Abstract
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-Å 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.
Original language | English (US) |
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Pages (from-to) | 562-578 |
Number of pages | 17 |
Journal | FEBS Journal |
Volume | 282 |
Issue number | 3 |
DOIs | |
State | Published - 2015 |
Keywords
- electron acceptor
- flavin oxygen reactivity
- FMN
- L-lactate oxidase
- α-hydroxy acid oxidizing flavoenzymes
ASJC Scopus subject areas
- Biochemistry
- Cell Biology
- Molecular Biology