5kil | pdb_00005kil

CmlA beta-hydroxylase E377D mutant

spinqualitylabelsall models 5kil, resolution 2.72Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5kil is a 1 chain structure with sequence from Strvp. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: ACT, FEO, K Related: 4jo0, 5kik Gene: SVEN_0921 (STRVP) Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the beta-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 (WTR) to initiate O2 activation. However, rapid O2 activation occurs only when WTR is bound to CmlP, the NRPS to which l-PAPA is covalently attached. Here the X-ray crystal structure of WTR 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 WTR cluster ligand structure as well as the structures of WTR in complex with a functional CmlP variant (CmlPAT) with and without l-PAPA attached. It is found that formation of the active WTR:CmlPAT-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 WTR is E377. The crystal structure of the CmlA variant E377D shows only monodentate carboxylate coordination. Reduced E377D reacts rapidly with O2 in the presence or absence of CmlPAT-l-PAPA, showing loss of regulation. However, this variant fails to catalyze hydroxylation, suggesting that E377 has the dual role of coupling regulation of O2 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. A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme beta-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase.,Jasniewski AJ, Knoot CJ, Lipscomb JD, Que L Jr Biochemistry. 2016 Oct 18;55(41):5818-5831. doi: 10.1021/acs.biochem.6b00834., Epub 2016 Oct 7. PMID:27668828 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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