Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase

The mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) is believed to have evolved from a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. To enable us to investigate the roles of these ions, a homology model of human NMDMC was constructed based on the structures of three homologous proteins. The model supports the hypothesis that the absolutely required Pi can bind in close proximity to the 2′-hydroxyl of NAD through interactions with Arg166 and Arg198. The characterization of mutants of Arg166, Asp190, and Arg198 show that Arg166 is primarily responsible for Pi binding, while Arg198 plays a secondary role, assisting in binding and properly orienting the ion in the cofactor binding site. Asp190 helps to properly position Arg166. Mutants of Asp133 suggest that the magnesium ion interacts with both Pi and the aspartate side chain and plays a role in positioning Pi and NAD. NMDMC uses Pi and magnesium to adapt an NADP binding site for NAD binding. This adaptation represents a novel variation of the classic Rossmann fold. The mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) is believed to have evolved from a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in its absolute requirement for inorganic phosphate and magnesium ions to support dehydrogenase activity. To enable us to investigate the roles of these ions, a homology model of human NMDMC was constructed based on the structures of three homologous proteins. The model supports the hypothesis that the absolutely required Pi can bind in close proximity to the 2′-hydroxyl of NAD through interactions with Arg166 and Arg198. The characterization of mutants of Arg166, Asp190, and Arg198 show that Arg166 is primarily responsible for Pi binding, while Arg198 plays a secondary role, assisting in binding and properly orienting the ion in the cofactor binding site. Asp190 helps to properly position Arg166. Mutants of Asp133 suggest that the magnesium ion interacts with both Pi and the aspartate side chain and plays a role in positioning Pi and NAD. NMDMC uses Pi and magnesium to adapt an NADP binding site for NAD binding. This adaptation represents a novel variation of the classic Rossmann fold. During embryogenesis and tumorigenesis mammalian mitochondria use a folate-dependent pathway to generate both glycine and one-carbon units to support cytoplasmic purine synthesis (1Christensen K.E. Patel H. Kuzmanov U. Mejia N.R. MacKenzie R.E. J. Biol. Chem. 2005; 280: 7597-7602Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 2Patel H. Di Pietro E. MacKenzie R.E. J. Biol. Chem. 2003; 278: 19436-19441Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). One of the enzymes in this pathway, the NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC), 5The abbreviations used are: NMDMC, NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase; methylene-THF, 5,10-methylenetetrahydrofolate; formyl-THF, 10-formyltetrahydrofolate; DCS, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase; DC, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase; GR, glutathione reductase; DH, dehydrogenase; PDB, Protein Data Bank; MOPS, 4-morpholinepropanesulfonic acid; WT, wild type. catalyzes the interconversion of 5,10-methylenetetrahydrofolate (methylene-THF) and 10-formyltetrahydrofolate (formyl-THF) in mammalian mitochondria. The mitochondrial formyl-THF is converted to formate by a monofunctional formyl-THF synthetase and is released to the cytoplasm for reconversion into formyl-THF to support purine biosynthesis (1Christensen K.E. Patel H. Kuzmanov U. Mejia N.R. MacKenzie R.E. J. Biol. Chem. 2005; 280: 7597-7602Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). NMDMC can use both NAD and NADP as a cofactor in its dehydrogenase activity, although the maximal activity with NADP is only about twenty percent of that with NAD (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar). NMDMC is thought to have evolved from a trifunctional NADP-dependent methylene-THF dehydrogenase-methenyl-THF cyclohydrolase-formyl-THF synthetase (DCS) through the loss of the synthetase domain and the change in cofactor specificity from NADP to NAD (4Patel H. Christensen K.E. Mejia N. MacKenzie R.E. Arch. Biochem. Biophys. 2002; 403: 145-148Crossref PubMed Scopus (18) Google Scholar). This change in cofactor specificity is important because the use of NAD rather than NADP in mitochondria shifts the equilibrium of the reaction to favor the production of formyl-THF (5Pelletier J.N. MacKenzie R.E. Biochemistry. 1995; 34: 12673-12680Crossref PubMed Scopus (38) Google Scholar). The increased production of formyl-THF is required to meet the demand for glycine and purines during embryogenesis (1Christensen K.E. Patel H. Kuzmanov U. Mejia N.R. MacKenzie R.E. J. Biol. Chem. 2005; 280: 7597-7602Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 2Patel H. Di Pietro E. MacKenzie R.E. J. Biol. Chem. 2003; 278: 19436-19441Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Di Pietro E. Sirois J. Tremblay M.L. MacKenzie R.E. Mol. Cell. Biol. 2002; 22: 4158-4166Crossref PubMed Scopus (84) Google Scholar). However, it is not known how this cofactor specificity change was accomplished. NMDMC is unique in its absolute requirement for magnesium and inorganic phosphate ions for NAD-dependent dehydrogenase activity and magnesium ions for NADP-dependent dehydrogenase activity (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar, 7Rios-Orlandi E.M. MacKenzie R.E. J. Biol. Chem. 1988; 263: 4662-4667Abstract Full Text PDF PubMed Google Scholar). However, neither ion is essential for the cyclohydrolase activity. The role of these ions in the dehydrogenase activity is not clear. The sequence of binding of the cofactors and substrates to NMDMC, as established kinetically by Yang and Mackenzie (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar) and Rios-Orlandi and MacKenzie (7Rios-Orlandi E.M. MacKenzie R.E. J. Biol. Chem. 1988; 263: 4662-4667Abstract Full Text PDF PubMed Google Scholar), suggests a role for Pi and Mg2+ in the binding of the cofactor; the ions bind to the protein first, followed by NAD and then the folate substrate. A preferred order of binding of the ions was not established; either ion appears to be able to bind to the enzyme and affect the binding of the other. These results suggested a possible interaction between the two ions in the binding site. The observation that Pi competitively inhibits the cofactor in NADP-dependent dehydrogenase assays of NMDMC led to the proposal that Pi may occupy a position adjacent to the 2′-hydroxyl of NAD, close to the space that would be occupied by the 2′-phosphate of NADP (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar). Previous work on the DC domain of the human NADP-dependent DCS identified two residues (Arg173 and Ser197) as being important to the binding of NADP to the enzyme through its 2′-phosphate (8Pawelek P.D. Allaire M. Cygler M. MacKenzie R.E. Biochim. Biophys. Acta. 2000; 1479: 59-68Crossref PubMed Scopus (21) Google Scholar). Sequence alignments of mitochondrial NAD-DCs with trifunctional NADP-DCSs suggest that Arg166 and Arg198 (numbered from the amino-terminal glutamate of the mature enzyme) may interact with Pi (9Pawelek P.D. MacKenzie R.E. Biochim. Biophys. Acta. 1996; 1296: 47-54Crossref PubMed Scopus (7) Google Scholar and Fig. 1). The crystal structure of the DC domain of the human NADP-dependent DCS has been determined both with bound NADP and with bound NADP and folate analogues (10Allaire M. Li Y. MacKenzie R.E. Cygler M. Structure (Camb.). 1998; 6: 173-182Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 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 (45) Google Scholar). The structure of the Escherichia coli NADP-dependent DC has been determined by x-ray crystallography in the absence of bound substrates (12Shen B.W. Dyer D.H. Huang J.-Y. D'ari L. Rabinowitz J. Stoddard B.L. Protein Sci. 1999; 8: 1342-1349Crossref PubMed Scopus (28) Google Scholar), and the structure of the Saccharomyces cerevisiae NAD-dependent dehydrogenase has been determined with and without bound NAD (13Monzingo A.F. Breska A. Ernst S. Appling D.R. Robertus J.D. Protein Sci. 2000; 9: PubMed Scopus Google Scholar). However, crystal structure of NMDMC has been constructed a homology model of the enzyme based on three structures and used this to the Pi and Mg2+ binding was to the of MacKenzie R.E. Arch. Biochem. Biophys. PubMed Scopus Google Scholar) and in was from was from and enzymes of and from and NAD, and from was from and from and of of the of structures for three NMDMC have been The DC domain of the human NADP-dependent DCS structure has been with NADP (10Allaire M. Li Y. MacKenzie R.E. Cygler M. Structure (Camb.). 1998; 6: 173-182Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and with NADP and three folate analogues 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 (45) Google Scholar). The E. coli NADP-dependent DC structure has been in the absence of (12Shen B.W. Dyer D.H. Huang J.-Y. D'ari L. Rabinowitz J. Stoddard B.L. Protein Sci. 1999; 8: 1342-1349Crossref PubMed Scopus (28) Google Scholar). The S. cerevisiae NAD-dependent dehydrogenase has been both without and in with NAD (13Monzingo A.F. Breska A. Ernst S. Appling D.R. Robertus J.D. Protein Sci. 2000; 9: PubMed Scopus Google Scholar). with bound cofactors and substrates to a model of human The three structures from the Protein Data the DC domain of the human NADP-dependent DCS in with NADP and a folate the E. coli NADP-dependent DC and the S. cerevisiae NAD-dependent dehydrogenase in with NAD the sequence these is not as the percent between the structures homologous Fig. 1). A sequence of the and was J.D. 22: PubMed Scopus Google Scholar). The was then to alignments to the structure a followed by of as in the Biol. 1996; PubMed Scopus Google Scholar). for the model from the by sequence to The from the and the side the M. A. PubMed Scopus Google Scholar) with the 2000; PubMed Scopus Google Scholar) The from to unique to mitochondrial NAD-dependent (9Pawelek P.D. MacKenzie R.E. Biochim. Biophys. Acta. 1996; 1296: 47-54Crossref PubMed Scopus (7) Google Scholar and Fig. and was not in the The position of Asp190 suggested that it a role in the cofactor binding it was in the and its position was determined by P.D. J. M. Biol. 1998; PubMed Scopus Google Scholar). The from to is in the structure and is not in the E. coli and S. cerevisiae This has been not to be required for activity cofactor binding S. MacKenzie R.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and was not in the The and of the model structure was P.D. J. M. Biol. 1998; PubMed Scopus Google Scholar). the residues and to position homologous to the NAD, and NADP into the model based on the position of the cofactors in the The crystal structures and show only of the enzymes and the model only of NMDMC, is known to be a N.R. Rios-Orlandi E.M. MacKenzie R.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). To model the two of the model on the structure Biol. 1996; PubMed Scopus Google Scholar), and the and of the side the was P.D. J. M. Biol. 1998; PubMed Scopus Google Scholar). The of the model was J. 1993; Google Scholar). of the model The Scholar). of a was to NMDMC by the of the required of the the sequence of the and an site. The was then into Mejia N.R. MacKenzie R.E. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) to The an of residues that have on enzyme activity. The and of to the NMDMC not and the not with the enzyme The of the protein was for this because it than NMDMC with and to that may from the to the in the results is to as wild into in as in and MacKenzie S. MacKenzie R.E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the was by to the of of the mutants in this affect the cofactor binding and only the dehydrogenase activity, the cyclohydrolase activity and folate binding as for of the protein of the mutants in this cyclohydrolase activity. between the for methylene-THF, with either NAD NADP as the for for of the This that of the protein structure in the mutants and that the folate binding site is by these Protein and NMDMC into E. coli of used to of with and with was an to by to a of was to an by for in a of to on and in of phosphate and by on of by of by in a for of was to the followed by an The was to and and to of a of in binding phosphate and The was for on a and the was by for in a The was then in of binding and into a The was with of binding followed by of binding The enzyme was with binding the enzyme identified by Biochem. PubMed Scopus Google Scholar). enzyme for by on Protein was determined by in as a and assays Yang and Mackenzie (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar) and and MacKenzie P.D. MacKenzie R.E. Biochemistry. 1998; PubMed Scopus Google Scholar). phosphate magnesium and NAD. NADP-dependent assays the was by the phosphate and NAD and activity assays as the of three in a The of was on mutants with activity a as in 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 (45) Google Scholar), and assays as in and MacKenzie P.D. MacKenzie R.E. Biochemistry. 1998; PubMed Scopus Google Scholar), with magnesium to the the of three in The of was as for the cyclohydrolase enzymes with activity, to the by The for Mg2+ with NAD as a cofactor determined in the by to the of the for and as the and of three to of activity by Pi was by assays Pi of to Yang and MacKenzie (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar). for Pi NADP from the of of the of Pi The homology model of NMDMC is in with the The of the between the model and is as structure structure coli and structure cerevisiae Fig. 1). The of the model that of residues in in and in residues in A of the homology with Pi and NAD, is in Fig. The for the model have been in the Protein Data with The of Pi NADP in dehydrogenase assays Yang and MacKenzie (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar) to that Pi to NMDMC in a position to the of the 2′-phosphate of NADP in The NAD binding site of the NMDMC model is with the NADP binding site of DCS in Fig. The Pi in the NMDMC structure is from NAD into a of Arg166 and Arg198. of the Pi of the and of the NAD Arg166 and Arg198 homologous to and of the human DCS, in NADP binding (8Pawelek P.D. Allaire M. Cygler M. MacKenzie R.E. Biochim. Biophys. Acta. 2000; 1479: 59-68Crossref PubMed Scopus (21) Google Scholar). of DCS with and the of the 2′-phosphate of NADP (10Allaire M. Li Y. MacKenzie R.E. Cygler M. Structure (Camb.). 1998; 6: 173-182Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). this is dehydrogenase activity of DCS is to than of activity, and of for NADP the of this in binding the cofactor (8Pawelek P.D. Allaire M. Cygler M. MacKenzie R.E. Biochim. Biophys. Acta. 2000; 1479: 59-68Crossref PubMed Scopus (21) Google Scholar). the homology model Arg166 appears to have the to interactions with Pi and can to the of to the role of in this was for of the human DCS with the 2′-phosphate of NADP (10Allaire M. Li Y. MacKenzie R.E. Cygler M. Structure (Camb.). 1998; 6: 173-182Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and was by to a role in cofactor binding (8Pawelek P.D. Allaire M. Cygler M. MacKenzie R.E. Biochim. Biophys. Acta. 2000; 1479: 59-68Crossref PubMed Scopus (21) Google Scholar). NMDMC Arg198 is the homologous to of the homology model of NMDMC it appears that this has the to with Pi and can to and it was for of the the side chain of Asp190, a unique to NMDMC, was Arg166 in a position that suggests an interaction of this the to the position of Arg166 in the protein and the without the of the side of of Arg166 dehydrogenase activity This is with the role of in is primarily responsible for NADP binding through the suggests that Arg166 has a role in binding Pi in activity of Arg166 activity not not in a of the of the position of Arg166 without the side chain Asp190 was The results in and of this that the interaction of Asp190 and Arg166 as and in a of the position of Arg166 in the binding site in the loss of activity. of activity with both This would have a interaction between residues and of Arg166, the position of Arg166 would not to the of the and side The of for as by its is not although of for substrates and ions These in the the of activity to the of positioning the properly in the binding site Arg166, and to The in the Mg2+ suggests that the Mg2+ and Pi binding the NAD and Pi binding interact with other. The for NADP is than that for NAD, because the 2′-phosphate is and positioning of a rather than two NADP to Arg166 in the These show that the role of Asp190 is to position Arg166 in the binding activity of Asp190 activity not not in a of to of in a of of Arg198 mutants in and the of the is with a that is not of both and an important role for this this is by is the homologous in DCS, activity is while the is The Pi as by the for is in this that Arg198 in Pi binding. The for Mg2+ and NADP not by this that the binding site of Mg2+ is not in and that the of Arg166 and Mg2+ is to for activity of activity in a of Arg198 mutants to of not determined to enzyme not determined to enzyme activity. in a The in the is with a and that has a of both NAD and NADP-dependent of the increased with wild type. The in this both the loss of for Pi by the for and the of properly positioning the Pi in the binding site to the increased of the side The Mg2+ as in by this that the two binding interact with other. this in to the side chain may be the Mg2+ site it in a that binding. The role of Arg198 is by the of on the for and have the for as by the activity, than binding the phosphate is it be properly for activity by an interaction with a Arg198 the phosphate that interacts with Arg166 and it the binding site to interact with NAD. with was used to for this as to the used (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar). This a in the binding of Mg2+ with NAD. The with NAD as a cofactor is and to the The of for was to be of binding. This is not in NADP as a cofactor is the substrate. both and for The of this is not clear. of Mg2+ binding identified by a sequence of methylene-THF to aspartate and glutamate residues that in and NMDMC not in NADP-dependent enzymes that not These residues in because the preferred of of being in known Mg2+ binding Chem. 2003; PubMed Scopus Google Scholar). This the residues to from The of these residues was in the homology model to to a Mg2+ binding site. The of a possible binding to the of the site between binding the of the and its to the the only that be of a Mg2+ binding site. three of these residues Asp133 as the only to to a Mg2+ binding site. of of the Asp133 mutants in and of aspartate by glutamate the activity with both NAD and NADP that the position of the is However, and without activity. The and mutants show in for as by the the of for Mg2+ These results support a role for Asp133 in to bind The loss of Mg2+ in these mutants the positioning of Pi in the binding site and results in for the This suggests that the role of the Mg2+ ion is to in the binding and positioning of the role of activity of Asp133 activity not not in a of Asp133 mutants to of in a The of Arg166 for model of NMDMC that residues Arg166 and Arg198 homologous to and that interact with the 2′-phosphate of NADP bound to the human NMDMC, Arg166 and Arg198 a for binding Arg166 is the that is primarily responsible for binding mutants of this the to dehydrogenase activity. The of activity of is not that that residues Pi because can interactions with the ion and can be J. Mol. Biol. Google Scholar, J. Mol. Biol. 1993; PubMed Scopus Google Scholar). of Asp190 that this is required to properly position Arg166 in the binding site. This of interaction to position the side chain has been in that bind phosphate ions Structure (Camb.). 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. PubMed Scopus Google Scholar, M. Biochemistry. PubMed Scopus Google Scholar). of Asp190 to the glutamate Arg166 and The activity. However, the interaction with Arg166 the to binding, the These show that the position of Arg166 is for cofactor binding to Arg198 in the Arg166, Arg198 can be to residues without dehydrogenase activity. The dehydrogenase and of Arg198 mutants with NAD and NADP show that this in Pi binding is not essential for the binding of A of the for Pi and dehydrogenase of and that the role of Arg198 is not only to in binding Pi to NMDMC to position Pi the binding site to the interactions that NAD to The Arg198 and Asp190 mutants both suggest that the position of the phosphate is it is not bound to the The of Arg166 and homologous to the 2′-phosphate binding residues of DCS, the hypothesis of Yang and MacKenzie (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar) that Pi to NMDMC the 2′-hydroxyl of NAD. Pi in this position can with the NAD The for NAD and NADP in the the for the NADP activity is only of that with NAD and NAD is bound to NMDMC the between and the is than the of the 2′-phosphate of bound to the Pi site of NMDMC, NADP in the binding the of the activity of the enzyme not the for The and of the residues of the Pi binding site supports the interaction of the Pi and Mg2+ binding suggested by the enzyme (3Yang X.-M. MacKenzie R.E. Biochemistry. 1993; 32: 11118-11123Crossref PubMed Scopus (37) Google Scholar). as and that the positioning of the Pi in the binding site in for Mg2+ and an interaction of the binding The has Pi without NADP binding, has on Mg2+ These results suggest that the phosphate ion of the Mg2+ binding site and suggests that the role of the Mg2+ ion is to in the binding and positioning of the role of Arg198. A Mg2+ ion with Asp133 with Pi the in Fig. The binding site of NMDMC is of Asp133 and and these the for the The of the Mg2+ Chem. 2003; PubMed Scopus Google Scholar) by the of NAD, and These interactions the Mg2+ a with the of the the of Arg166, and the of NAD. The position of Mg2+ suggests that the ion the position of Pi and NAD in the binding site through and The Pi role in NMDMC is to the role of Mg2+ in that use substrates PubMed Scopus Google Scholar). to Pi and Mg2+ in and use of Pi to bind NAD in NMDMC is to binding to glutathione Biochemistry. 1988; PubMed Scopus Google Scholar). uses to glutathione to to through interactions with two and can use as a cofactor; the of for is than the for and it can only bind to the protein in the of Pi Biochemistry. 1988; PubMed Scopus Google Scholar). The Pi in in the position as the 2′-phosphate of with the two side the use of the two is between and NMDMC, these not it is not possible to these in the the of the that interacts with the of J. 1996; PubMed Scopus Google Scholar), in NMDMC Arg166 is the of the has been by A. PubMed Scopus Google Scholar) to use that protein the to the cofactor binding to a classic binding site. The interaction of Mg2+ and Pi with a a is to the crystal structure of the with bound and Mg2+ ions S. L. J. Mol. Biol. 2002; PubMed Scopus Google Scholar). this structure three Mg2+ ions bind the with the and of the The Structure of the to of NADP the cofactor binding site of NMDMC is with a classic Rossmann NAD binding site and to the NADP binding site of DCS, the role for the ions The classic NAD site has interactions between the cofactor and the protein to cofactor binding. The sequence interacts with the and close proximity to the cofactor to the glycine in this is thought to be important for close because side chain this position would cofactor binding J. 1996; PubMed Scopus Google Scholar). have a aspartate that with the of the of NAD J. 1996; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). NADP binding on the interaction of the 2′-phosphate of NADP with an side chain PubMed Scopus Google Scholar), as is the with DCS (8Pawelek P.D. Allaire M. Cygler M. MacKenzie R.E. Biochim. Biophys. Acta. 2000; 1479: 59-68Crossref PubMed Scopus (21) Google Scholar, M. Li Y. MacKenzie R.E. Cygler M. Structure (Camb.). 1998; 6: 173-182Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The sequence is not as in and the aspartate is NMDMC and DCS the sequence of of DCS and of The of the glycine by in this the close interactions required for NAD binding NMDMC the aspartate the cofactor binding site of NMDMC an NADP binding site than a classic NAD binding site. on DCS that NADP binding to the protein is on the interaction between the 2′-phosphate and (8Pawelek P.D. Allaire M. Cygler M. MacKenzie R.E. Biochim. Biophys. Acta. 2000; 1479: 59-68Crossref PubMed Scopus (21) Google the interactions between the protein and the cofactor not for NADP binding. the of the NMDMC and DCS cofactor binding it that the role of the ions in NMDMC is to for the of a bound phosphate on the The Mg2+ and Pi ions interactions that adapt an NADP site to bind NAD. have to to use NAD A. PubMed Scopus Google Scholar, J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. A. J. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). These have used a to side the cofactor binding site to the binding site of a homologous protein for NAD. through used an to change the cofactor specificity of the mitochondrial methylene-THF a protein specificity for NAD to these A. PubMed Scopus Google Scholar, J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. A. J. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). This use of Mg2+ and Pi to bind NAD to the site of NMDMC represents a novel variation of the Rossmann fold. and for

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