Identifying and Engineering Flavin Dependent Halogenases for Selective Biocatalysis

Abstract

Organohalogen compounds are extensively used as building blocks, intermediates, pharmaceuticals, and agrochemicals due to their unique chemical and biological properties. Installing halogen substituents, however, frequently requires functionalized starting materials and multistep functional group interconversion. Several classes of halogenases evolved in nature to enable halogenation of a different classes of substrates; for example, site-selective halogenation of electron rich aromatic compounds is catalyzed by flavin-dependent halogenases (FDHs). Mechanistic studies have shown that these enzymes use FADH₂ to reduce O₂ to water with concomitant oxidation of X^- to HOX (X = Cl, Br, I). This species travels through a tunnel within the enzyme to access the FDH active site. Here, it is believed to interact with an active site lysine proximal to bound substrate, enabling electrophilic halogenation with selectivity imparted via molecular recognition, rather than directing groups or strong electronic activation.The unique selectivity of FDHs led to several early biocatalysis efforts, preparative halogenation was rare, and the hallmark catalyst-controlled selectivity of FDHs did not translate to non-native substrates. FDH engineering was limited to site-directed mutagenesis, which resulted in modest changes in site-selectivity or substrate preference. To address these limitations, we optimized expression conditions for the FDH RebH and its cognate flavin reductase (FRed), RebF. We then showed that RebH could be used for preparative halogenation of non-native substrates with catalyst-controlled selectivity. We reported the first examples in which the stability, substrate scope, and site selectivity of a FDH were improved to synthetically useful levels via directed evolution. X-ray crystal structures of evolved FDHs and reversion mutations showed that random mutations throughout the RebH structure were critical to achieving high levels of activity and selectivity on diverse aromatic substrates, and these data were used in combination with molecular dynamics simulations to develop predictive model for FDH selectivity. Finally, we used family wide genome mining to identify a diverse set of FDHs with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds.The diversity of our evolved and mined FDHs allowed us to pursue synthetic applications beyond simple aromatic halogenation. For example, we established that FDHs catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. These results highlight the ability of FDH active sites to tolerate different substrate topologies. This utility was further expanded by our recent studies on the single component FDH/FRed, AetF. While we were initially drawn to AetF because it does not require a separate FRed, we found that it halogenates substrates that are not halogenated efficiently or at all by other FDHs and provides high enantioselectivity for reactions that could only be achieved using RebH variants after extensive mutagenesis. Perhaps most notably, AetF catalyzes site-selective aromatic iodination and enantioselective iodoetherification. Together, these studies highlight the origins of FDH engineering, the utility and limitations of the enzymes developed to date, and the promise of FDHs for an ever-expanding range of biocatalytic halogenation reactions.

Figure 1.

Enzyme catalysis proceeding via (A) substrate activation vs (B) reactive intermediate formation. (C) Simplified mechanism of FDH catalysis.

Figure 2.

(A) Representative RebH substrate scope with isolated yields. ^aThe cofactor regeneration system consists of 0.5 mol % RebF and 50 U mL⁻¹ glucose dehydrogenase. ^b10 mol % RebH loading was used. A nearly 1:1 mixture of 5- and 6-halogenation was observed. (B) Preparative chlorination using RebH in cell lysate.

Figure 3.

(A) CD melting curves for evolved RebH variants. (B) Improved tryptophan chlorination yields for evolved RebH variants at increased temperatures. (C) Improved yields of substrates using stabilized RebH variants.

Figure 4.

(A) Substrates used for evolution of RebH substrate scope. (B) Representative substrate scope of evolved RebH variants with isolated yields.

Figure 5.

(A/B) MALDI-MS screen for altered site selectivity using deuterated probe substrates 8 and 9 (not shown). (C) Yields and site selectivities of evolved RebH variants 0S, 8F, and 10S.

Figure 6.

(A) Chemoenzymatic halogenation/cross-coupling. (B) Representative substrate scope of FDHs from activity profiling with “x” noting electronically activated sites that are not halogenated.

Figure 7.

Treemap of FDH sequence space showing (A) reported substrate preferences, (B) characterized and mined sequences, (C) enzyme solubility, (D/E) chlorination and bromination activity. (F) Representative scope of mined FDHs.

Figure 8.

Representative substrate scope for enantioselective FDH catalysis involving (A) desymmetrization and (B) atroposelective halogenation.

Figure 9.

(A) Halocyclization of 14 catalyzed by engineered and mined FDHs. (B) High pH or glutathione increases the e.r. for halocyclization of 14 by 4V+S. Representative products for halocyclization involving (C) carboxylate nucleophiles and (D) alcohol nucleophiles.

Figure 10.

(A) Native products of the single component FDHs Bmp5 and AetF. (B) Representative substrate scope of AetF. (C/D) Selective alkene and alkyne halogenation by AetF.

Figure 11.

Structure of RebH (PDB ID 2OA1) with C_α of mutated residues shown as spheres and colored based on (A) goal of directed evolution campaign in which mutations were identified or (B) distance (d) from bound tryptophan.