Mutation of Trp93 of MauG to tyrosine causes loss of bound Ca2+ and alters the kinetic mechanism of tryptophan tryptophylquinone cofactor biosynthesis

摘要

pThe dihaem enzyme MauG catalyses a six-electron oxidation required for post-translational modification of preMADH (precursor of methylamine dehydrogenase) to complete the biosynthesis of its TTQ (tryptophan tryptophylquinone) cofactor. Trpsup93/sup of MauG is positioned midway between its two haems, and in close proximity to a Casup2+/sup that is critical for MauG function. Mutation of Trpsup93/sup to tyrosine caused loss of bound Casup2+/sup and changes in spectral features similar to those observed after removal of Casup2+/sup from WT (wild-type) MauG. However, whereas Casup2+/sup-depleted WT MauG is inactive, W93Y MauG exhibited TTQ biosynthesis activity. The rate of TTQ biosynthesis from preMADH was much lower than that of WT MauG and exhibited highly unusual kinetic behaviour. The steady-state reaction exhibited a long lag phase, the duration of which was dependent on the concentration of preMADH. The accumulation of reaction intermediates, including a diradical species of preMADH and quinol MADH (methylamine dehydrogenase), was detected during this pre-steady-state phase. In contrast, steady-state oxidation of quinol MADH to TTQ, the final step of TTQ biosynthesis, exhibited no lag phase. A kinetic model is presented to explain the long pre-steady-state phase of the reaction of W93Y MauG, and the role of this conserved tryptophan residue in MauG and related dihaem enzymes is discussed./p