Catalytic loop closure governs substrate selectivity in the rhizocticin biosynthetic ligases RhiM and RhiC.

RhiM and RhiC are ATP-dependent l-amino acid ligases involved in the biosynthesis of the antifungal peptide rhizocticin. RhiM catalyzes the ligation of l-arginine with a phosphonic amino acid, and RhiC subsequently ligates the RhiM product with hydrophobic amino acids. Although biochemical studies have clarified the enzymatic functions of these ligases, the molecular basis of the catalytic mechanisms and substrate selectivity is unknown because there is no structural information for nucleotide- and substrate-bound states or direct structural evidence for the proposed acyl phosphate intermediate. In this study, we performed X-ray crystallographic and biochemical analyses of RhiM and RhiC. Crystal structures were determined for the nucleotide-bound forms of the enzymes as well as the substrate- and intermediate-analog-bound forms of RhiC. The structure of RhiC bound to an intermediate analog provides structural evidence supporting a mechanism where catalysis proceeding via an acyl phosphate intermediate. Structural comparisons of the previously reported apo form of RhiM and the multiple reaction-state structures determined here indicate that closure of three catalytic loops is essential for forming the substrate binding pocket and the correct positioning of the substrate and intermediates in the active site. The hydrophilic and hydrophobic pockets formed by these loops are optimally configured to accommodate the RhiM and RhiC substrates. Mutational analyses confirm that residues involved in substrate binding and Mg2+ coordination are critical for the ligase activity. These findings show that conformational rearrangements of the catalytic loops govern catalytic reaction and ligand selectivity in these enzymes.