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Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2.

ABSTRACT: The alpha-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His(2)(Asp/Glu)(1) "facial triad" of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X(-)) coordinates at the vacated site. Halogenation is thought to result from "rebound" of the halogen radical from the X-Fe(III)-OH intermediate produced by hydrogen (H(*)) abstraction to the substrate radical. The alternative decay pathway for the X-Fe(III)-OH intermediate, rebound of the hydroxyl radical to the substrate radical (as occurs in the hydroxylases), reportedly does not compete. Here we show for the halogenase SyrB2 that positioning of the alkyl group of the substrate away from the oxo/hydroxo ligand and closer to the halogen ligand sacrifices H(*)-abstraction proficiency for halogen-rebound selectivity. Upon replacement of L-Thr, the C4 amino acid tethered to the SyrB1 carrier protein in the native substrate, by the C5 amino acid L-norvaline, decay of the chloroferryl intermediate becomes 130x faster and the reaction outcome switches to primarily hydroxylation of C5, consistent with projection of the methyl group closer to the oxo/hydroxo by the longer side chain. Competing H(*) abstraction from C4 results primarily in chlorination, as occurs at this site in the native substrate. Consequently, deuteration of C5, which slows attack at this site, switches both the regioselectivity from C5 to C4 and the chemoselectivity from hydroxylation to chlorination. Thus, substrate-intermediate disposition and the carboxylate --> halide ligand swap combine to specify the halogenation outcome.

SUBMITTER: Matthews ML

PROVIDER: S-EPMC2764930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aliphatic halogenases activate O(2), cleave alpha-ketoglutarate (alphaKG) to CO(2) and succinate, and form haloferryl [X-Fe(IV)O; X = Cl or Br] complexes that cleave aliphatic C-H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related alphaKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O(2) to form the C-H bond-cleaving ferryl complex is "triggered" by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogenases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via a thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presence of free l-Thr or its analogues, but SyrB1 charged either by l-Thr (l-Thr-S-SyrB1) or by any of several non-native amino acids does trigger the reaction by as much as 8000-fold (for the native substrate). Triggering efficacy is sensitive to the structures of both the amino acid and the carrier protein, being diminished by 5-24-fold when the native l-Thr is replaced with another amino acid and by approximately 40-fold when SyrB1 is replaced with the heterologous carrier protein, CytC2. The directing effect of the carrier protein and consequent tolerance for profound modifications to the target amino acid allow the chloroferryl state to be formed in the presence of substrates that perturb the ratio of its two putative coordination isomers, lack the target C-H bond (l-Ala-S-SyrB1), or contain a C-H bond of enhanced strength (l-cyclopropylglycyl-S-SyrB1). For the latter two cases, the SyrB2 chloroferryl state so formed exhibits unprecedented stability (t(1/2) = 30-110 min at 0 degree C), can be trapped at high concentration and purity by manual freezing without a cryosolvent, and represents an ideal target for structural characterization. As initial steps toward this goal, extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the Fe-O and Fe-Cl distances and density functional theory (DFT) calculations have been used to confirm that the measured distances are consistent with the anticipated structure of the intermediate.

Project description:We present here a computational study of reactions at a model complex of the SyrB2 enzyme active site. SyrB2, which chlorinates L-threonine in the syringomycin biosynthetic pathway, belongs to a recently discovered class of alpha-ketoglutarate (alphaKG), non-heme Fe(II)-dependent halogenases that share many structural and chemical similarities with hydroxylases. Namely, halogenases and hydroxylases alike decarboxylate the alphaKG co-substrate, facilitating formation of a high-energy ferryl-oxo intermediate that abstracts a hydrogen from the reactant complex. The reaction mechanisms differ at this point, and mutation of active site residues (Asp for the hydroxylase to Ala or Ala to Asp/Glu for halogenase) fails to reproduce hydroxylating activity in SyrB2 or halogenating activity in similar hydroxylases. Using a density functional theory approach with a recently implemented Hubbard U correction for accurate treatment of transition-metal chemistry, we explore probable reaction pathways and mechanisms via a model complex consisting of only the iron center and its direct ligands. We show that the first step, alphaKG decarboxylation, is barrierless and exothermic, but the subsequent hydrogen abstraction step has an energetic barrier consistent with that accessible under biological conditions. In the model complex we use, radical chlorination is barrierless and exothermic, whereas the analogous hydroxylation is found to have a small energetic barrier. The hydrogen abstraction and radical chlorination steps are strongly coupled: the barrier for the hydrogen abstraction step is reduced when carried out concomitantly with the exothermic chlorination step. Our work suggests that the lack of chlorination in mutant hydroxylases is most likely due to poor binding of chlorine in the active site, whereas mutant halogenases do not hydroxylate for energetic reasons. Although secondary shell residues undoubtedly modulate the overall reactivity and binding of relevant substrates, we show that a small model compound consisting exclusively of the direct ligands to the metal can help explain reactivity heretofore not yet understood in the halogenase SyrB2.

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Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2.

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