Characterization of recombinant biosynthetic precursors of the cysteine tryptophylquinone cofactors of l-lysine-epsilon-oxidase and glycine oxidase from Marinomonas mediterranea.

The lysine-ε-oxidase, LodA, and glycine oxidase, GoxA, from Marinomonas mediteranea each possesses a cysteine tryptophylquinone (CTQ) cofactor. This cofactor is derived from posttranslational modifications which are covalent crosslinking of tryptophan and cysteine residues and incorporation of two oxygen atoms into the indole ring of Trp. In this manuscript, it is shown that the recombinant synthesis of LodA and GoxA containing a fully synthesized CTQ cofactor requires coexpression of a partner flavoprotein, LodB for LodA and GoxB for GoxA, which are not interchangeable. An inactive precursor of LodA or GoxA which contained a monohydroxylated Trp residue and no crosslink to the Cys was isolated from the soluble fraction when they were expressed alone. The structure of LodA revealed an Asp residue close to the cofactor which is conserved in quinohemoprotein amine dehydrogenase (QHNDH), containing CTQ, and methylamine dehydrogenase (MADH) containing tryptophan tryptophylquinone (TTQ) as cofactor. To study the role of this residue in the synthesis of the LodA precursor, Asp-512 was mutated to Ala. When the mutant protein was coexpressed with LodB an inactive protein was isolated which was soluble and contained no modifications at all, suggesting a role for this Asp in the initial LodB-independent hydroxylation of Trp. A similar role had been proposed for this conserved Asp residue in MADH. It is noteworthy that the formation of TTQ in MADH from the precursor also requires an accessory enzyme for its biosynthesis but it is a diheme enzyme MauG and not a flavoprotein. The results presented reveal novel mechanisms of post-translational modification involved in the generation of protein-derived cofactors. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.