title: 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.
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 × 10(6) 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 × 10(6) 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.
authors.affiliation: Research Center Pharmaceutical Engineering, Graz, Austria; Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stoisser
authors.forename: Daniela
authors.initials: D
authors.lastname: Rainer
authors.forename: Stefan
authors.initials: S
authors.lastname: Leitgeb
authors.forename: David K
authors.initials: DK
authors.lastname: Wilson
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D056570:Aerococcus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase; D010100:Oxygen
keywords: FMN; electron acceptor; flavin oxygen reactivity; l-lactate oxidase; α-hydroxy acid oxidizing flavoenzymes