The Roles of Two Amino Acid Residues in the Active Site of l-Lactate Monooxygenase

Sanders, Stephen A.; Williams, Charles H.; Massey, Vincent

doi:10.1074/jbc.274.32.22289

Cited by 13 publications

(13 citation statements)

References 31 publications

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“…Similar mutations have been carried out with lactate monooxygenase (25,26), mandelate dehydrogenase (27), and flavocytochrome b 2 (6,28). In each case, it is clear that the pairs of arginine residues are involved in substrate binding and catalysis.…”

Section: Discussionmentioning

confidence: 90%

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

Yorita

Matsuoka²,

Misaki³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

confidence: 90%

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

Yorita

Matsuoka²,

Misaki³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…In contrast to the wild-type DAAO, the anionic semiquinone species of the Arg 285 mutants is unstable. On the anaerobic addition of benzyl viologen, the semiquinone form of Arg 285 DAAO mutants slowly disproportionated to the oxidized and reduced forms with the end point containing the thermodynamically stabilized amount of semiquinone (24). After 24 h, when eventually the equilibrium was reached, ϳ20% of the photoreduced R285K and R285A remained in the semiquinone form (and ϳ0% for the R285D and R285Q, see Table I).…”

Section: Resultsmentioning

confidence: 99%

“…The thermodynamic stability of the semiquinone was determined by the addition of 5 M benzyl viologen from a side arm of the cuvette after the photoreduction was complete. Disproportionation of the semiquinone was then followed until equilibration was reached (for up to 24 h) at 15°C (24).…”

Section: Methodsmentioning

confidence: 99%

Role of Arginine 285 in the Active Site of Rhodotorula gracilis d-Amino Acid Oxidase

Molla

Porrini

Job

et al. 2000

Journal of Biological Chemistry

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(1, 2). The precise mechanism of substrate dehydrogenation of this well studied enzyme (3) has not yet been solved, even if recently two groups have reported the crystal structure of the enzyme purified from pig kidney (pkDAAO) at a resolution of 2.6 and 3.0 Å, respectively (4, 5). Over the years, three main different mechanisms have been proposed for the reaction catalyzed by this flavoenzyme (see Mattevi et al. (6) for a recent review). (i) The hypothesis that the reductive half-reaction of DAAO involves the initial formation of a carbanion by abstraction of the ␣-H of the substrate as a proton comes from the elimination of halide from ␤-chloro-D-alanine (7). (ii) The observation of a transfer of ␣-hydrogen of the substrate to the C(5) position of the enzyme reconstituted with 5-deaza-FAD provides evidence in favor of a direct hydride-transfer mechanism (8). (iii) Finally a concerted mechanism (consistent with the experimental evidence for a carbanion mechanism) in which ␣-H ϩ abstraction is coupled with the transfer of a hydride from the amino group of the substrate has been put forward (9).As a model for DAAO to have a better understanding of this crucial issue, we have used the enzyme from the red yeast Rhodotorula gracilis (RgDAAO). Actually, RgDAAO possesses peculiar properties, as the high catalytic efficiency and the tight binding with the coenzyme FAD, which distinguish it from the mammalian enzyme (10 -12). These properties are most probably related to its physiological role (yeast can metabolize D-amino acids and use them as the sole nitrogen and carbon source) and to an evolutionary drive. From comparison of the primary sequences of the known DAAOs, it is evident that only three residues, among those identified in or near the active site (13), are conserved (namely two tyrosines and one arginine). The presence of an arginine residue located at the active site and directly involved in DAAO catalysis was in fact previously proposed by various chemical modification studies (for a review, see Ref. 3 and references therein). The possibility that the arginine residue could be involved in substrate binding by electrostatic interaction with the substrate was inferred by Nishino et al. (14) from 2,3-butanedione modification of the mammalian enzyme, followed by reaction with dansylchloride. On the other hand, the reactivity with sulfite and the spectral properties of the native and cyclohexandione-modified pk-DAAO reconstituted with 8-mercapto-FAD suggested that the active site arginine could act as the positively charged group near the flavin N(1)-C(2)ϭO locus and responsible for stabilization of anionic flavin forms (15). This basic residue has been identified in the primary sequence of RgDAAO, by irreversible inhibition using phenylglyoxal (16), although the question regarding its role was not solved. More recently, the resolution of

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“…1). Subsequent modeling studies, combined with site-directed mutagenesis studies, led to the idea that the carboxylate group of the substrates bind to these same side chains, as well as probably to that of Arg289 (12,15,(44)(45)(46)(47). In HAO, these residues are Phe23, Arg164, and Arg250.…”

Section: Ph Profiles Of the Hao Kinetic Parameters For Mandelate And mentioning

confidence: 96%

“…The positioning of lactate is adapted from that of pyruvate observed in the crystal structure (15). Arg289 is not pictured here; it lies close to Arg376, above the plane of the figure, and in the unliganded enzyme its guanido group is turned away from the active site and forms an ion pair with invariant Asp292 (7); crystallographic evidence, confirmed by site-directed mutagenesis, indicates it can move toward the substrate and interact with it (8,(44)(45)(46)(47)53). The numbering of equivalent residues is for Flb2: Tyr143 Tyr254 Asp282 Arg289 Asp292 Lys349 His373 Arg376; for HAO: Phe23 Tyr129 Asp157 Arg164 Asp167 Lys223 His247 Arg250; for GOX: Tyr24 Tyr129 Asp157 Arg164 Asp167 Lys230 His254 Arg257; for MDH: Tyr26 Tyr131 Asp158 Arg165 Asp168 Lys250 His274 Arg277.…”

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confidence: 95%

Hydroxamates as Substrates and Inhibitors for FMN-Dependent 2-Hydroxy Acid Dehydrogenases

Amar

North

Miškinienė

et al. 2002

Bioorganic Chemistry

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The Roles of Two Amino Acid Residues in the Active Site of l-Lactate Monooxygenase

Cited by 13 publications

References 31 publications

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

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

Role of Arginine 285 in the Active Site of Rhodotorula gracilis d-Amino Acid Oxidase

Hydroxamates as Substrates and Inhibitors for FMN-Dependent 2-Hydroxy Acid Dehydrogenases

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