The crystal structure of l-lactate oxidase from Aerococcus viridans at 2.1 Å resolution reveals the mechanism of strict substrate recognition

Umena, Yasufumi; Yorita, Kazuko; Matsuoka, Takeshi; Kita, Akiko; Fukui, Kiyoshi; Morimoto, Yukio

doi:10.1016/j.bbrc.2006.09.025

Cited by 51 publications

(63 citation statements)

References 16 publications

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“…L-lactate, glycolate, L-mandelate) via C-H hydrogen abstraction [8][9][10][11][12][13][14], probably via hydride transfer to the oxidized form of FMN [15,16]. The residues involved in FMN binding and forming the active site are highly conserved [3][4][5][6][7], suggesting a common catalytic mechanism of substrate oxidation in the first (reductive) half-reaction of the overall conversion ( Fig. 1, Scheme 1).…”

Section: Introductionmentioning

confidence: 99%

“…Small differences in microenvironment of the reactive C4a-N5 locus of FMN might be responsible [17,18], in part at least, for these starkly varied O 2 reactivities. Enzyme structures [3][4][5][6][7] reveal that strand b1 of the b barrel provides close contact contact with the re-face, the side of the flavin ring opposite to where the substrate binds (Fig. 1).…”

mentioning

confidence: 99%

“…1). A naturally occurring Ala/Gly residue variation within strand b1 is a prominent factor of structural variability around the FMN N5 atom [3][4][5][6][7]. Evidence from sitedirected mutagenesis reveals that Ala/Gly residue exchange in different enzymes impacts significantly upon the O 2 reactivity, in addition to having a major effect on the a-hydroxy acid substrate specificity [19][20][21][22][23].…”

mentioning

confidence: 99%

“…They share a common (b/a) 8 TIM barrel fold, with the FMN binding site located at the C-terminal end of the b barrel [3][4][5][6][7]. Enzymes are functional oligomers, typically homotetramers.…”

Section: Introductionmentioning

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: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

Section: Introductionmentioning

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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“…The X-ray structure of the enzyme indicates that the C-terminal is in close vicinity to the entrance of the active site and thus an extended C-terminus (His-tag) may be detrimental [23].…”

Section: Spectrophotometry Studymentioning

confidence: 99%

Comparative study of three lactate oxidases from Aerococcus viridans for biosensing applications

Taurino

Reiss

Richter

et al. 2013

Electrochimica Acta

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Keywords:l-Lactate oxidases from Aerococcus viridans Engineered and commercial enzymes Multi-walled carbon nanotubes Enzyme biosensor a b s t r a c t A comparison between engineered and commercially available l-lactate oxidases from Aerococcus viridans was conducted for biosensing applications. Enzymes were adsorbed onto the surfaces of graphite electrodes modified with multi-walled carbon nanotubes. Thermostable l-lactate oxidases were cloned with a (i) N-, (ii) a C-terminal His-tag and (iii) a wild-type enzyme. Subsequently to the heterologous expression in Escherichia coli and purification, we determined the kinetic parameters of these enzymes in solution. The kinetics of the wild-type, of the N-terminally His-tagged enzyme and of the commercial l-lactate oxidase from A. viridans were studied with a classical Michaelis-Menten as well as with a substrate inhibition model, while the enzyme carrying a C-terminal His-tag showed no activity. The active enzymes were used to fabricate and comparatively investigate multi-walled carbon nanotubes-based biosensors. The enzyme kinetic results were compared with electrochemical studies. By using both spectrophotometric and amperometric techniques, the inhibition phenomenon fits better to the data especially those data related with Lox-His-N. The electrochemical data of the fabricated enzymatic biosensors showed that the N-terminally His-tagged l-lactate oxidase performed best on carboxyl-modified carbon nanotubes. The sensor based on this engineered enzyme showed the highest sensitivity and lowest detection limit in the range of l-lactate concentration 0-1 mM as well as long term stability over one month.

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Dual Activities of Oxidation and Oxidative Decarboxylation by Flavoenzymes

et al. 2022

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Specific flavoenzyme oxidases catalyze oxidative decarboxylation in addition to their classical oxidation reactions in the same active sites. The mechanisms underlying oxidative decarboxylation by these enzymes and how they control their two activities are not clearly known. This article reviews the current state of knowledge of four enzymes from the l‐amino acid oxidase and l‐hydroxy acid oxidase families, including l‐tryptophan 2‐monooxygenase, l‐phenylalanine 2‐oxidase and l‐lysine oxidase/monooxygenase and lactate monooxygenase which catalyze substrate oxidation and oxidative decarboxylation. Apart from specific interactions to allow substrate oxidation by the flavin cofactor, specific binding of oxidized product in the active sites appears to be important for enabling subsequent decarboxylation by these enzymes. Based on recent findings of l‐lysine oxidase/monooxygenase, we propose that nucleophilic attack of H2O2 on the imino acid product is the mechanism enabling oxidative decarboxylation.

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The crystal structure of l-lactate oxidase from Aerococcus viridans at 2.1 Å resolution reveals the mechanism of strict substrate recognition

Cited by 51 publications

References 16 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

Comparative study of three lactate oxidases from Aerococcus viridans for biosensing applications

Dual Activities of Oxidation and Oxidative Decarboxylation by Flavoenzymes

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The crystal structure of l-lactate oxidase from Aerococcus viridans at 2.1 Å resolution reveals the mechanism of strict substrate recognition

Cited by 51 publications

References 16 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

Comparative study of three lactate oxidases from Aerococcus viridans for biosensing applications

Dual Activities of Oxidation and Oxidative Decarboxylation by Flavoenzymes

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Product

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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