Crystallographic studies of FMN and vitamin B(12) dependent enzymes: Flavodoxin and methionine synthase.

Abstract

The goal of this research has been to investigate the structural basis for modulation of reactivities of protein-bound cofactors. Two different systems have been studied: flavodoxin from Anacystis nidulans and methionine synthase from Escherichia coli. To examine the role of conformational changes in modulating redox properties of noncovently bound FMN, the structures of semiquinone and hydroquinone A. nidulans flavodoxin have been determined by x-ray crystallography to 1.8 A and 2.0 A resolution, respectively. As documented for Clostridium beijerinckii and D. vulgaris flavodoxin, formation of semiquinone is accompanied by an inversion of a peptide unit. A new hydrogen bond is formed between the carbonyl oxygen and the N(H)5 of the flavin, stabilizing the semiquinone. Unlike the case for other flavodoxins, the peptide flips back in the hydroquinone structure. This implies that the "inverted" conformation is less favorable in A. nidulans flavodoxin than in D. vulgaris or clostridial flavodoxin. The 3.0 A structure of the B$\sb{12}$-binding domains of methionine synthase has revealed motifs and interactions responsible for the recognition of the cofactor. The corrin macrocycle lies between a helical domain and an $\alpha/\beta$ domain that is a variant of a Rossmann fold. The methylcobalamin (methylB$\sb{12}$) undergoes a conformational change on binding the protein; the dimethylbenzimidazole to open up the lower face of the corrin to coordination by a histidine residue from the protein. The Asp-x-His-x-x-Gly motif surrounding the histidine ligand is conserved among the adenosylcobalamin enzymes that catalyze carbon skeletal rearrangements, suggesting that displacement of the dimethylbenzimidazole may be a common feature of cobalamin-binding proteins. A hydrogen bonding network that includes the histidine$\sp{759}$ ligand and neighboring residues, Asp$\sp{757}$, and Ser$\sp{810}$, has prompted speculation that a catalytic quartet, Co-His-Asp-Ser, modulates the reactivity of the B$\sb{12}$ prosthetic group in methionine synthase.