Enzymatic Hydroxylation of Aliphatic C-H Bonds by a Mn/Fe Cofactor

Abstract

The aerobic oxidation of carbon-hydrogen (C-H) bonds in biology is currently known to be accomplished by a limited set of cofactors that typically include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C-H bond activation, and those that do catalyze this important reaction display limited intrinsic reactivity. Here we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis, AibH1H2, requires manganese to functionalize a strong, aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site at the locus of O₂ activation and substrate coordination. This result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to single-electron transfer or stoichiometric protein oxidation. Furthermore, the AibH1H2 cofactor is supported by a protein fold distinct from typical bimetallic oxygenases, and bioinformatic analyses identify related proteins abundant in microorganisms. This suggests that many uncharacterized monooxygenases may similarly require manganese to perform oxidative biochemical tasks.

Figure 1

(A) The enzymatic reaction catalyzed by AibH1H2 and its active site cofactor described in this work. (B) Cartoon and surface representation of the AibH1H2 (αβ)₂ heterotetramer, made up of AibH1 (green) and AibH2 (blue) subunits alongside their respective metal binding sites.

Figure 2

Catalytic activity of 25 μM AibH1H2 under different metalation conditions. (A) GC-MS chromatograms filtered at m/z = 218 (Figures S2 and S3) of representative catalytic activity assays of AibH1H2 under different growth conditions with no additional metal added during the activity assay. (B) Catalytic activity of ^FeAibH1H2 and ^MnAibH1H2 (Table 1, entries 1 and 3, respectively) with one additional equivalent of metal or an equal volume of water (denoted with “–”), added to the reaction mixture. (C) Time-dependent generation of d-MeSer by ^Mn*AibH1H2 (black traces, Table 1 entry 4) and ^Fe*AibH1H2 (red traces, Table 1 entry 5) with one additional equivalent of Mn (dashed trace) or Fe (solid trace) added to the reaction mixture.

Figure 3

Characterization of the AibH2 Mn/Fe cofactor (PDB: 8FUN). Cartoon representations of the ^Mn*AibH1H2 protein structure at Site 0 (A), View 1 (B), and View 2 (C) of the dinuclear cofactor are shown overlaid with the anomalous density difference map at the Mn K-edge (black) and the dual-wavelength isomorphous anomalous density difference map near the Fe K-edge (yellow), each contoured at 5.0σ.

Scheme 1. Proposed Metalation Scheme of AibH1H2

Site 0 is depicted with a Mn^II ion to reflect the crystallographic data (PDB: 8FUN), but the physiological identity of the metal is unknown.

Figure 4

(a) High-resolution crystal structure of ^FeAibH1H2 crystallized in the presence of Tris. 2F_o – F_c electron density is contoured at 2.5σ and shown in gray mesh. (b) Continuous-wave X-band EPR spectra of ^MnAibH1H2 in 1 M Tris with one additional equivalent of natural abundance Fe (top, blue) or ⁵⁷Fe (bottom, purple) collected at 15 K and 20 mW power. (c) EPR spectra of ^MnAibH1H2 in CHES without (top, black) and with (bottom, red) 100 mM AIB taken at 12 K and 63.25 mW power. Simulations are shown in gray dashed lines. (d) The extracted components used to simulate the CHES/AIB sample. Component 1 has identical parameters to the 20 mM CHES samples without AIB, and Component 2 represents a new species. Refer to Table S2 for simulated spin Hamiltonian parameters. Tris = tris(hydroxymethyl)aminomethane, CHES = N-cyclohexyl-2-aminoethanesulfonic acid.

Figure 5

Bioinformatic identification of an uncharacterized family of AibH2-like proteins. (A) Unrooted, neighbor-joining phylogenetic tree of 555 Uniprot Reference Proteome sequences with at least 24% identity to AibH2 and conserved metal-coordinating residues. Biochemically characterized representatives of the PF04909 protein family were included for context. Branch thickness is proportional to the bootstrap values. The branches are colored according to the taxonomic group: red: actinobacteria; magenta: proteobacteria; blue: bacilli; orange: chloroflexi; yellow: eukaryote; teal: haloarchaea; green: cyanobacteria; black: other. The underlying shading reflects the enzymatic reactions (inset) known or expected to be catalyzed by these enzymes. Tree scale, 1.0 amino acid substitution per site. (B) Multiple sequence alignment of representative candidate monooxygenase sequences and characterized monometallic enzymes highlighting the metal binding residues found in AibH2. (C) Ribbon and surface representation of AibH1H2 illustrating the localization of variable (cyan) and conserved (purple) regions of 300 randomly chosen AibH2-like sequences. (D) Genome neighborhood diagrams of AibH2 and AibH2-like proteins. Genes are colored according to an inferred function: black: AibH2-like monooxygenase; red: Rieske-type ferredoxin; blue; small-molecule permease components.