Kinetic and structural evidence that Asp-678 plays multiple roles in catalysis by the quinoprotein glycine oxidase

Abstract

PlGoxA from Pseudoalteromonas luteoviolacea is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor. A notable feature of its catalytic mechanism is that it forms a stable product-reduced CTQ adduct that is not hydrolyzed in the absence of O2. Asp-678 resides near the quinone moiety of PlGoxA, and an Asp is structurally conserved in this position in all tryptophylquinone enzymes. In those other enzymes, mutation of that Asp results in no or negligible CTQ formation. In this study, mutation of Asp-678 in PlGoxA did not abolish CTQ formation. This allowed, for the first time, studying the role of this residue in catalysis. D678A and D678N substitutions yielded enzyme variants with CTQ, which did not react with glycine, although glycine was present in the crystal structures in the active site. D678E PlGoxA was active but exhibited a much slower kcat. This mutation altered the kinetic mechanism of the reductive half-reaction such that one could observe a previously undetected reactive intermediate, an initial substrate-oxidized CTQ adduct, which converted to the product-reduced CTQ adduct. These results indicate that Asp-678 is involved in the initial deprotonation of the amino group of glycine, enabling nucleophilic attack of CTQ, as well as the deprotonation of the substrate-oxidized CTQ adduct, which is coupled to CTQ reduction. The structures also suggest that Asp-678 is acting as a proton relay that directs these protons to a water channel that connects the active sites on the subunits of this homotetrameric enzyme.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor

“…Formation of the initial mono-hydroxy-Trp intermediate in MADH and LodA/GoxA is thought to be an autocatalytic process 36 , 45 , which appears to be copper-ion dependent in LodA 45 , with the participation of an Asp residue 40 , 46 , 47 located close to the cofactor, and strictly conserved in all tryptophylquinone enzymes. However, a recent study on GoxA has shown that mutation of the corresponding Asp678 does not abolish CTQ formation 48 . In addition, the corresponding Asp33 in QhpC may be placed in the equivalent position only after the formation of the CTQ thioether bond (Supplementary Fig.…”

Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase

“…Among these are direct hydrogen bond interactions between these residues and bound substrate as well as pi-stacking interactions between Tyr-766 and Phe-316. We had previously identified a hydrated channel connecting the two active sites, into which the loops bearing His-767 and Tyr-766 project (13). This suggested the binding of substrate to one active site may control access to the other.…”

“…It was previously shown that D678A and D678N PlGoxA variant proteins were not reduced by glycine, but did have glycine present in the active sites in the glycinesoaked crystals (13). In the structures of the glycine-reduced CTQ adduct of WT PlGoxA and the glycine-soaked crystals of D678A and D678N PlGoxA, a His-583 ring nitrogen interacts with a carboxyl oxygen of glycine.…”

“…Recently, it was shown that mutation of Asp-678 to Glu in the active site of PlGoxA eliminated the observed cooperativity of steady-state kinetic behavior, but it did not diminish the observed cooperativity of glycine binding (13). The explanation for this was that the mutation slowed the rate of one or more reaction steps such that binding of glycine was no longer even partially rate limiting in the overall reaction.…”

“…R signifies the point of attachment to the protein. (B) Reaction scheme and structures of intermediates for glycine oxidation by PlGoxA.This is based on the results of previous studies(13,17) and the current study.…”