4yl2

Aerococcus viridans L-lactate oxidase Y191F mutant

spinqualitylabelsall models 4yl2, resolution 1.90Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 4yl2 is a 4 chain structure with sequence from Aerococcus viridans. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.9Å Ligands: FMN, PYR Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function LOX_AERVM Catalyzes the oxidation of (S)-lactate (L-lactate) to pyruvate, with a reduction of O2 to H2O2 (Ref.1, PubMed:27302031, PubMed:25423902, PubMed:2818595, PubMed:8589073, PubMed:26260739). Cannot oxidize D-lactate, glycolate, and D,L-2-hydroxybutanoate (PubMed:2818595). May be involved in the utilization of L-lactate as an energy source for growth (By similarity).[UniProtKB:O33655] [UniProtKB:O33655] Publication Abstract from PubMed Among alpha-hydroxy acid-oxidizing flavoenzymes L-lactate oxidase (LOX) is unique in featuring a second-sphere tyrosine (Tyr191 in Aerococcus viridans LOX; avLOX) at the binding site for the substrate's carboxylate group. Y191F, Y191L and Y191A variants of avLOX were constructed to affect a hydrogen bond network connecting Tyr191 to the carboxylate of the bound ligand via the conserved Tyr40 and to examine consequent effects on enzymatic reactivity. Kinetic studies at 20 degrees C and pH 6.5 revealed that release of pyruvate product was decreased 4.7-fold (Y191F), 19-fold (Y191L) and 28-fold (Y191A) compared to wild-type enzyme (~141 s-1 ) and thus became mainly rate limiting for L-lactate oxidation by the variants at steady state under air-saturated conditions. In the Y191L and the Y191A but not in the Y191F variant, L-lactate binding was also affected strongly by the site-directed substitution. Reduction of the flavin cofactor by L-lactate and its reoxidation by molecular oxygen were however comparatively weakly affected by the replacements of Tyr191. Unlike the related lactate monooxygenase, which prevents fast dissociation of pyruvate to promote its oxidative decarboxylation by H2 O2 into acetate, CO2 and water as final reaction products, all avLOX variants retained their native oxidase activity where catalytic turnover results in the equivalent formation of H2 O2 . The 1.9 A crystal structure of the Y191F variant bound with FMN and pyruvate revealed a strictly locally disruptive effect of the site-directed substitution. Product off-rates appear to be dictated by partitioning of residues including Tyr191 from an active-site lid loop into bulk solvent and modulation of the hydrogen bond strength that links Tyr40 with the pyruvate's carboxylate group. Overall, this study emphasizes the possibly high importance of contributions from second-sphere substrate binding residues to the fine tuning of reactivity in alpha-hydroxy acid-oxidizing flavoenzymes, requiring that the catalytic steps of flavin reduction and oxidation are properly timed with the physical step of alpha-keto-acid product release. This article is protected by copyright. All rights reserved. Speeding up the product release: a second-sphere contribution from Tyr191 to the reactivity of L-lactate oxidase revealed in crystallographic and kinetic studies of site-directed variants.,Stoisser T, Klimacek M, Wilson DK, Nidetzky B FEBS J. 2015 Aug 11. doi: 10.1111/febs.13409. PMID:26260739 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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