Roles of Conserved Residues of the Glycine Oxidase GoxA in Controlling Activity, Cooperativity, Subunit Composition, and Cysteine Tryptophylquinone Biosynthesis

Abstract

GoxA is a glycine oxidase that possesses a cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications that are catalyzed by a modifying enzyme GoxB. It is the second known tryptophylquinone enzyme to function as an oxidase, the other being the lysine ϵ-oxidase, LodA. All other enzymes containing CTQ or tryptophan tryptophylquinone (TTQ) cofactors are dehydrogenases. Kinetic analysis of GoxA revealed allosteric cooperativity for its glycine substrate, but not O This is the first CTQ- or TTQ-dependent enzyme to exhibit cooperativity. Here, we show that cooperativity and homodimer stabilization are strongly dependent on the presence of Phe-237. Conversion of this residue, which is a Tyr in LodA, to Tyr or Ala eliminates the cooperativity and destabilizes the dimer. These mutations also significantly affect the k and K values for the substrates. On the basis of structural and modeling studies, a mechanism by which Phe-237 exerts this influence is presented. Two active site residues, Asp-547 and His-466, were also examined and shown by site-directed mutagenesis to be critical for CTQ biogenesis. This result is compared with the results of similar studies of mutagenesis of structurally conserved residues of other tryptophylquinone enzymes. These results provide insight into the roles of specific active-site residues in catalysis and CTQ biogenesis, as well as describing an interesting mechanism by which a single residue can dictate whether or not an enzyme exhibits cooperative allosteric behavior toward a substrate.

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

“…Those characterized to date catalyze the oxidative deamination of the ε-amino group of lysine (LodA) or the amine group of glycine (GoxA). Some features of the reductive half-reaction are similar to those of MADH, AADH and QHNDH, but some are different 18,19 . The LodA-like proteins are oxidases, using molecular oxygen as an electron acceptor 18 .…”

Following the Evolutionary Track of a Highly Specific l -Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes

“…L AAO (L-amino acid oxidase) is an amino acid-metabolizing enzyme that catalyzes the oxidation of the main-chain amino group of L-amino acids, thereby generating ␣-keto acid and ammonia (1). Two types of LAAOs have been reported: one is flavin adenine dinucleotide (FAD) dependent (2)(3)(4) while the other is not FAD dependent (5)(6)(7)(8). The former type of LAAO is the main target of this study.…”

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

“…Previous studies of tryptophylquinone-bearing enzymes have shown that mutation of active site residues is not well-tolerated. Mutation of the active site Asp residue that corresponds to Asp-678 in the TTQ-dependent methylamine dehydrogenase (16) and the CTQ-dependent MmLodA (14) and MmGoxA (12) prevented the formation of the quinone cofactor. Mutation of some other active site residues in these enzymes also prevented or reduced the levels of cofactor biosynthesis.…”

“…The inter-subunit projection of these residues suggests a role in cooperativity. It was previously shown that Phe-237 in MmGoxA plays a role in its cooperative kinetic behavior (12). The corresponding residue in PlGoxA is Phe-316, which suggests that it may also play a role in cooperativity of PlGoxA.…”

“…A second LodAlike protein from the same bacterium is a glycine oxidase, MmGoxA (11), which forms a homodimer. Analysis of the steadystate kinetics of glycine oxidation by MmGoxA did reveal positive cooperativity, with an h value of 1.7 (12). With MmLodA and MmGoxA, it was not possible to monitor substrate binding because no stable product-CTQ adduct with distinct spectroscopic features was detectable.…”