Trifluorosubstrates as mechanistic probes for an FMN-dependent l-2-hydroxy acid-oxidizing enzyme

Lederer, Florence; Vignaud, Caroline; North, Paul; Bodevin, Sabrina

doi:10.1016/j.bbapap.2016.05.001

Cited by 3 publications

(9 citation statements)

References 94 publications

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“…In view of this facilitating effect of the fluorine atoms in a carbanion mechanism, a faster rate and a higher catalytic efficiency for F3Lac compared to lactate could have been expected (Table 1 ). In our previous work with Fcb2 for the same lactate/F3Lac comparison [ 49 ], the difference between the two substrates was much larger (about four orders of magnitude for the rate). But in that case, the F3Lac catalytic efficiency was still higher than that of mandelate, a very poor Fcb2 substrate [ 43 , 49 ].…”

Section: Discussionmentioning

confidence: 99%

“…In our previous work with Fcb2 for the same lactate/F3Lac comparison [ 49 ], the difference between the two substrates was much larger (about four orders of magnitude for the rate). But in that case, the F3Lac catalytic efficiency was still higher than that of mandelate, a very poor Fcb2 substrate [ 43 , 49 ]. The F3Lac low rate and efficiency can possibly arise from several factors: a steric problem due to the replacement of the methyl group by the trifluoromethyl group (lactate itself is a poor substrate compared to glycolate, Table 1 ), a possible distortion of substrate binding or transition state geometry due to potential halogen bonds and, importantly, a higher redox potential of the F3Lac/F3Pyr couple compared to the lactate/pyruvate couple.…”

Section: Discussionmentioning

confidence: 99%

“…In some cases, experimental results led to ambiguous conclusions. But in other cases, pieces of evidence were provided that are hardly compatible or even incompatible with a hydride transfer mechanism [ 43 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ].…”

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confidence: 99%

“…Indeed, F3Lac has been shown not to be a substrate but an inhibitor of NAD‐dependent lactate dehydrogenases from four different species [ 58 , 59 , 60 , 61 ]. We recently showed that F3Lac is a substrate for Fcb2 [ 49 ]. Here, we show that F3Lac is also a substrate for glycolate oxidase, another piece of evidence pointing to a carbanion mechanism.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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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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“…For CA, the abstraction of a relatively acidic -proton by an active-site base results in a dianionic/enolate intermediate, and electron transfer to this intermediate or covalent association with FMN ox takes place. While the majority of relevant biochemical and kinetic studies support HT, unequivocal structural evidence remains lacking (Cao et al, 2014;Dijkman et al, 2013;Gillet et al, 2016;Lederer et al, 2016;Walsh & Wencewicz, 2013).…”

Section: Hmomentioning

confidence: 99%

Biochemical and structural explorations of α-hydroxyacid oxidases reveal a four-electron oxidative decarboxylation reaction

Yeh

Lin

Lyu

et al. 2019

Acta Cryst Sect D Struct Biol

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p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallography, were exploited to reach these conclusions and provide additional insights.

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