X-ray Structures of Aerococcus viridans Lactate Oxidase and Its Complex with d-Lactate at pH 4.5 Show an α-Hydroxyacid Oxidation Mechanism

Furuichi, Makio; Suzuki, Nobuhiro; Dhakshnamoorhty, Balasundaresan; Minagawa, Hiroko; Yamagishi, Ryosuke; Watanabe, Yuta; Goto, Yukari; Kaneko, Hiroki; Yoshida, Yoshihito; Yagi, Hisao; Waga, Iwao; Kumar, Penmetcha K. R.; Mizuno, Hiroshi

doi:10.1016/j.jmb.2008.02.062

Cited by 39 publications

(52 citation statements)

References 35 publications

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“…The region of residues near the range 199-204 is a good candidate for participation in such a conformational rearrangement as a result of its high apparent mobility. Various residues in this segment are often missing in crystal structures and those that are present usually exhibit high temperature factors [3][4][5][6][7][27][28][29]. In the A95G mutant subunits, certain residues in this region cannot be observed in the electron density: A, 203-204; B, 199-204; C, 203-204; D, 203-204.…”

Section: Reactivity With O 2 and Alternative Electron Acceptors In Thmentioning

confidence: 99%

“…In the present study, we have focused on the LOX from Aerococcus viridans (avLOX), a prototypical oxidase-type member of the a-hydroxy acid-oxidizing flavoenzyme family that has been well characterized biochemically [9,21,25,26] and structurally [6,[27][28][29]. The enzyme is biotechnologically important for its commercialized use in analytical devices for blood L-lactate determination [30,31].…”

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confidence: 99%

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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

et al. 2014

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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.

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Section: Reactivity With O 2 and Alternative Electron Acceptors In Thmentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 2014

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“…All these residues are very well conserved in the ␣-hydroxyoxidase family members (29). It has also been suggested that the hydrogen bond network is important for the enzymatic activity (10). Changes in the lactate oxidase structure have also been reported to broaden the active site of the lactate oxidase enzyme (17).…”

Section: Discussionmentioning

confidence: 99%

“…The protein sequence of lactate oxidase in A. viridans (BAA09172), the predicted protein sequence of the lactate oxidase previously described in S. iniae (accession number Y07622), and the predicted protein sequences of the corrected type 1 lactate oxidase and the novel type 2 variant from S. iniae described in this paper were aligned in Clustal X (28). Enzymatically active sites for A. viridans as described in previous publications (10,16,17,35,36) that are conserved in S. iniae were marked in the alignment. The Protein Data Bank (PDB) file of 2DU2 (30) on the resolved crystal structure of lactate oxidase in A. viridans was downloaded from the Research Collaboratory for Structural Bioinformatics PDB (http://www.pdb.org/pdb/home/home.do) and subunits 2 and 3 were visualized using PyMOL, version 1.0 (8).…”

Section: Methodsmentioning

confidence: 99%

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Host-Directed Evolution of a Novel Lactate Oxidase in Streptococcus iniae Isolates from Barramundi ( Lates calcarifer )

Nawawi

Baiano

Kvennefors

et al. 2009

Appl Environ Microbiol

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In Streptococcus iniae, lactate metabolism is dependent upon two proteins, lactate permease that mediates uptake and lactate oxidase, a flavin mononucleotide-dependent enzyme that catalyzes oxidation of ␣-hydroxyacids. A novel variant of the lactate oxidase gene, lctO, in Australian isolates of S. iniae from diseased barramundi was found during a diagnostic screen using LOX-1 and LOX-2 primers, yielding amplicons of 920 bp instead of the expected 869 bp. Sequencing of the novel gene variant (type 2) revealed a 51-nucleotide insertion in lctO, resulting in a 17-amino-acid repeat in the gene product, and three-dimensional modeling indicated formation of an extra loop in the monomeric protein structure. The activities of the lactate oxidase enzyme variants expressed in Escherichia coli were examined, indicating that the higher-molecular-weight type 2 enzyme exhibited higher activity. Growth rates of S. iniae expressing the novel type 2 enzyme were not reduced at lactate concentrations of 0.3% and 0.5%, whereas a strain expressing the type 1 enzyme exhibited reduced growth rates at these lactate concentrations. During a retrospective screen of 105 isolates of S. iniae from Australia, the United States, Canada, Israel, Réunion Island, and Thailand, the type 2 variant arose only in isolates from a single marine farm with unusually high tidal flow in the Northern Territory, Australia. Elevated plasma lactate levels in the fish, resulting from the effort of swimming in tidal flows of up to 3 knots, may exert sufficient selective pressure to maintain the novel, high-molecular-weight enzyme variant.

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Further evidence in favour of a carbanion mechanism for glycolate oxidase

Pasquier

Lederer

2023

FEBS Open Bio

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The flavoenzyme glycolate oxidase oxidizes glycolic acid to glyoxylate and the latter, more slowly, to oxalate. It is a member of an FMN‐dependent enzyme family that oxidizes l ‐2‐hydroxy acids to keto acids. There has been a controversy concerning the chemical mechanism of substrate oxidation by these enzymes. Do they proceed by hydride transfer, as observed for NAD‐dependent enzymes, or by initial formation of a carbanion that transfers the electrons to the flavin? The present work describes a comparison of the reactivity of glycolate, lactate and trifluorolactate with recombinant human glycolate oxidase, by means of rapid‐kinetics experiments in anaerobiosis. We show that trifluorolactate is a substrate for glycolate oxidase, whereas it is known as an inhibitor for NAD‐dependent enzymes, as is trifluoroethanol for NAD‐dependent alcohol dehydrogenases. Unexpectedly, it was observed that, once reduced, a flavin transfers an electron to an oxidized flavin, so that the end species is a flavin semiquinone, whatever the substrate. This phenomenon has not previously been described for a glycolate oxidase. Altogether, considering that another member of this flavoenzyme family (flavocytochrome b 2 , a lactate dehydrogenase) has also been shown to oxidize trifluorolactate (Lederer F et al. (2016) Biochim Biophys Acta 1864, 1215–21), this work provides another important piece of evidence which is hardly compatible with a hydride transfer mechanism for this flavoenzyme family.

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X-ray Structures of Aerococcus viridans Lactate Oxidase and Its Complex with d-Lactate at pH 4.5 Show an α-Hydroxyacid Oxidation Mechanism

Cited by 39 publications

References 35 publications

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

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

Host-Directed Evolution of a Novel Lactate Oxidase in Streptococcus iniae Isolates from Barramundi ( Lates calcarifer )

Further evidence in favour of a carbanion mechanism for glycolate oxidase

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X-ray Structures of Aerococcus viridans Lactate Oxidase and Its Complex with d-Lactate at pH 4.5 Show an α-Hydroxyacid Oxidation Mechanism

Cited by 39 publications

References 35 publications

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

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

Host-Directed Evolution of a Novel Lactate Oxidase in Streptococcus iniae Isolates from Barramundi ( Lates calcarifer )

Further evidence in favour of a carbanion mechanism for glycolate oxidase

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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

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