Site-Selective C-H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling

Abstract

Enzymes are powerful catalysts for site-selective C-H bond functionalization. Identifying suitable enzymes for this task and for biocatalysis in general remains challenging, however, due to the fundamental difficulty of predicting catalytic activity from sequence information. In this study, family-wide activity profiling was used to obtain sequence-function information on flavin-dependent halogenases (FDHs). This broad survey provided a number of insights into FDH activity, including halide specificity and substrate preference, that were not apparent from the more focused studies reported to date. Regions of FDH sequence space that are most likely to contain enzymes suitable for halogenating small-molecule substrates were also identified. FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds were characterized and used for preparative-scale late-stage C-H functionalization. In many cases, these enzymes provide activities that required several rounds of directed evolution to accomplish in previous efforts, highlighting that this approach can achieve significant time savings for biocatalyst identification and provide advanced starting points for further evolution.

Figure 1

(A) Sequence-similarity network for flavin-dependent halogenases. Each circle is a representative node, grouping protein sequences with >50% sequence identity as determined by CD-HIT. Edge detection threshold set at alignment score of 70 (≈30% sequence identity). Nodes are filled according to native substrate functional group of at least one sequence in the representative node; colored stroke indicates domain (thin black stroke = bacterial). Subnetworks with ≥15 sequences but without any characterized protein are labeled numerically. Level 2 subnetworks formed from the Indole (B) and Phenol (C) subnetwork using a stricter alignment score cutoff of 140 (≈40% sequence identity). Level 2 subnetworks are labeled based on known sequences in the subnetwork. For Indole Subnetwork sequences, nodes containing known tryptophan halogenases are filled according to their regioselectivity, and subnetworks with ≥15 sequences are labeled numerically. For Phenol Subnetwork sequences, nodes are filled according to the halogenase variant type (A = free small molecule native substrate, B = ACP-tethered native substrate).

Figure 2

(A) Sequence-similarity network for flavin-dependent halogenases, drawn at the less stringent edge detection threshold (≈30% identity), colored according to subnetwork. Subnetworks within the Indole and Phenol Subnetworks at the more stringent threshold are colored differently. Subnetworks with fewer than 15 members are colored white; subnetworks without sequences of known or inferred function are colored light gray. (B) Treemap illustrating the SSN with the same coloring as part A. (C) Treemap comparing FDHs previously studied as biocatalysts with FDHs investigated in this study. (D) Treemap illustrating solubility of genome-mined enzymes in each subnetwork of the SSN. Color gradient represents the fraction of enzymes within the subnetwork that was soluble; diagonal bars indicate subnetworks wherein no enzyme was tested. Treemaps illustrating the fraction of enzymes in each subnetwork that were capable of chlorinating (E) or brominating (F) at least one substrate in the high-throughput screen (8% conversion threshold).

Figure 3

(A) Probe substrates included in initial high-throughput screen. (B) Scheme summarizing LC-MS-based high-throughput screening method employed.

Figure 4

Heatmap of high-throughput screening results, with hierarchical clustering dendrograms for substrate/halide activity similarity and enzyme activity similarity at the top and left, respectively. Substrate functional groups and halide used in the reaction are color-coded with bars at the tips of the dendrograms. Only reactions with >8% conversion, a value selected that removed false positives (see the SI).

Figure 5

(A) Representative compounds included in expanded high-throughput substrate screen, each of which was halogenated by at least one genome-mined FDH. (B) Heatmap of expanded substrate screen data with 10 of the most active enzymes from the probe high-throughput substrate screen.

Figure 6

Preparative-scale bioconversions of larger substrates.

Figure 7

Regiocomplementary halogenation of large molecules.

Figure 8

(A) Comparison of isolated RebH and 1-F11 protein yields after Ni-NTA purification from 50 mL expression cultures. (B) Comparison of CD thermal melts of RebH, thermostable RebH variant 3-LSR, and genome-mined halogenase 1-F11. Curves shown are the best fit for thermal unfolding monitored at 222 nm using CDPal. (C) Wild-type RebH required several rounds of directed evolution before yohimbine halogenation was detectable. Halogenase 1-F11 can halogenate yohimbine without directed evolution (HPLC conversion shown).