and the two and are to and an from the barrel that of are in II Taq FBP aldolase as a in solution with a of H. J. PubMed Google Scholar) consistent with The mutually are as a of and where the is the 2-fold whereas the and are the 2-fold The of the tetramer are in the in the and in the is in with the subunits the The the are more than the a the as a surface of surface as the or of surface whereas the between the subunits are minor of in a of dimers the The the on and as well as in the loop regions and and that in and and that and are between structural to that in van der the on the and as well as on the and that in and that and between and and and and one is the related between and are also the subunits and and FBP aldolase as the at in or at in Sygusch J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: PubMed Scopus Google Scholar). The are with two in the and to to from Taq FBP aldolase with in the The two in the are in the closed conformation, one loop and one loop that the active site. two loop regions that and and are in the In contrast, in the of the cofactor one in the closed in with for and and in the open for in of the 2-fold thus one tetramer in the open and one tetramer with two in the open and two in the closed conformation. in the in Taq FBP aldolase on its and in from as The in the the are the for the binding sites of the in the subunits in their open the the in the closed two mutually exclusive binding sites for metal binding is mediated by a conformational transition side rotations that by histidine and these two mutually exclusive are only their corresponding in the are only the the of that of the found in the subunits the that display the open conformation. for the two mutually exclusive in the closed from and and and and and one from a and In contrast, for the cation found in the open not only of and and and and one from and but also the of in of Taq FBP aldolase active site. for to the The of the is and the is The of the are in whereas the of the enzyme are in For as are not to a 2-fold related are in the closed in with the anions that with the phosphate-binding sites of the two mutually exclusive as and the cation as a and cation binding to the active site as in the subunits in their open conformation. by with respect to to that with the monovalent FBP into the active site the sites of the closed as in the Taq FBP aldolase with The novel metal-binding site is as a The and of the FBP are of the site by such as or are to class II FBP aldolases and in the of Taq aldolase, only the enzyme Sygusch J. 2001; PubMed Scopus Google Scholar). in the for cation sites in subunits in the with from to the The cation sites with The cation is by the of the of from to in the and the of the side chains of to to and a to FBP aldolase in the of an an metal-binding site metal-binding site not previously and is found in the in its closed conformation. The metal is by two of the side of one of the of and the of and a The metal to a The site a a to that of Sulfate FBP aldolase from of as the Sygusch J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: PubMed Scopus Google Scholar). these with or for binding to of the are found in the that to two anions to the active site of the in the closed conformation. in to the of the of and as well as to two on the loop that the active site in The second is and an with the side of as well as to the of and The two anions are also found in the subunits in the in their open of the closed is not for binding to the not as as in the in the closed conformation. for only a of the to to the closed The also to to a of with in the closed where contrast, the second in the active site to the open in a to that in the closed and at the of a in the The site is at the surface of and is from the active site. the also for and the to in subunits to a of In the the from the side chains of and as well as from two and in In the closed conformation, an with side one and the in of are the in of a H. J. PubMed Google Scholar, A. P. Structure. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Taq aldolase, is stable at for several A. R. Biochem. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google in its only are in the E. coli FBP aldolase crystal In an of between and in the Taq FBP aldolase crystal structure is by two in the E. coli to in E. coli in E. coli in E. coli and in E. coli lysine in E. coli and in E. coli that in In E. coli aldolase, two and formation of an In a of class II FBP aldolase C. Sygusch J. J. Biochem. 1996; PubMed Scopus Google only of in in the Taq aldolase structure are in Taq FBP aldolase are between subunits related by the one of these are found in the E. coli structure In the is in a of and and the 2-fold related and also found subunits related by the In the side chains of and are and the of the 2-fold related in van der found in the Taq FBP aldolase structure the related by the a of and structural that to thermostability are and as by A. P. Structure. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of the crystal structures of class II FBP aldolase from E. coli and T. aquaticus with A. R. J. Mol. 1997; Google Scholar) is in the for in between E. coli aldolase and Taq aldolase in closed conformation. The that the Taq aldolase structure and a higher of a more structure that is consistent with structural found in loop in the crystal structure of the E. coli enzyme, whereas is to in Taq aldolase. For instance, the loop and in the E. coli structure of whereas only are in a on the In the loop in the E. coli structure not in Taq aldolase. The E. coli structure also structure at its with the Taq contrast, and the in Taq aldolase are not in the E. coli from and that are to and an that from the barrel The to of the of van der with the The in on of the with on The of thus not only but also formation and is consistent with the thermostability of Taq aldolase. in Class II FBP II FBP aldolases during catalysis. for Taq aldolase in open and closed in that the loop and a loop conformational active site ligand Comparison of the crystal structure of E. coli class II FBP aldolase to S.J. Leonard G.A. McSweeney S.M. Thompson A.W. Naismith J.H. Qamar S. Plater A. Berry A. Hunter W.N. Structure. 1996; 4: 1303-1315Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and T. N. J. 2000; PubMed Scopus (51) Google Scholar) as well as in with to D.R. Leonard G.A. Berry A. Hunter W.N. J. Mol. Biol. 1999; PubMed Scopus Google the transition of the a closure mechanism mediating ligand In the E. coli structures, these to and and display an open conformation, in In the of the its conformation, a closed position with the more open in the In the E. coli the loop the active site in and a in the Taq as in the Taq aldolase closed conformation, a of of the loop of the E. coli enzyme not in the ligand by in the loop and most from as a of the conformational change by the adjacent is the mechanism that is for The loop to of the site as a by to the of the loop in its closed and closure. The in their open only to the that with the loop in the closed position as by and consistent with a used in In contrast, the in its closed binding of The in the closed open its of of with the the position in the subunits an open is also by crystal In with the and the carbonyl of is to the side of the The crystal by distinct loop that closure the two distinct conformational states are not with an mechanism closure is by ligand The sites have the of the FBP binding in the Taq aldolase active site. The position of the in the structure of the E. coli enzyme in with the transition with that of a in of structure and that closure. of in E. coli aldolase FBP binding S. K. Berry A. Sci. 1996; PubMed Scopus Google that the with the in Taq aldolase the binding of the FBP and with the sites is consistent with binding by the acyclic keto of FBP in open and closed The FBP in is of and loop closure the whereas the is to with FBP and bond with and The in the E. coli and FBP as well as binding A.R. S.M. G.J. Qamar S. Berry A. J. Mol. Biol. 1999; PubMed Scopus Google Scholar) the of the acyclic keto of FBP in The FBP corresponding to the of FBP also the binding for the transition in the active site of E. coli aldolase, in D.R. Leonard G.A. Berry A. Hunter W.N. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). to the closed conformation, FBP by the keto with the cation from the binding site, consistent with a reaction mechanism where the cation is to polarize the keto of FBP not of with the binding sites for the The loop by in the closed in a conformational change with the open position in that the active site. in the open is on to the active site and side the active site a with the side on the as in the open in closure thus of and The by the side into the closed remodels the site with respect to the binding site in in the open the cation at the interior site to only with an a and are to binding of the cation with the that in of class II aldolases by the of the closed conformation. by the transition metal cation between the two mutually exclusive binding sites is not side rotations by the chelating histidine residues. The of the in class II aldolases is that of a the a conformational as by that remodels the active site. The active site the cation to exchange between two sites in Taq of the site the cation to as a Lewis acid by the keto during the that the conformational change active site cation exchange is the basis for metal cation by class II FBP aldolases. In the enzyme, the with metal and whereas in the enzyme, of the active sites that the cation is in the open in Taq aldolase, whereas at the interior site in the E. coli aldolase the cation is the of from histidine but as to the of The cation an and in a with the side than binding in a as in the The of the of two thus a for by cation that is consistent with of the cation as a Lewis acid with J. in Chemistry. New Scholar). the active site and the and are in the and class II and of the active sites reveals structural The cation binding thus to by of the metal cation as Lewis acid and by structural between the two active site by the cation in the enzyme with suggests active site at higher also to the reaction The cation in E. coli aldolase the transition by the and as well as the active site histidine D.R. Leonard G.A. Berry A. Hunter W.N. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). of a transition in the enzyme at higher by with the active site is by the in as a metal cofactor in the enzyme H. J. PubMed Google Scholar). the higher by with in Taq aldolase H. J. PubMed Google Scholar) with the of the cation, the that of the metal cofactor active site at for Taq aldolase and for on the The of in and is by is

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