title: Role of aspartate-37 in determining cofactor specificity and binding in rat liver dihydropteridine reductase.
abstract: Full-length rat dihydropteridine reductase (DHPR) cDNAs have been combined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express dihydropteridine reductase immunoreactive proteins and demonstrate conversion of quinonoid dihydropteridines to their tetrahydro forms. Several recombinant enzymes have been purified to homogeneity and biochemical studies have been carried out comparing their properties with those exhibited by the rat liver enzyme. The optimal reaction conditions, kinetic constants, and stability are similar for the recombinant and naturally occurring enzyme. The results indicate that the nonmutant recombinant rat DHPR is an authentic replica of the natural protein and that the characteristics of DHPR activity are determined by a single gene product and do not require specific modification via the eukaryotic cell. In addition to the wild type, three specific mutagenic forms of the reductase, A-6-V, W-104-F, and D-37-I, and an additional abbreviated structure have also been formed. Each of the products exhibits reductase activity, although they show varied affinities for their cofactor, NADH, and less stability to chromatography, dialysis, and concentration than the wild-type enzyme. The N-terminal sequence contains a classic NADH binding region between amino acids 9 and 36, and Asp 37 is essential for binding the cofactor as is shown by the approximately 20-fold increase in dissociation constant for the D-37-I mutant and diminished kcat (approximately 43 s-1 compared to 156 s-1 for the wild-type enzyme). The results indicate that the DHPR cofactor binding site is similar to typical dinucleotide requiring dehydrogenases such as lactic acid and liver alcohol dehydrogenase.
authors.affiliation: Agouron Pharmaceuticals Inc., La Jolla, California 92037.
authors.forename: D A
authors.initials: DA
authors.lastname: Matthews
authors.forename: K I
authors.initials: KI
authors.lastname: Varughese
authors.forename: M
authors.initials: M
authors.lastname: Skinner
authors.forename: N H
authors.initials: NH
authors.lastname: Xuong
authors.forename: J
authors.initials: J
authors.lastname: Hoch
authors.forename: K
authors.initials: K
authors.lastname: Trach
authors.forename: M
authors.initials: M
authors.lastname: Schneider
authors.forename: T
authors.initials: T
authors.lastname: Bray
authors.forename: J M
authors.initials: JM
authors.lastname: Whiteley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D004093:Dihydropteridine Reductase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D009243:NAD; D001224:Aspartic Acid; D004093:Dihydropteridine Reductase