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) |
|---|---|
| Pages (from-to) | 562-578 |
| Number of pages | 17 |
| Journal | FEBS Journal |
| Volume | 282 |
| Issue number | 3 |
| DOIs | |
| State | Published - 2015 |
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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 journal › Article
}
TY - JOUR
T1 - 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
AU - Stoisser, Thomas
AU - Rainer, Daniela
AU - Leitgeb, Stefan
AU - Wilson, David K.
AU - Nidetzky, Bernd
PY - 2015
Y1 - 2015
N2 - 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.
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.
KW - electron acceptor
KW - flavin oxygen reactivity
KW - FMN
KW - L-lactate oxidase
KW - α-hydroxy acid oxidizing flavoenzymes
UR - http://www.scopus.com/inward/record.url?scp=84921800843&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84921800843&partnerID=8YFLogxK
U2 - 10.1111/febs.13162
DO - 10.1111/febs.13162
M3 - Article
C2 - 25423902
AN - SCOPUS:84921800843
VL - 282
SP - 562
EP - 578
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 3
ER -