Dinuclear Iron Cluster‐Containing OxygenaseCmlA

Abstract

CmlA is a tailoring enzyme from the nonribosomal peptide synthetase (NRPS)‐based biosynthetic pathway for chloramphenicol expressed in Streptomyces venezuelae . The enzyme is a monooxygenase that catalyzes the β‐hydroxylation of l ‐ para ‐aminophenylalanine ( l ‐PAPA) covalently tethered to the NRPS CmlP. Spectroscopic and X‐ray crystallographic studies reveal an oxo‐bridged dinuclear iron cluster in the active site that binds and activates O 2 . The structure shows that the subunits of the homodimeric CmlA are composed of a 248 residue N‐terminal domain with a novel fold and a 284 residue C‐terminal domain with a metallo‐β‐lactamase (MBL) fold where the diiron cluster is bound. Oxygenase activity has not been previously observed for a diiron cluster in an MBL fold and no other diiron cluster enzyme is known to catalyze β‐hydroxylation. In the diferrous state, the diiron cluster is reactive with O 2 , but the reaction is very slow in the absence of the CmlP– l ‐PAPA complex. Thus, CmlA is regulated so that O 2 is activated only when substrate is bound. CmlA is the first diiron cluster‐containing enzyme known to be active in an NRPS pathway, but a gene search reveals numerous homologs of CmlA that are likely to catalyze similar reactions during biosynthesis of other natural products.

Diiron monooxygenases in natural product biosynthesis

“…The X-ray crystal structure of CmlA was subsequently solved at 2.17 Å resolution using phasing from a selenomethionine-substituted protein. 14, 16 In accord with the sequence predictions, the enzyme was found to fold into two domains as shown in Figure 2A with a short linker region (residues 236-248) that connects these two domains. The N-terminal (residues 1-235) domain has no structural homologs.…”

“…As illustrated in Figure 4C, computational docking studies suggested that the binding orientation of L-PAPA in the active site would place the β-carbon immediately over the newly opened coordination site on Fe1 where O 2 would bind and be activated. 14, 16 This placement would also allow a hydrogen bond to form between one of the carboxylate oxygens of E377 and the amine of L-PAPA if E377 would rotate into a monodentate binding orientation. Thus, substrate binding both initiates O 2 activation and fixes the substrate in the correct orientation to be hydroxylated.…”

“…A BLAST search of the genome data base reveals at least fifty homologs of CmlA that are likely to contain dinuclear iron clusters and catalyze O 2 activation and insertion reactions. 7, 14, 16 These homologs are found in the genomes of many different types of bacteria, including some cyanobacteria. The CmlA homologues identified thus far are of similar size and have 30–45% sequence identity.…”

“…Recent reviews summarize the technical aspects of preparation, characterization, and structure determination of CmlI and CmlA. 15, 16…”

CmlI N-Oxygenase Catalyzes the Final Three Steps in Chloramphenicol Biosynthesis without Dissociation of Intermediates

“…2, 3 Structurally similar clusters are found in many enzymes that activate O 2 and then catalyze reactions such as functionalization of unactivated C-H bonds, desaturation reactions, aromatic hydroxylation, and radical formation, but notably, not aryl-amine oxygenation. 4-14 Conversely, CmlI and its structural and function homolog AurF 15-19 do not catalyze the types of reactions common to other diiron oxygenases. Kinetic and spectroscopic studies of CmlI and AurF have shown that, despite the structural similarities to other diiron oxygenases, a different type of reactive oxygen intermediate is generated during catalysis.…”