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

Abstract

Among a-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°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 with wild-type enzyme (~141 s À1) and thus became mainly rate limiting for L-lactate oxidation by the variants at a steady-state under air-saturated conditions. In the Y191L and the Y191A variants, 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 the fast dissociation of pyruvate to promote its oxidative decarboxylation by H 2 O 2 into acetate, CO 2 and water as final reaction products, all avLOX variants retained their native oxidase activity where catalytic turnover results in the equivalent formation of H 2 O 2 . The 1.9A 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 a-hydroxy acid-oxidizing flavoenzymes, requiring that the catalytic steps of flavin reduction and oxidation are properly timed with the physical step of a-keto acid product release. DatabaseThe structure of Y191F variant of A. viridans L-lactate oxidase was deposited in the wwPDB under the accession code 4YL2.Abbreviations avLOX, Aerococcus viridans LOX; FMN, flavin mononucleotide; LMO, L-lactate monooxygenase; LOX, L-lactate oxidase.

Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr215 in Aerococcus viridans lactate oxidase

“…Several Ramachandran outliers involving Gln269 and Ser297 were noted which have strong corresponding electron density. Similar conformations have been seen for these residues in other high resolution avLOX structures 12 14 23 24 35 36 37 .…”

“…Moreover, partitioning of the phenylalanine sidechain into solvent is much more unfavorable than a tyrosine when the lid is in the open conformation. Similar effects of partitioning lid residues on product off rates have been noted before in avLOX variants 37 . The change to a histidine at this position also results in a reduction of product off rate.…”

“…3 , indicating reduction of the FMN cofactor over time. The wild-type enzyme was analyzed in previous studies 12 37 and it shows similar behavior (see Figure S1, Supporting Information ). Decrease in absorbance at 455 nm was used to monitor the enzyme reduction ( Fig.…”

“…Parameters are from the data in Figs 3, 4, 5 ( k red , k slow ) or Fig. S2 ( k ox ). c The data for wild-type enzyme at pH 6.5 are from our previous paper37. d Parameters shown in squared brackets are from stopped-flow measurements at 530 nm.…”

“…Moreover, it is also partially responsible for closing loop 4, the sequence of residues consisting of the residue range 173–222 which connect β4 and α4 in the core (β/α) 8 barrel, over the active site via its interaction with the lactate and side chains on the core of the protein such as Arg 181 . Movement of all or part of this flexible loop is necessary for substrate binding and product release 12 14 24 35 36 37 . It was therefore particularly interesting to determine what specific role Tyr 215 might play in the catalytic reaction of avLOX and especially, in the proposed loop conformational change involved in product dissociation.…”