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Residues Involved in the Mechanism of the Bifunctional Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase

2002; Elsevier BV; Volume: 277; Issue: 21 Linguagem: Inglês

10.1074/jbc.m200127200

1083-351X

Saravanan Sundararajan, Robert E. MacKenzie,

Metabolism and Genetic Disorders

The human bifunctional dehydrogenase-cyclohydrolase domain catalyzes the interconversion of 5,10-methylene-H4folate and 10-formyl-H4folate. Although previous structure and mutagenesis studies indicated the importance of lysine 56 in cyclohydrolase catalysis, the role of several surrounding residues had not been explored. In addition to further defining the role of lysine 56, the work presented in this study explores the functions of glutamine 100 and aspartate 125 through the use of site-directed mutagenesis and chemical modification. Mutants at position 100 are inactive with respect to cyclohydrolase activity while preserving significant dehydrogenase levels. We succeeded in producing a K56Q/Q100K double mutant, which has no cyclohydrolase yet retains more than two-thirds of wild type dehydrogenase activity. Neither activity is detectable in aspartate 125 mutants with the exception of D125E. The results indicate that the function of glutamine 100 is to activate lysine 56 for cyclohydrolase catalysis and that aspartate 125 is involved in the binding of the H4folate substrates. In highlighting the importance of these residues, catalytic mechanisms are proposed for both activities as well as an explanation for the differences in channeling efficiency in the forward and reverse directions. The human bifunctional dehydrogenase-cyclohydrolase domain catalyzes the interconversion of 5,10-methylene-H4folate and 10-formyl-H4folate. Although previous structure and mutagenesis studies indicated the importance of lysine 56 in cyclohydrolase catalysis, the role of several surrounding residues had not been explored. In addition to further defining the role of lysine 56, the work presented in this study explores the functions of glutamine 100 and aspartate 125 through the use of site-directed mutagenesis and chemical modification. Mutants at position 100 are inactive with respect to cyclohydrolase activity while preserving significant dehydrogenase levels. We succeeded in producing a K56Q/Q100K double mutant, which has no cyclohydrolase yet retains more than two-thirds of wild type dehydrogenase activity. Neither activity is detectable in aspartate 125 mutants with the exception of D125E. The results indicate that the function of glutamine 100 is to activate lysine 56 for cyclohydrolase catalysis and that aspartate 125 is involved in the binding of the H4folate substrates. In highlighting the importance of these residues, catalytic mechanisms are proposed for both activities as well as an explanation for the differences in channeling efficiency in the forward and reverse directions. The human bifunctional dehydrogenase-cyclohydrolase domain (DC301) 1The abbreviations used are: DC301dehydrogenase-cyclohydrolase domainDfforward dehydrogenaseCfforward cyclohydrolaseH4folatetetrahydrofolateDEPCdiethylpyrocarbonate 1The abbreviations used are: DC301dehydrogenase-cyclohydrolase domainDfforward dehydrogenaseCfforward cyclohydrolaseH4folatetetrahydrofolateDEPCdiethylpyrocarbonate of the human NADP+-dependent trifunctional methylene-H4folate dehydrogenase/methenyl-H4folate cyclohydrolase/formyl-H4folate synthetase catalyzes two sequential reactions involved in the interconversion of substituted tetrahydrofolates (Scheme I). The interconversion of 5,10-methylene-H4folate with 5,10-methenyl-H4folate is accomplished through the NADP+-dependent dehydrogenase activity, whereas 5,10-methenyl-H4folate and 10-formyl-H4folate are interconverted by the cyclohydrolase activity. A significant amount of the labile methenyl-H4folate intermediate is channeled between the two activities in the forward direction (50–60%), whereas in the reverse direction, channeling is complete (1Pawelek P.D. MacKenzie R.E. Biochemistry. 1998; 37: 1109-1115Crossref PubMed Scopus (29) Google Scholar). Because 5,10-methylene-H4folate and 10-formyl-H4folate are important contributors to methionine, serine, thymidylate, and purine syntheses (reviewed in Ref. 2MacKenzie R.E. Blakley R. Benkovic S. Folates and Pterins: Chemistry and Biochemistry of Folates. 1. John Wiley & Sons, Inc., New York1984: 255-306Google Scholar), their interconversion within eukaryotic and prokaryotic cells is crucial to maintain a proper balance of one-carbon precursors.Several lines of evidence have pointed to the fact that both these activities share a common active site. Proteolysis experiments that isolated the bifunctional domain from the trifunctional enzyme failed to resolve the dehydrogenase from the cyclohydrolase activities (3Tan L.U.L. MacKenzie R.E. Biochim. Biophys. Acta. 1977; 485: 52-59Crossref PubMed Scopus (21) Google Scholar, 4Villar E. Schuster B. Peterson D. Schirch V. J. Biol. Chem. 1985; 260: 2245-2252Abstract Full Text PDF PubMed Google Scholar). Chemical modification of the enzyme with DEPC, phenylglyoxal, and carbodiimide-activated folate showed that both activities could be simultaneously inactivated and that DEPC and phenylglyoxal modification could be protected with folate and NADP+ (5Smith D.D.S. MacKenzie R.E. Can. J. Biochem. Cell Biol. 1983; 61: 1166-1171Crossref PubMed Scopus (17) Google Scholar, 6Smith D.D.S. MacKenzie R.E. Biochem. Biophys. Res. Commun. 1985; 128: 148-154Crossref PubMed Scopus (11) Google Scholar, 7Pelletier J.N. MacKenzie R.E. Biochemistry. 1995; 34: 12673-12680Crossref PubMed Scopus (38) Google Scholar). The kinetic parameters for the cyclohydrolase activity are affected by NADP+ and 2′,5′ADP (1Pawelek P.D. MacKenzie R.E. Biochemistry. 1998; 37: 1109-1115Crossref PubMed Scopus (29) Google Scholar, 8Pelletier J.N. MacKenzie R.E. Biochemistry. 1994; 33: 1900-1906Crossref PubMed Scopus (14) Google Scholar). Finally, the covalent incorporation of 1 mol of 3H-folate/mol of enzyme (6Smith D.D.S. MacKenzie R.E. Biochem. Biophys. Res. Commun. 1985; 128: 148-154Crossref PubMed Scopus (11) Google Scholar) and equilibrium dialysis studies (7Pelletier J.N. MacKenzie R.E. Biochemistry. 1995; 34: 12673-12680Crossref PubMed Scopus (38) Google Scholar, 8Pelletier J.N. MacKenzie R.E. Biochemistry. 1994; 33: 1900-1906Crossref PubMed Scopus (14) Google Scholar) further indicated that only one NADP+ and one tetrahydrofolate binding site exist per monomer of the dimeric enzyme.The crystal structure of DC301 with bound NADP+ revealed that each subunit of the homodimer consists of two domains connected by two long α-helices, creating a single cleft between them. The relative positions of the two domains is different in each subunit of the dimer, indicating that these domains can move around two well defined hinge regions and adapt the size of the cleft to accommodate substrates (9Allaire M., Li, Y. MacKenzie R.E. Cygler M. Structure. 1998; 6: 173-182Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). NADP+ binds to the C-terminal domain on one side of the active site cleft through interactions of the 2′-phosphate with Arg173 and Ser197 (Fig. 1). Mutagenesis studies confirmed the importance of these two residues in dinucleotide cofactor binding (10Pawelek P.D. Allaire M. Cygler M. MacKenzie R.E. Biochim. Biophys. Acta. 2000; 1479: 59-68Crossref PubMed Scopus (21) Google Scholar). The structure of DC301 with bound folate analogs localized the folate binding site to the other side of the cleft (11Schmidt A., Wu, H. MacKenzie R.E. Chen V.J. Bewly J.R. Ray J.E. Toth J.E. Cygler M. Biochemistry. 2000; 39: 6325-6335Crossref PubMed Scopus (44) Google Scholar) and involved a conserved Y XXXK motif. Tyr52 was found to only contribute to substrate positioning through hydrophobic stacking interactions, whereas Lys56 mutants completely lost cyclohydrolase activity and retained residual dehydrogenase activity. From these structures and existing mutagenesis data, a preliminary mechanism was proposed for DC301 catalysis in the forward direction (11Schmidt A., Wu, H. MacKenzie R.E. Chen V.J. Bewly J.R. Ray J.E. Toth J.E. Cygler M. Biochemistry. 2000; 39: 6325-6335Crossref PubMed Scopus (44) Google Scholar) and revealed several other substructures that might be involved in the function of this enzyme. A Ser49-Gln100-Pro102 motif was suggested to be important in catalysis, because the three residues apparently coordinate the binding of a water molecule and Gln100 makes a hydrogen bond contact with the catalytic lysine. Moreover, the structure of DC301 with the bound Ly345899 inhibitor suggests a role for Asp125 in folate binding (Fig. 1b). A flexible loop spanning positions 241–250 positioned at roughly 10 Å from the active site may be involved in closing over the active site upon substrate binding and protecting the labile methenyl-H4folate intermediate from release to the solvent. Through site-directed mutagenesis and chemical modification, this study probes the roles of Gln100, Asp125, and the flexible loop (residues 241–150) in substrate binding, channeling, and catalysis.Figure 1Three-dimensional structures.a, the putative folate binding site and the flexible loop structure (residues 241–250). Glutamine 100, lysine 56, and aspartate 125 are shown as well as previously mutated residues and the water molecule whose position is coordinated by glutamine 100 and lysine 56 (9). b, active site showing folate analog Ly345899 and NADP+. Figures were produced using Protein Data Bank identification number 1A4I with Swiss Protein Database Viewer version 3.6 (30Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9464) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The human bifunctional dehydrogenase-cyclohydrolase domain (DC301) 1The abbreviations used are: DC301dehydrogenase-cyclohydrolase domainDfforward dehydrogenaseCfforward cyclohydrolaseH4folatetetrahydrofolateDEPCdiethylpyrocarbonate 1The abbreviations used are: DC301dehydrogenase-cyclohydrolase domainDfforward dehydrogenaseCfforward cyclohydrolaseH4folatetetrahydrofolateDEPCdiethylpyrocarbonate of the human NADP+-dependent trifunctional methylene-H4folate dehydrogenase/methenyl-H4folate cyclohydrolase/formyl-H4folate synthetase catalyzes two sequential reactions involved in the interconversion of substituted tetrahydrofolates (Scheme I). The interconversion of 5,10-methylene-H4folate with 5,10-methenyl-H4folate is accomplished through the NADP+-dependent dehydrogenase activity, whereas 5,10-methenyl-H4folate and 10-formyl-H4folate are interconverted by the cyclohydrolase activity. A significant amount of the labile methenyl-H4folate intermediate is channeled between the two activities in the forward direction (50–60%), whereas in the reverse direction, channeling is complete (1Pawelek P.D. MacKenzie R.E. Biochemistry. 1998; 37: 1109-1115Crossref PubMed Scopus (29) Google Scholar). Because 5,10-methylene-H4folate and 10-formyl-H4folate are important contributors to methionine, serine, thymidylate, and purine syntheses (reviewed in Ref. 2MacKenzie R.E. Blakley R. Benkovic S. Folates and Pterins: Chemistry and Biochemistry of Folates. 1. John Wiley & Sons, Inc., New York1984: 255-306Google Scholar), their interconversion within eukaryotic and prokaryotic cells is crucial to maintain a proper balance of one-carbon precursors. dehydrogenase-cyclohydrolase domain forward dehydrogenase forward cyclohydrolase tetrahydrofolate diethylpyrocarbonate dehydrogenase-cyclohydrolase domain forward dehydrogenase forward cyclohydrolase tetrahydrofolate diethylpyrocarbonate Several lines of evidence have pointed to the fact that both these activities share a common active site. Proteolysis experiments that isolated the bifunctional domain from the trifunctional enzyme failed to resolve the dehydrogenase from the cyclohydrolase activities (3Tan L.U.L. MacKenzie R.E. Biochim. Biophys. Acta. 1977; 485: 52-59Crossref PubMed Scopus (21) Google Scholar, 4Villar E. Schuster B. Peterson D. Schirch V. J. Biol. Chem. 1985; 260: 2245-2252Abstract Full Text PDF PubMed Google Scholar). Chemical modification of the enzyme with DEPC, phenylglyoxal, and carbodiimide-activated folate showed that both activities could be simultaneously inactivated and that DEPC and phenylglyoxal modification could be protected with folate and NADP+ (5Smith D.D.S. MacKenzie R.E. Can. J. Biochem. Cell Biol. 1983; 61: 1166-1171Crossref PubMed Scopus (17) Google Scholar, 6Smith D.D.S. MacKenzie R.E. Biochem. Biophys. Res. Commun. 1985; 128: 148-154Crossref PubMed Scopus (11) Google Scholar, 7Pelletier J.N. MacKenzie R.E. Biochemistry. 1995; 34: 12673-12680Crossref PubMed Scopus (38) Google Scholar). The kinetic parameters for the cyclohydrolase activity are affected by NADP+ and 2′,5′ADP (1Pawelek P.D. MacKenzie R.E. Biochemistry. 1998; 37: 1109-1115Crossref PubMed Scopus (29) Google Scholar, 8Pelletier J.N. MacKenzie R.E. Biochemistry. 1994; 33: 1900-1906Crossref PubMed Scopus (14) Google Scholar). Finally, the covalent incorporation of 1 mol of 3H-folate/mol of enzyme (6Smith D.D.S. MacKenzie R.E. Biochem. Biophys. Res. Commun. 1985; 128: 148-154Crossref PubMed Scopus (11) Google Scholar) and equilibrium dialysis studies (7Pelletier J.N. MacKenzie R.E. Biochemistry. 1995; 34: 12673-12680Crossref PubMed Scopus (38) Google Scholar, 8Pelletier J.N. MacKenzie R.E. Biochemistry. 1994; 33: 1900-1906Crossref PubMed Scopus (14) Google Scholar) further indicated that only one NADP+ and one tetrahydrofolate binding site exist per monomer of the dimeric enzyme. The crystal structure of DC301 with bound NADP+ revealed that each subunit of the homodimer consists of two domains connected by two long α-helices, creating a single cleft between them. The relative positions of the two domains is different in each subunit of the dimer, indicating that these domains can move around two well defined hinge regions and adapt the size of the cleft to accommodate substrates (9Allaire M., Li, Y. MacKenzie R.E. Cygler M. Structure. 1998; 6: 173-182Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). NADP+ binds to the C-terminal domain on one side of the active site cleft through interactions of the 2′-phosphate with Arg173 and Ser197 (Fig. 1). Mutagenesis studies confirmed the importance of these two residues in dinucleotide cofactor binding (10Pawelek P.D. Allaire M. Cygler M. MacKenzie R.E. Biochim. Biophys. Acta. 2000; 1479: 59-68Crossref PubMed Scopus (21) Google Scholar). The structure of DC301 with bound folate analogs localized the folate binding site to the other side of the cleft (11Schmidt A., Wu, H. MacKenzie R.E. Chen V.J. Bewly J.R. Ray J.E. Toth J.E. Cygler M. Biochemistry. 2000; 39: 6325-6335Crossref PubMed Scopus (44) Google Scholar) and involved a conserved Y XXXK motif. Tyr52 was found to only contribute to substrate positioning through hydrophobic stacking interactions, whereas Lys56 mutants completely lost cyclohydrolase activity and retained residual dehydrogenase activity. From these structures and existing mutagenesis data, a preliminary mechanism was proposed for DC301 catalysis in the forward direction (11Schmidt A., Wu, H. MacKenzie R.E. Chen V.J. Bewly J.R. Ray J.E. Toth J.E. Cygler M. Biochemistry. 2000; 39: 6325-6335Crossref PubMed Scopus (44) Google Scholar) and revealed several other substructures that might be involved in the function of this enzyme. A Ser49-Gln100-Pro102 motif was suggested to be important in catalysis, because the three residues apparently coordinate the binding of a water molecule and Gln100 makes a hydrogen bond contact with the catalytic lysine. Moreover, the structure of DC301 with the bound Ly345899 inhibitor suggests a role for Asp125 in folate binding (Fig. 1b). A flexible loop spanning positions 241–250 positioned at roughly 10 Å from the active site may be involved in closing over the active site upon substrate binding and protecting the labile methenyl-H4folate intermediate from release to the solvent. Through site-directed mutagenesis and chemical modification, this study probes the roles of Gln100, Asp125, and the flexible loop (residues 241–150) in substrate binding, channeling, and catalysis. We thank Dr. R. Kazlauskas and for helpful discussions and C. Pickett for preparation and preliminary characterization of the K56Q/Q100K and D125A mutants.