Dataset Information

A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme ?-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase.

ABSTRACT: The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the ?-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.

SUBMITTER: Jasniewski AJ

PROVIDER: S-EPMC5258830 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme β-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase.

Jasniewski Andrew J AJ Knoot Cory J CJ Lipscomb John D JD Que Lawrence L

Biochemistry 20161007 41

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 (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. H ...[more]

PMID: 27668828

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Project description:Progress toward the development of functional models of the carboxylate-bridged diiron active site in soluble methane monooxygenase is described in which potential substrates are introduced as substituents on bound pyridine ligands. Pyridine ligands incorporating a thiol, sulfide, sulfoxide, or phosphine moiety were allowed to react with the preassembled diiron(II) complex [Fe(2)(mu-O(2)CAr(R))(2)(O(2)CAr(R))(2)(THF)(2)], where (-)O(2)CAr(R) is a sterically hindered 2,6-di(p-tolyl)- or 2,6-di(p-fluorophenyl)benzoate (R = Tol or 4-FPh). The resulting diiron(II) complexes were characterized crystallographically. Triply and doubly bridged compounds [Fe(2)(mu-O(2)CAr(Tol))(3)(O(2)CAr(Tol))(2-MeSpy)] (4) and [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(2-MeS(O)py)(2)] (5) resulted when 2-methylthiopyridine (2-MeSpy) and 2-pyridylmethylsulfoxide (2-MeS(O)py), respectively, were employed. Another triply bridged diiron(II) complex, [Fe(2)(mu-O(2)CAr(4)(-)(FPh))(3)-(O(2)CAr(4)(-)(FPh))(2-Ph(2)Ppy)] (3), was obtained containing 2-diphenylphosphinopyridine (2-Ph(2)Ppy). The use of 2-mercaptopyridine (2-HSpy) produced the mononuclear complex [Fe(O(2)CAr(Tol))(2)(2-HSpy)(2)] (6a). Together with that of previously reported [Fe(2)(mu-O(2)CAr(Tol))(3)(O(2)CAr(Tol))(2-PhSpy)] (2) and [Fe(2)(mu-O(2)CAr(Tol))(3)(O(2)CAr(Tol))(2-Ph(2)Ppy)] (1), the dioxygen reactivity of these iron(II) complexes was investigated. A dioxygen-dependent intermediate (6b) formed upon exposure of 6a to O(2), the electronic structure of which was probed by various spectroscopic methods. Exposure of 4 and 5 to dioxygen revealed both sulfide and sulfoxide oxidation. Oxidation of 3 in CH(2)Cl(2) yields [Fe(2)(mu-OH)(2)(mu-O(2)CAr(4)(-)(FPh))(O(2)CAr(4)(-FPh))(3)(OH(2))(2-Ph(2)P(O)py)] (8), which contains the biologically relevant {Fe(2)(mu-OH)(2)(mu-O(2)CR)}(3+) core. This reaction is sensitive to the choice of carboxylate ligands, however, since the p-tolyl analogue 1 yielded a hexanuclear species, 7, upon oxidation.

Project description:BackgroundHemerythrins, are the non-heme, diiron binding respiratory proteins of brachiopods, priapulids and sipunculans; they are also found in annelids and bacteria, where their functions have not been fully elucidated.ResultsA search for putative Hrs in the genomes of 43 archaea, 444 bacteria and 135 eukaryotes, revealed their presence in 3 archaea, 118 bacteria, several fungi, one apicomplexan, a heterolobosan, a cnidarian and several annelids. About a fourth of the Hr sequences were identified as N- or C-terminal domains of chimeric, chemotactic gene regulators. The function of the remaining single domain bacterial Hrs remains to be determined. In addition to oxygen transport, the possible functions in annelids have been proposed to include cadmium-binding, antibacterial action and immunoprotection. A Bayesian phylogenetic tree revealed a split into two clades, one encompassing archaea, bacteria and fungi, and the other comprising the remaining eukaryotes. The annelid and sipunculan Hrs share the same intron-exon structure, different from that of the cnidarian Hr.ConclusionThe phylogenomic profile of Hrs demonstrated a limited occurrence in bacteria and archaea and a marked absence in the vast majority of multicellular organisms. Among the metazoa, Hrs have survived in a cnidarian and in a few protostome groups; hence, it appears that in metazoans the Hr gene was lost in deuterostome ancestor(s) after the radiata/bilateria split. Signal peptide sequences in several Hirudinea Hrs suggest for the first time, the possibility of extracellular localization. Since the alpha-helical bundle is likely to have been among the earliest protein folds, Hrs represent an ancient family of iron-binding proteins, whose primary function in bacteria may have been that of an oxygen sensor, enabling aerophilic or aerophobic responses. Although Hrs evolved to function as O2 transporters in brachiopods, priapulids and sipunculans, their function in annelids remains to be elucidated. Overall Hrs exhibit a considerable lack of evolutionary success in metazoans.

Dataset Information

A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme ?-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase.

Publications

A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme β-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase.

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