Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis

Abstract

The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the auto-catalytic hydroxylation of a specific Trp residue at the C-7 position on the indole side-chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis, as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provide evidence that copper is required for the initial hydroxylation of the precursor protein; and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed the fluorescence appears and when it is added back to the protein the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the un-activated Trp side-chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor

“…Thus, QhpG inserts both oxygen atoms into the Trp of CTQ, whereas in the biogenesis of TTQ in MADH and CTQ in LodA and GoxA, the substrate for the modifying enzyme has the first hydroxyl present and only the second is added along with the formation of the Trp–Trp or Trp–Cys crosslink. Formation of the initial mono-hydroxy-Trp intermediate in MADH and LodA/GoxA is thought to be an autocatalytic process 36 , 45 , which appears to be copper-ion dependent in LodA 45 , with the participation of an Asp residue 40 , 46 , 47 located close to the cofactor, and strictly conserved in all tryptophylquinone enzymes. However, a recent study on GoxA has shown that mutation of the corresponding Asp678 does not abolish CTQ formation 48 .…”

Protein-Derived Cofactors Revisited: Empowering Amino Acid Residues with New Functions

“…A long-standing question initially raised during studies of the biosynthesis of TTQ in MADH is how this seemingly impossible reaction occurs, the autocatalytic hydroxylation of a specific tryptophan residue of a precursor protein at a specific position on the indole side chain. Study of an D512A variant of LodA provided a clue as to how this may happen . The majority of the purified protein was the completely unmodified form without the initial hydroxylation, but a small fraction contained active protein with CTQ.…”

“…Study of an D512A variant of LodA provided a clue as to how this may happen. 37 The majority of the purified protein was the completely unmodified form without the initial hydroxylation, but a small fraction contained active protein with CTQ. Metal analysis revealed that the active form contained weakly bound copper.…”

Diversity of structures, catalytic mechanisms and processes of cofactor biosynthesis of tryptophylquinone-bearing enzymes

“…Metal analysis and spectroscopic analysis of the inactive and active variants strongly suggested that the initial hydroxylation is a copper-dependent process, and that after cofactor maturation the copper is no longer present or required for catalysis. It was proposed that an active site Asp residue dictates the selectivity for copper binding in the unmodified protein and that the hydroxylation proceeds via a Cu 3+ -OH intermediate, which is stabilized by a Cys sulfur and oxidizes the Trp [38].…”