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

Abstract

Aerococcus viridans L-lactate oxidase (avLOX) is a biotechnologically important flavoenzyme that catalyzes the conversion of L-lactate and O 2 into pyruvate and H 2 O 2. The enzymatic reaction underlies different biosensor applications of avLOX for blood L-lactate determination. The ability of avLOX to replace O 2 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 O 2 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 AE 0.1 9 10 6 M À1 Ás À1 ) than O 2 , whereas avLOX wildtype under the same conditions was 14-fold more reactive towards O 2 (1.8 AE 0.1 9 10 6 M À1 Ás À1 ) 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.Abbreviations avLOX, Aerococcus viridans L-lactate oxidase; DCIP, 2,6-dichlorophenol-indophenol; GOX, glycolate oxidase; LOX, L-lacate oxidase; PDB, Protein Data Bank.

Speeding up the product release: a second‐sphere contribution from Tyr191 to the reactivity of l‐lactate oxidase revealed in crystallographic and kinetic studies of site‐directed variants

“…, the dependence of k obs on the l ‐lactate concentration passed through the origin. Therefore, this indicates that the rate constant k 4 (enzyme oxidation by pyruvate) was not significantly different from zero in each reaction , in agreement with previous studies of the wild‐type enzyme . For the mechanism in Scheme when k 4 = 0s −1 , the amplitude of the stopped‐flow traces is expected to correspond to a completely reduced enzyme, independent of l ‐lactate concentration .…”

“…The kinetic evidence from this study is consistent with a reaction pathway for the mutated lactate oxidases that is similar to that of the wild‐type enzyme . In this pathway, as shown in Scheme , the enzyme–lactate complex is converted to a reduced enzyme–pyruvate complex from which product dissociates (steps k 1 to k 6 ).…”

“…of 0.21 Å. As previously described for A95G variant , additional amino acid differences from the wild‐type enzyme are present (T102A, S163G, G232A and R255A) but all are on the surface and are distant from the active site. The Y191F mutation is clearly supported by the unbiased electron density (Fig.…”

Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidase

“…An extinction coefficient of protein-bound FMN of 12500 M −1 cm −1 was determined using a reported protocol12. Enzyme turnover numbers ( k cat ) are determined using the molar concentration of protein-bound FMN.…”

“…Despite high similarity in their active-site architectures, other α-hydroxy-acid oxidases catalyze distinctly different chemical transformations (e.g. oxidase and dehydrogenase types of oxidation; oxidative decarboxylation)12312132728. Determinants of reaction selectivity are therefore likely to be the non-conserved residues in the active site such as Tyr 215 .…”