Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN

Abstract

T he enzyme L-lactate oxidase from Aerococcus viridans (LOX) is a member of the family of flavoproteins that catalyze the oxidation of ␣-hydroxyacids (1). The kinetic characteristics of the enzyme have been reported by our group, but still the mechanism of the reductive half-reaction by ␣-hydroxyacids is one of central interest. Our previous study of the reactivity of LOX with a series of para-substituted mandelates indicated little development of charge in the transition state of the reductive half reaction (2). This result is compatible with either a hydride mechanism or with a synchronous mechanism in which the ␣-CH-of the substrate is formally abstracted as a proton, whereas the resulting charge is simultaneously neutralized by another event. We proposed the participation of two arginine residues, Arg-181 and Arg-268, which are located around the FMN and which could be part of the substrate binding site, based on the x-ray crystal structures of glycolate oxidase (3, 4) and flavocytochrome b 2 (5, 6). These two arginine residues are conserved in all family members, as shown in Fig. 1. It should be noted that there is a 20-to 30-aa residue insertion in L-lactate monooxygenase and L-mandelate dehydrogenase between these two arginine residues.In this paper, we have replaced these two residues by either lysine as a relatively mild perturbation or methionine as removing positive charge (R268K, R181K, R181M, and R181K ϩ R268K) and studied the characteristics of the resulting enzymes to try to elucidate the functions of these arginine residues. We found that both residues are involved in the reductive half reaction and in the interaction of substrates and ligands with the FMN prosthetic group. Materials and MethodsSite-Directed Mutagenesis. Amino acid substitutions R181K, R268K, and their double substitution were carried out by a site-directed mutagenesis of the lactate oxidase gene in the expression vector, pAOX8, which is composed of a 1.2-kb insert containing the coding region for lactate oxidase in pACYC184. Two fragments were generated by PCR: one contained the sequence from the HindIII site of the pACYC184 plasmid to the site of mutagenesis and the other the sequence from the site of mutagenesis to the BamHI site of the expression plasmid. These two fragments were annealed at the site of mutagenesis and extended enzymatically to yield the coding region with the expected mutation. The resulting sequence was amplified by PCR using the HindIII and BamHI site primers. After treatment with HindIII and BamHI, the DNA product was ligated into these restriction sites in pACYC184 to yield the expression plasmid, pAOX8, with the appropriate mutation. The double mutant was generated by using the primers for both mutations together with the primers that generated the HindIII and BamHI sites, resulting in three DNA fragments, which were annealed, extended, and ligated into the expression vector. The mutation of R181M was carried out by the method of Kunkel et al. (7).Flavin Extraction from the Enzymes. Buffer salts in sol...

Expression inEscherichia coliof an Unnamed Protein Gene fromAspergillus oryzaeRIB40 and Cofactor Analyses of the Gene Product as Formate Oxidase

“…This spectrum of the reduced extract is similar to those of 8-formyltetraacetyl riboflavin reduced with dithionite and photochemically reduced 8-formyl FMN. 11,13) In the cases of 8-formyltetraacetyl riboflavin and 8-formyl-FAD, absorbance maxima at 390 and 520 nm and at 320, 392, 520 nm are observed, respectively. This is to be ascribed to their semiquinone forms.…”

Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

“…Oxidation of position 8α was previously observed in a flavoenzyme. 40 In the R268K mutant of lactate oxidase from Aerococcus viridans, FMN was almost completely transformed into 8-formyl-FMN upon aging. However, the positional identification relied on the spectral properties being similar to those of the synthesized analogue 8-formyl tetraacetylriboflavin.…”

Formate Oxidase, an Enzyme of the Glucose-Methanol-Choline Oxidoreductase Family, Has a His-Arg Pair and 8-Formyl-FAD at the Catalytic Site

“…6,16,17) The molecular mass (MM) of the 8-replaced FAD was estimated to be 799 by LC-MS analysis. The spectra, spectral changes, and MM suggest the possibility that the wild and recombinant FODs have 8-formyl-FAD as a cofactor.…”