Interaction of GoxA with Its Modifying Enzyme and Its Subunit Assembly Are Dependent on the Extent of Cysteine Tryptophylquinone Biosynthesis

Abstract

GoxA is a glycine oxidase bearing a protein-derived cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications catalyzed by a flavoprotein, GoxB. Two forms of GoxA were isolated. An active form with mature CTQ and an inactive precursor protein that lacked CTQ. The active GoxA was present as a homodimer with no detectable affinity for GoxB, whereas the precursor was isolated as a monomer in a tight complex with one GoxB. Thus, the interaction of GoxA with GoxB and subunit assembly of mature GoxA are each dependent on the extent of CTQ biosynthesis.

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

“…WT GoxA (Fig. 6A) elutes from the column primarily as a dimer, as was reported previously (11). One observes a minor peak at ϳ76 kDa (16%), which corresponds to the predicted mass of a monomer, and a major peak of the dimer at ϳ140 kDa (84%).…”

“…The analysis yielded values of k cat ϭ 39 Ϯ 2 s Ϫ1 , K 0.5 ϭ 514 Ϯ 42 M for glycine, and a Hill coefficient of 1.7 Ϯ 0.2, which is indicative of positive cooperativity. Given the previous finding that GoxA is a homodimer (11), the h value of 1.7 (where h is the Hill coefficient) is consistent with a kinetic mechanism in which the binding of the first glycine molecule to one active site enhances the affinity of a second active site for glycine. The concentration of O 2 was varied in the presence of a saturating concentration of glycine (5 mM).…”

“…Construction, Expression, and Purification of GoxA and Its Variant Proteins-WT GoxA and GoxA variants that were generated by site-directed mutagenesis were expressed by coexpression of the goxA gene with goxB in Escherichia coli Rosetta cells as described previously (11). The GoxA proteins possess an N-terminal His 6 tag, which was engineered into the gene.…”

“…Furthermore, the inactive protein was isolated primarily in complex with the modifying enzyme GoxB. It was shown previously that a precursor of WT GoxA, which lacks CTQ, forms a tight complex with GoxB (11). As indicated in that previous study, the presence or absence of CTQ in GoxA when in complex with GoxB could be determined using a staining protocol that is specific for covalent quinoproteins (17) after transfer of proteins, which were run on SDS-PAGE by electroblot to a nitro-cellulose membrane.…”

“…Although LodA exhibits typical Michaelis-Menten behavior, the current steady-state kinetic study of GoxA reveals that it exhibits allosteric cooperativity toward the glycine substrate. It was shown previously that purified active GoxA eluted primarily as a dimer during size exclusion chromatography (11). As the crystal structure of GoxA has yet to be determined, a homology model has been constructed from the GoxA sequence and the LodA structure.…”