Structural and Spectroscopic Characterization of a Product Schiff Base Intermediate in the Reaction of the Quinoprotein Glycine Oxidase, GoxA

Abstract

The LodA-like proteins are a recently identified family of enzymes that rely on a cysteine tryptophylquinone (CTQ) cofactor for catalysis. They differ from other tryptophylquinone enzymes in that they are oxidases rather than dehydrogenases. GoxA is a member of this family that catalyzes the oxidative deamination of glycine. Our previous work with GoxA from Pseudoalteromonas luteoviolaceae demonstrated that this protein forms a stable intermediate upon anaerobic incubation with glycine. The spectroscopic properties of this species were unique among those identified for tryptophylquinone enzymes characterized to date. Here we use X-ray crystallography and resonance Raman spectroscopy to identify the GoxA catalytic intermediate as a product Schiff-base. Structural work additionally highlights features of the active site pocket that confer substrate specificity, intermediate stabilization and catalytic activity. The unusual properties of GoxA are discussed within the context of the other tryptophylquinone enzymes.

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

“…The resulting crystals were yellow in color and isomorphous to the WT (Table 2) with very similar overall structures (rmsd < 0.3 Å across backbone atoms). Upon soaking in cryoprotection solution containing 10 mM glycine, F316A and Y766F turned deep blue in color, consistent with the formation of a product-reduced CTQ Schiff base in the crystal (17). H583C exhibited no color change while H767A became nearly transparent, which would be consistent with reduced CTQ (7).…”

“…An unusual feature of the reaction of WT PlGoxA is that addition of glycine to the enzyme under anaerobic conditions yields a stable Schiff base adduct with the product bound to the reduced CTQ (17). Spectral changes associated with formation of this adduct upon anaerobic titration of PlGoxA with glycine yielded an h value of 3.7 indicative of strong cooperative binding (7).…”

“…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.…”

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

“…The extent of CTQ biosynthesis in the WT and variant proteins was similar, as evidenced by a 280/400 nm absorbance ratio of ϳ16. It was previously shown that the addition of glycine to PlGoxA under anaerobic conditions caused a significant change in the absorbance spectrum due to conversion of oxidized CTQ to a substrate-reduced CTQ Schiff base adduct (8). The addition of glycine to D678E PlGoxA caused a similar change in absorbance consistent with formation of the substrate-reduced CTQ Schiff adduct.…”

The Redox Properties of a Cysteine Tryptophylquinone-Dependent Glycine Oxidase Are Distinct from Those of Tryptophylquinone-Dependent Dehydrogenases

“…It was subsequently shown that the absorbance spectrum of the glycine-reduced GoxA was that of the reduced CTQ product Schiff base adduct. 14 The reduction of GoxA with glycine was repeated using incremental additions of glycine ( Figure 2A) under anaerobic conditions and unexpected results were obtained. As noted before, 6 it was not possible to reduce GoxA stoichiometrically with glycine.…”