The Ala95‐to‐Gly substitution in Aerococcus viridans l‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors

Aerococcus viridansl-lactate oxidase (avLOX) is a biotechnologically important flavoenzyme that catalyzes the conversion of l-lactate and O2 into pyruvate and H2O2. 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) allows the possiblity of analytical and practical applications. The A95G variant of avLOX was previously shown to exhibit lowered reactivity with O2 compared to wild-type enzyme and therefore was employed in a detailed investigation with respect to the specificity for different electron acceptor substrates. From stopped-flow experiments performed at 20 °C (pH 6.5), we determined that the A95G variant (fully reduced by l-lactate) was approximately three-fold more reactive towards DCIP (1.0 ± 0.1 × 106 M−1·s−1) than O2, whereas avLOX wild-type under the same conditions was 14-fold more reactive towards O2 (1.8 ± 0.1 × 106 m−1·s−1) than DCIP. Substituted 1,4-benzoquinones were up to five-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 revealed that the steric volume created by removal of the methyl side chain of Ala95 and a slight additional shift in the main chain at position Gly95 together enable the 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 related glycolate oxidase in electron acceptor substrate specificities, despite the latter containing an alanine at the analogous position. Database Atomic coordinates and related experimental data concerning the structure of the A95G variant of A. viridans lacate oxidase have been deposited in the Protein Data Bank under the identifier 4RJE.