Structure of a Dinuclear Iron Cluster-Containing β-Hydroxylase Active in Antibiotic Biosynthesis

A family of dinuclear iron cluster-containing oxygenases that catalyze beta-hydroxylation tailoring reactions in natural product biosynthesis by nonribosomal peptide synthetase (NRPS) systems was recently described [Makris, T. M., Chakrabarti, M., Mfinck, E., and Lipscomb, J. D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 15391-15396]. Here, the 2.17 A X-ray crystal structure of the archetypal enzyme from the family, Cm1A, is reported. Cm1A catalyzes /3-hydroxylation of L-paminophenylalanine during chloramphenicol biosynthesis. The fold of the N-terminal domain of Cm1A is unlike any previously reported, but the C-terminal domain has the afifia fold of the metallo-beta-lactamase (MBL) superfamily. The diiron cluster bound in the C-terminal domain is coordinated by an acetate, three His residues, two Asp residues, one Glu residue, and a bridging oxo moiety. One of the Asp ligands forms an unusual monodentate bridge. No other oxygen-activating diiron enzyme utilizes this ligation or the MBL protein fold. The N-terminal domain facilitates dimerization, but using computational docking and a sequence-based structural comparison to homologues, we hypothesize that it likely serves additional roles in NRPS recognition and the regulation of 02 activation.