Characterization and Biocatalytic Potential of an Enzymatic Friedel–Crafts Alkylation from Cylindrocyclophane Biosynthesis

Abstract

The cyanobacterial enzyme CylK assembles the cylindrocylophane natural products by performing two sequential Friedel–Crafts alkylation reactions, forming new alkyl–aryl carbon–carbon bonds between resorcinol nucleophiles and secondary alkyl chloride electrophiles. Other enzymatic transformations analogous to the Friedel–Crafts alkylation have been reported, however they require highly activated electrophilic substrates. Remarkably, the substitution reaction catalyzed by CylK occurs in a selective fashion with inversion of configuration at the alkyl chloride stereocenter. This transformation is one of only a few known biological examples of ‘cryptic halogenation’, a biosynthetic logic that produces a halogenated intermediate and leverages its increased reactivity to form a new bond, releasing the corresponding halide anion as a side product. Although multiple classes of halogenating enzymes are mechanistically characterized, the cognate halide-utilizing enzymes involved in cryptic halogenation are underexplored. Moreover, the reaction catalyzed by CylK is the only known biological example of aromatic ring alkylation with an alkyl halide electrophile, and broadly, structures and biochemical mechanisms for enzymatic activation of alkyl halide substrates as biosynthetic intermediates are unknown. Here, we describe our efforts to characterize CylK in order to evaluate its potential as a biocatalyst and to enhance our fundamental knowledge of this enzyme family. In Chapter 2 we describe our work to improve CylK enzyme preparations and to elucidate the substrate scope and selectivity of this reaction. These efforts were motivated by the biocatalytic potential of CylK and the desire to develop an understanding of mechanistic and substrate binding requirements that might influence substrate tolerance and reactivity. Our results reveal that CylK can mediate Friedel–Crafts alkylations with an array of non-biological substrate pairs while maintaining the exquisite regio- and stereoselectivity of the native biological reaction; however, CylK has a strict requirement for nucleophilic resorcinol substrates with free phenolic substituents. Subsequently, collaborators discovered that an enzyme homologous to CylK instead catalyzes carbon–oxygen bond formation between carboxylate nucleophiles and secondary alkyl chloride electrophiles, suggesting that while the broader enzyme family likely shares an alkyl halide activating strategy, CylK may have a distinct active site architecture that enables resorcinol engagement. In Chapter 3, in collaboration with the Boal Lab at Penn State University, we determine the structural basis for the CylK-catalyzed reaction in order to understand how it orients and activates resorcinol nucleophiles and alkyl halide electrophiles for stereospecific nucleophilic substitution. Our X-ray crystal structures reveal that CylK is a distinctive fusion of Ca2+-binding and β-propeller domains, and site-directed mutagenesis and anomalous diffraction with bound bromide ions locate the active site to the domain interface. This implicates hydrogen bonding interactions with Arg105 and Tyr473 in CylK’s alkyl halide activation strategy, and additional density functional theory and molecular dynamics modeling suggests that the nucleophilicity of resorcinol substrates is enhanced via deprotonation by Asp440. Notably, free halide anions do not inhibit the reaction catalyzed by CylK, suggesting that this enzyme precisely calibrates the concerted activation of both reaction partners. Bioinformatic analysis of the enzyme family establishes that these three key catalytic residues are conserved in homologous enzymes from other cyanobacteria that likely use additional nucleophilic substrates beyond resorcinols and carboxylates. Finally, in Chapter 4 we apply our knowledge of CylK’s structure and substrate scope to begin preliminary protein engineering efforts. Motivated by the therapeutic potential of alkylated stilbenes accessed in trace quantities with wild-type CylK, we develop screening methodology for enzyme evolution that leverages the putative biological activity of the CylK reaction products. This strategy, dubbed bioactivity-guided directed evolution, seeks to enable biocatalyst development in parallel with optimization of biological activity. Additionally, we construct libraries of CylK mutants and identify two enhanced variants that may serve as starting points for additional rounds of mutagenesis and screening. Future efforts are focused on applying insights from our structural, mechanistic, and bioinformatic work to guide protein engineering, natural product discovery, and further enzyme characterization.