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.

Lactococcus lactis Dihydroorotate Dehydrogenase A Mutants Reveal Important Facets of the Enzymatic Function

“…Mutations in the pyrDa gene were introduced by the polymerase chain reaction (PCR) as described by Björnberg et al (7) using the plasmid pFN1 as template and mutant primers. The PCR products were digested with BamHI and HindIII and cloned into BamHI-and HindIIIdigested pUHE23-2 to generate plasmids similar to pFN1, but with a mutation in the pyrD gene.…”

“…When consider- ing these data, it should be kept in mind that the first halfreaction for the wild-type enzyme, i.e. the reduction of the flavin by DHO, is much faster (Ͼ50-fold) than the second half-reaction, which is the reoxidation of the flavin by the electron acceptor (7). This means that changes of the amino acid sequence that affect the first half-reaction may not necessarily impair the steady state activity of a mutant enzyme.…”

“…Based on presently known sequences, DHODs can be divided into two main classes (7). Class 1 enzymes, which are subdivided into class 1A and 1B enzymes, are cytosolic proteins, whereas class 2 enzymes are membrane-associated.…”

“…Based on structural comparisons of class 1 and class 2 enzymes we have suggested that the mechanism for the first half-reaction is highly conserved, and two highly conserved lysines are essential for the catalytic function (17). In the class 1 enzymes the active site base is a cysteine residue (7) while in class 2 enzymes it is a serine (14).…”

“…In addition to the two loop regions, the alignment of DHOD sequences and structures revealed a number of other residues conserved between the DHODs. Two of these (Asn-132 and Lys-43 in DHODA) are important for catalysis (7). Their side chains are hydrogenbonded to the product orotate, and in addition Lys-43 interacts with O4 of FMN (9).…”