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.
Highlights
PlGoxA from Pseudoalteromonas luteoviolacea is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor
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 productreduced 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 D678A, D678N, and D678E PlGoxA variants each were isolated as a homotetrameric protein that eluted at essentially the same volume as WT PlGoxA during size-exclusion chromatography
Summary
PlGoxA catalyzes the oxidative deamination of glycine (Reaction 1). LodA-like proteins are named after the CTQ-bearing lysine ⑀-oxidase, which is encoded by the lodA gene from the Marinomonas mediterranea [2, 3]. In the reductive half-reaction of PlGoxA (Fig. 1B), glycine is deprotonated to form a neutral amino group that allows a nucleophilic attack of the CTQ to form a substrate Schiff base adduct This is followed by deprotonation of the ␣-carbon to yield a product-reduced CTQ Schiff base. Asp-678 in PlGoxA is conserved in sequence and structure in the other CTQ-dependent oxidases that have been characterized, GoxA and LodA from M. mediterranea [3, 9] In each of those enzymes, the mutation of that Asp residue abolished or greatly reduced CTQ biosynthesis [6, 10]. The results suggest that Asp678 has multiple roles in the kinetic mechanism It is critical for deprotonation of the glycine substrate to allow formation of the substrate-oxidized CTQ Schiff base adduct and for subsequent deprotonation of that species to form the product-reduced CTQ Schiff base adduct. The structure suggests that Asp-678 is acting as a proton relay, which directs protons to a water channel that connects subunits of this homotetrameric enzyme
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