5KIL

CmlA beta-hydroxylase E377D mutant

Summary for 5KIL

• Entry DOI: 10.2210/pdb5kil/pdb
• Related: 4JO0 5kik
• Descriptor: CmlA protein, MU-OXO-DIIRON, POTASSIUM ION, ... (5 entities in total)
• Functional Keywords: oxygen activation, diiron cluster, antibiotic biosynthesis, beta-hydroxylase, oxidoreductase
• Biological source: Streptomyces venezuelae (strain ATCC 10712 / CBS 650.69 / DSM 40230 / JCM 4526 / NBRC 13096 / PD 04745)
• Total number of polymer chains: 1
• Total formula weight: 62488.71

Authors

Knoot, C.J.,Lipscomb, J.D. (deposition date: 2016-06-16, release date: 2017-04-26, Last modification date: 2023-09-27)

Primary citation

Jasniewski, A.J.,Knoot, C.J.,Lipscomb, J.D.,Que, L.
A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme beta-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase.
Biochemistry, 55:5818-5831, 2016

PubMed Abstract: The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the β-hydroxylation of the precursor l-p-aminophenylalanine (l-PAPA) catalyzed by the monooxygenase CmlA. The active site of CmlA contains a dinuclear iron cluster that is reduced to the diferrous state (WT) to initiate O activation. However, rapid O activation occurs only when WT is bound to CmlP, the NRPS to which l-PAPA is covalently attached. Here the X-ray crystal structure of WT is reported, which is very similar to that of the as-isolated diferric enzyme in which the irons are coordinately saturated. X-ray absorption spectroscopy is used to investigate the WT cluster ligand structure as well as the structures of WT in complex with a functional CmlP variant (CmlP) with and without l-PAPA attached. It is found that formation of the active WT:CmlP-l-PAPA complex converts at least one iron of the cluster from six- to five-coordinate by changing a bidentately bound amino acid carboxylate to monodentate on Fe1. The only bidentate carboxylate in the structure of WT is E377. The crystal structure of the CmlA variant E377D shows only monodentate carboxylate coordination. Reduced E377D reacts rapidly with O in the presence or absence of CmlP-l-PAPA, showing loss of regulation. However, this variant fails to catalyze hydroxylation, suggesting that E377 has the dual role of coupling regulation of O reactivity with juxtaposition of the substrate and the reactive oxygen species. The carboxylate shift in response to substrate binding represents a novel regulatory strategy for oxygen activation in diiron oxygenases.

PubMed: 27668828
DOI: 10.1021/acs.biochem.6b00834

Experimental method

X-RAY DIFFRACTION (2.72 Å)