Beta-Hydroxylation in Natural Product Biosynthesis: A New Activity and a
New Protein Fold for Diiron Cluster Oxygenases
Cory J. Knoot, Thomas M. Makris, Carrie M. Wilmot, and John D. Lipscomb*
Department of Biochemistry, Molecular Biology and Biophysics, University of
Minnesota, Minneapolis, Minnesota 55455 USA
Natural product antibiotics are often biosynthesized via NRPS-based systems.1
Tailoring reactions such as -hydroxylation shape the final antibiotic. In past
studies, we described a previously unrecognized type of NRPS-associated hydroxylase that utilizes a diiron cluster as found in oxygenases such as methane
monooxygenase (MMO).2,3 The archetypal enzyme, CmlA, catalyzes -hydroxylation of L-p-amino-phenylalanine, the precursor of chloramphenicol. Recently,
we solved the 2.17 Å X-ray crystal structure of CmlA.4 The structure shows that
the enzyme has an N-terminal domain with a novel fold and a diiron clusterbinding C-terminal domain with a metallo--lactamase fold (Fig. 1). No other
diiron monooxygenase utilizes this fold. The diiron cluster is in a cavity that
extends 10 Å down from a long crease formed by the interface of the N- and Cterminal domains. The cluster in CmlA is bound by 3 His and 3 Asp/Glu ligands.
One iron is coordinated by a solvent in an unusual location away from the open
substrate cavity (Fig. 2). In contrast, MMO has 2 His and 4 carboxylate ligands,
and the sites away from the substrate cavity are occupied by protein ligands. We
speculate that there is a correlation between the carboxylate/His ratio and the
strength of the CH bond that can be broken; CmlA may be tuned to break the
relatively weak -carbon bond. Reduced CmlA will not bind O2 or react with its
substrate unless the latter is covalently bound to the phosphopantetheine arm of
the thiolation domain of the NRPS, CmlP. Computational docking of this domain
to CmlA shows that it binds in the interdomain crease. The covalent arm bearing
the L-p-amino-phenylalanine extends into the active site channel and places the carbon of the substrate immediately over the cluster. The lack of activity of CmlA
in the absence of substrate-loaded CmlP is mysterious. However, the structure
reveals a Glu residue adjacent to the CmlP binding site which is poised near the
unusual solvent-occupied site on the cluster. It is possible that complex formation
with CmlP shifts this residue into the ligation, thereby providing the MMO-like
4th carboxylate to potentiate O2 activation (Fig. 3). Supported by NIH GM100943
(1) Fischbach, M. A.; Walsh, C. T. Chem. Rev. 2006, 106, 3468.
(2) Makris, T. M.; Chakrabarti, M.; Münck, E.; Lipscomb, J. D. Proc. Natl.
Acad. Sci. U. S. A. 2010, 107, 15391.
(3) Vu, V. V.; Makris, T. M.; Lipscomb, J. D.; Que, L. JACS 2011, 133, 6938.
(4) Makris, T. M.; Knoot, C. J.; Wilmot, C. M.; Lipscomb, J. D. Biochemistry
2013, 52, 6662.
Figure 1. CmlA overall fold
Figure 2. CmlA diiron cluster
Figure 3. Proposed role of E430
in triggering O2 activation