PDBsum entry 6vmf

Oxidoreductase

PDB id: 6vmf

Contents

Protein chains

754 a.a.

Ligands

SO4 ×3

Metals

_MG ×4

Waters ×1006

PDB id:

6vmf

Name:

Oxidoreductase

Title:

Crystal structure of the y766f mutant of goxa soaked with glycine

Structure:

Glycine oxidase. Chain: a, b, c, d. Engineered: yes. Mutation: yes

Source:

Pseudoalteromonas luteoviolacea dsm 6061. Organism_taxid: 1365250. Gene: n475_19905. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.24Å R-factor: 0.202 R-free: 0.250

Authors:

E.T.Yukl

Key ref:

K.J.Mamounis et al. (2020). Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase. J Biol Chem, 295, 6472-6481. PubMed id: 32234764 DOI: 10.1074/jbc.RA120.013198

Date:

27-Jan-20 Release date: 08-Apr-20

Protein chains

A0A161XU12  (A0A161XU12_9GAMM) -  Uncharacterized protein from Pseudoalteromonas luteoviolacea DSM 6061

Seq: 816 a.a.

Struc: 754 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DOI no: 10.1074/jbc.RA120.013198

J Biol Chem 295:6472-6481 (2020)

PubMed id: 32234764

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

K.J.Mamounis, E.T.Yukl, V.L.Davidson.

ABSTRACT

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on k_cat and K_0.5 for catalysis, K_0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.