The chemical mechanism of flavoprotein-catalysed α-hydroxy acid dehydrogenation: A mutational analysis

Lederer, Florence; Belmouden, Ahmed; Gondry, Muriel

doi:10.1042/bst0240077

Cited by 19 publications

(26 citation statements)

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“…Six amino acids are conserved within the family, except for long-chain a-hydroxy-acid oxidase where the residue corresponding to Tyr 24 in GOX is replaced with a phenylalanine (LE . Mutational analysis of GOX and several of the related enzymes has proposed a role for many of the conserved residues (for a review, see Lederer et al ., 1996). The active site of the GOX holoenzyme contains three water molecules, which, combined with the narrow substrate channel and pocket, have been used as a starting point for modeling the binding of substrate (Lindqvist & Brhdkn, 1989).…”

Section: Discussionmentioning

confidence: 99%

Three‐dimensional structures of glycolate oxidase with bound active‐site inhibitors

Stenberg

Lindqvist

1997

Protein Science

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A key step in plant photorespiration, the oxidation of glycolate to glyoxylate, is canied out by the peroxisomal flavoprotein glycolate oxidase (EC 1.1.3.15). The three-dimensional structure of this a@ barrel protein has been refined to 2 8, resolution (Lindqvist Y. 1989. J Mol Biol209151-166). FMN dependent glycolate oxidase is a member of the family of a-hydroxy acid oxidases. Here we describe the crystallization and structure determination of two inhibitor complexes of the enzyme, TKP (3-Decyl-2,5-dioxo-4-hydroxy-3-pyrroline) and TACA (4-Carboxy-5-( 1-pentyl)hexylsulfanyl-1,2,3-triazole). The structure of the TACA complex has been refined to 2.6 8, resolution and the TKP complex, solved with molecular replacement, to 2.2 8, resolution. The Rf,, for the TACA and TKP complexes are 24.2 and 25.1%, respectively. The overall structures are very similar to the unliganded holoenzyme, but a closer examination of the active site reveals differences in the positioning of the flavin isoalloxazine ring and a displaced flexible loop in the TKF' complex. The two inhibitors differ in binding mode and hydrophobic interactions, and these differences are reflected by the very different K, values for the inhibitors, 16 nM for TACA and 4.8 p M for TKP. Implications of the structures of these enzyme-inhibitor complexes for the model for substrate binding and catalysis proposed from the holo-enzyme structure are discussed.Keywords: drug design; flavin enzyme; glycolate oxidase; inhibitor binding; molecular replacement; protein crystallography Glycolate oxidase (EC 1.1.3.15, GOX) is a FMN-dependent a-hydroxy acid-oxidizing protein. In plants, the enzyme is located in the peroxisomes and performs a key step in photorespiration, the oxidation of glycolate to glyoxylate. In animals, the enzyme is located in the liver peroxisomes and is involved in oxalate production. The reaction catalyzed by the enzyme can be divided into two half-reactions. In the first half-reaction glycolate is oxidized by the flavin, and in the second part of the reaction, FMN is reoxidized by oxygen and hydrogen peroxide is formed (Macheroux et al., 1991).The three-dimensional structure for GOX from spinach with bound FMN has been determined to 2 A resolution (Lindqvist, 1989). The enzyme belongs to the class of eight stranded a@-barrels, the most common structural motif found so far, with over 45 known examples. The known a/P-barrel folds do not share any recognizable sequence identity fingerprints, but do show extensive structural similarity.Reprint requests to: Ylva Lindqvist, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Doktorsringen 4, S17177 Stockholm, Sweden; e-mail: ylva@alfa.mbh.ki.se. However, a significant proportion of the residues in GOX are highly conserved in several other FMN-dependent a-hydroxy acidoxidizing enzymes. These proteins include (sequence identity % to GOX in brackets, values from L i ? & Lederer, 1991) flavocytochrome bZ (37.2) from Saccharomyces cerevisiae and Hansenula anomala (41.1), lactate oxi...

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Section: Discussionmentioning

confidence: 99%

Three‐dimensional structures of glycolate oxidase with bound active‐site inhibitors

Stenberg

Lindqvist

1997

Protein Science

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“…They catalyze the oxidation of their a-hydroxy acid substrates (e.g. 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.…”

Section: Introductionmentioning

confidence: 99%

“…) [8] and another group of dehydrogenases barely reactive with O 2 and including mandelate dehydrogenase [14] and flavocytochrome b 2 [11,12]. 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.…”

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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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“…Thus, the present results probably have mechanistic implications for the whole family. Although the carbanion mechanism hypothesis rests on abundant evidence (for reviews, see Ghisla & Massey, 1991;Lederer, 1991bLederer et al, 1996), this mechanism is not generally considered as rigorously proven. It has been in particular pointed out that a somewhat different substrate conformation in the modeled flavocytochrome b2-lactate Michaelis complex might lead to a hydride transfer mechanism (Dubois et al, 1990;Lederer, 1991a).…”

Section: H373 K349mentioning

confidence: 99%

About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

Rao

Lederer

1998

Protein Science

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Flavocytochrome b2 or L-lactate dehydrogenase from yeasts catalyzes the oxidation of L-lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L-lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate a-hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on NE of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2-'H] lactate and the keto acid reverse substrate concentration enables the determination of the first-order rate constant, k:, for exchange of the substrate-derived protein-bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J BioZ Chem 260: 1 1 1 15-1 1 122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second-order rate constant for exchange is then 0.16 (kO.03) M" s". Using the Eigen equation, this figure yields a pK, of 9.1 f 0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, JBiochem 67:291-295). The mechanistic significance of these results is discussed.

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The chemical mechanism of flavoprotein-catalysed α-hydroxy acid dehydrogenation: A mutational analysis

Abstract: We are indebted to Drs M. Tegoni and C. Cambillau for communication of a manuscript before publication.

Cited by 19 publications

References 15 publications

Three‐dimensional structures of glycolate oxidase with bound active‐site inhibitors

Three‐dimensional structures of glycolate oxidase with bound active‐site inhibitors

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

About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

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The chemical mechanism of flavoprotein-catalysed α-hydroxy acid dehydrogenation: A mutational analysis

Abstract: We are indebted to Drs M. Tegoni and C. Cambillau for communication of a manuscript before publication.

Cited by 19 publications

References 15 publications

Three‐dimensional structures of glycolate oxidase with bound active‐site inhibitors

Three‐dimensional structures of glycolate oxidase with bound active‐site inhibitors

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

About the pKa of the active‐site histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)

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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

About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)