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Publication Abstract from PubMed

Aerococcus viridans L-lactate oxidase (avLOX) is a biotechnologically important flavoenzyme that catalyzes conversion of L-lactate and O2 into pyruvate and H2 O2. The enzymatic reaction underlies different biosensor applications of avLOX for blood L-lactate determination. The ability of avLOX to replace O2 with other electron acceptors such as 2,6-dichlorophenol-indophenol (DCIP) provides possiblities for analytical and practical uses. The A95G variant of avLOX was previously shown to exhibit lowered reactivity with O2 as compared to wild-type enzyme, and it was used here for detailed evaluation of effects on the specificity for different electron acceptor substrates. From stopped-flow experiments performed at 20 degrees C and pH 6.5, we determined that A95G variant (fully reduced by L-lactate) was ~3-fold more reactive towards DCIP (1.0 +/- 0.1 x 106 M-1 s-1 ) than O2 while avLOX wild type under the same conditions was 14-fold more reactive towards O2 (1.8 +/- 0.1 x 106 M-1 s-1 ) than DCIP. Substituted 1,4-benzoquinones were up to 5-fold better electron acceptors for reaction with L-lactate-reduced A95G variant than wild type. A 1.65 A crystal structure of oxidized A95G variant bound with pyruvate was determined and reveals that steric volume created by removal of the methyl side chain of Ala95 and a slight additional shift in the main chain at position Gly95 enable accomodation of a new active-site water molecule within hydrogen bond distance to the N5 of the FMN cofactor. The increased steric volume available in the active site allows the A95G variant to exhibit a similar trend with the the related glycolate oxidase in electron acceptor substrate specificities despite the fact that the latter contains an alanine at the analogous position. This article is protected by copyright. All rights reserved.

The Ala -to-Gly substitution in Aerococcus viridans L-lactate oxidase revisited: structural consequences at the catalytic site and effect on reactivity with O and other electron acceptors.,Stoisser T, Rainer D, Leitgeb S, Wilson DK, Nidetzky B FEBS J. 2014 Nov 25. doi: 10.1111/febs.13162. PMID:25423902^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.