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

Thomas Stoisser, Daniela Rainer, Stefan Leitgeb, David K. Wilson, Bernd Nidetzky

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)562-578
Number of pages17
JournalFEBS Journal
Volume282
Issue number3
DOIs
StatePublished - 2015

Fingerprint

lactate 2-monooxygenase
Aerococcus
2,6-Dichloroindophenol
Catalytic Domain
Substitution reactions
Electrons
Lactic Acid
Pyruvic Acid
Benzoquinones
Flavin Mononucleotide
Biosensing Techniques
Substrates
Substrate Specificity
Biosensors
Alanine
Hydrogen
Hydrogen bonds
Blood
Crystal structure

Keywords

  • electron acceptor
  • flavin oxygen reactivity
  • FMN
  • L-lactate oxidase
  • α-hydroxy acid oxidizing flavoenzymes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

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. / Stoisser, Thomas; Rainer, Daniela; Leitgeb, Stefan; Wilson, David K.; Nidetzky, Bernd.

In: FEBS Journal, Vol. 282, No. 3, 2015, p. 562-578.

Research output: Contribution to journalArticle

Stoisser, Thomas ; Rainer, Daniela ; Leitgeb, Stefan ; Wilson, David K. ; Nidetzky, Bernd. / 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. In: FEBS Journal. 2015 ; Vol. 282, No. 3. pp. 562-578.
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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-{\AA} 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.",
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AU - Leitgeb, Stefan

AU - Wilson, David K.

AU - Nidetzky, Bernd

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AB - 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.

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