title: Active site of dihydroorotate dehydrogenase A from Lactococcus lactis investigated by chemical modification and mutagenesis.
abstract: The flavin-containing enzyme dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate (DHO) to orotate, the first aromatic intermediate in pyrimidine biosynthesis. The first structure of a DHOD, the A form of the enzyme from Lactococcus lactis, has recently become known, and some conserved residues were suggested to have a role in the active site [Rowland et al. (1997) Structure 2, 239-252]. In particular, Cys 130 was hypothesized to work as a base, which activates dihydroorotate (DHO) for hydride transfer. By chemical modification and site-directed mutagenesis we have obtained results consistent with this proposal. Cys 130 was susceptible to alkylating reagents, and mutants of Cys 130 (C130A and C130S) showed hardly detectable enzyme activity at pH 8.0, while at pH 10 the C130S mutant enzyme had approximately 1% of wild-type activity. Mutants of Lys 43, Asn 132, and Lys 164 were also constructed. Exchange of Lys 43 to Ala or Glu (K43A and K43E) and of Asn 132 to Ala (N132A) affected both catalysis and substrate binding. Expressed as kcat/KM for DHO, the deterioration of these three mutant enzymes was 10(3)-10(4)-fold. Flavin spectra of the mutant enzymes were not, like the wild-type enzyme, bleached by DHO in stopped-flow experiments, showing that they were deficient with respect to the first half-reaction, namely reduction of FMN by DHO, which was not rate limiting for the wild-type enzyme. The binding interaction between flavin and the reaction product, orotate, could be monitored by a red shift of the flavin absorbance in the wild-type enzyme. The C130A, C130S, and N132A mutant enzymes displayed similar capacity to bind orotate. In contrast, orotate did not change the absorption spectra of the K43 mutant enzymes, although it did inhibit their activity. All of the mutant enzymes, except K164A, contained normal levels of flavin. The results are discussed in relation to the structures of DHODA and other flavoenzymes. The possible acid-base chemistry of Cys 130 is compared to previous work on mammalian dihydropyrimidine dehydrogenases, flavoenzymes, which catalyze the reversed reaction, namely the reduction of pyrimidine bases.
authors.affiliation: Center for Enzyme Research, Institute of Molecular Biology, and Centre for Crystallograpic Studies, Department of Chemistry, University of Copenhagen, Copenhagen, Denmark.
authors.forename: O
authors.initials: O
authors.lastname: Björnberg
authors.forename: P
authors.initials: P
authors.lastname: Rowland
authors.forename: S
authors.initials: S
authors.lastname: Larsen
authors.forename: K F
authors.initials: KF
authors.lastname: Jensen
mesh_terms: D000477:Alkylating Agents / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D000090722:Dihydroorotate Dehydrogenase; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D019143:Evolution, Molecular; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013053:Spectrophotometry
chemical_list: D000477:Alkylating Agents; D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors; D005420:Flavoproteins; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; D004228:Dithionitrobenzoic Acid; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D003545:Cysteine