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.

Fig. 1.

Absorbance (320 nm) versus time traces obtained after O₂-saturated reaction buffer (20 mM Hepes, pH 7.5) was mixed with an equal volume of an O₂-free solution containing SyrB2 (0.38–0.60 mM), Fe(II) (0.30 mM), αKG (10 mM), Cl⁻ (100 mM), and the indicated substrate [≥3 equiv or 0.90 mM; Nva (squares), 5-d₃-Nva (triangles), and 4,5-d₅-Nva (circles)]. Solid lines are fits to the data as previously described (16), giving parameters quoted in the text.

Fig. 2.

Representative, reconstructed mass spectra depicting the relative intensities of substrate and product iminium daughter ions obtained with Thr (A), Aba (B), and Nva (C) under the conditions defined along the z axis: rows 1–4, αKG dependence for the reaction with unlabeled substrate (concentrations of αKG displayed as equivalents relative to the substrate concentration); row 5, sample in which the unlabeled substrate was used as the standard and the deuterated substrate (2,3-d₂-Thr, 3-d₂-Aba, or 4,5-d₅-Nva) in the reaction; and row 6, reaction with the SyrB2-A118E variant. Color-coded icons show the structure of each daughter ion of the substrate (in blue), the labeled standard (in black), and the major products (hydroxylated in red and chlorinated in green) that could be unequivocally detected. The m/z values for each hydroxamate parent ion → iminium daughter ion transition are defined in Tables S1–S3. The fraction of the total peak intensity attributable to products that is contributed by each daughter ion is displayed below the appropriate icon. Note that these are not mole fractions. Note also that in C, the regiochemistry of the hydroxyl- and chloro-Nva products cannot be determined. Sample preparation and analysis are described in SI Text.

Fig. 3.

MS analysis of SyrB2 reactions with deuterated Nva substrates. (A) Representative, reconstructed mass spectra depicting the relative intensities of substrate and product daughter ions obtained with 4-d₂-Nva (4-d₂), 5-d₃-Nva (5-d₃), and 4,5-d₅-Nva (4,5-d₅) substrates in the absence and presence of αKG (equivalents of αKG relative to the substrate concentration are shown along the z axis). For each reaction, unlabeled Nva was used as the normalization standard, but its peak is not shown for esthetic purposes. Peaks correspond to the iminium daughter ions of each labeled substrate (blue), hydroxylated product (red), and chlorinated product (green). (B) Interpretation of the MS data in A in terms of relative intensities of peaks (labeled ①–⑧) that correspond to the reaction products for each substrate. Site of modification (chlorination or hydroxylation) in the daughter ions was inferred from whether H (1 atomic mass unit) or D (2 atomic mass units) was lost from the specifically deuterated Nva substrate. The m/z values for each hydroxamate parent ion → iminium daughter ion fragmentation are defined in Table S3. As for the reaction with unlabeled Nva in Fig. 1C, regiochemistry of the products in the 4,5-d₅-Nva reaction cannot be determined (C4 and C5 hydroxylated and chlorinated products shown in brackets). Sample preparation and analysis are described in SI Text.

Scheme 1.

Mechanistic and structural rationale for this study. (A) Proposed mechanisms for the last two steps in the reactions of αKG-dependent aliphatic hydroxylases (red) and halogenases (green). (B) Cartoon of active site with proposed positioning of Thr (blue) and Aba (red) substrates indicated by previously reported observations on the kinetics of chloroferryl intermediate decay (16).

Scheme 2.

Interpretation of the kinetic and mass spectral data with protium- and deuterium-containing Nva substrates. The rate constants for H^• abstraction are only estimates that are consistent with the observed rate constants for decay of the chloroferryl state in the presence of the various substrates. In particular, the rate constant for D^• abstraction from C4 is very poorly determined and could differ by as much as a factor of 3.