Abstract
The cyanobacterial enzyme CylK assembles the cylindrocyclophane natural products by performing two unusual alkylation reactions, forming new carbon-carbon bonds between aromatic rings and secondary alkyl halide substrates. This transformation is unprecedented in biology, and the structure and mechanism of CylK are unknown. Here, we report X-ray crystal structures of CylK, revealing a distinctive fusion of a Ca2+-binding domain and a β-propeller fold. We use a mutagenic screening approach to locate CylK's active site at its domain interface, identifying two residues, Arg105 and Tyr473, that are required for catalysis. Anomalous diffraction datasets collected with bound bromide ions, a product analog, suggest that these residues interact with the alkyl halide electrophile. Additional mutagenesis and molecular dynamics simulations implicate Asp440 in activating the nucleophilic aromatic ring. Bioinformatic analysis of CylK homologs from other cyanobacteria establishes that they conserve these key catalytic amino acids, but they are likely associated with divergent reactivity and altered secondary metabolism. By gaining a molecular understanding of this unusual biosynthetic transformation, this work fills a gap in our understanding of how alkyl halides are activated and used by enzymes as biosynthetic intermediates, informing enzyme engineering, catalyst design, and natural product discovery.
Highlights
36 Introduction 38 Chemists utilize alkyl halides as key synthetic reagents because of their accessibility and favorable reactivity [1]; examples of their use as intermediates in biological systems are rare [2]
Natural products associated with select CylK homologs are displayed; the bonds highlighted in blue are constructed by their respective CylK. 373 Inspired by the biosynthetic logic of the cyl pathway, we looked for co-localized monoalkylresorcinol (MAR) forming enzymes encoded near putative CylK homologs in order to ascertain if resorcinols are likely to serve as their native nucleophilic substrates
The observation that the majority of CylK homologs do not have proposed alkyl chloride activating residues, partner halogenases, nor resorcinol forming enzymes, indicates that this enzyme family may catalyze other reactions not involving alkyl halides or resorcinols. This information will enable future studies by prioritizing organisms that might produce more distantly related natural products and/or CylK-like enzymes with alternate substrate scopes. 389 Discussion 392 Our results provide a structural basis for understanding the unusual enzymatic Friedel–Crafts alkylation 393 catalyzed by CylK in cylindrocyclophane biosynthesis
Summary
51 Interrogating cylindrocyclophane biosynthesis by the cyanobacterium Cylindrospermum licheniforme ATCC 29412 presents an exciting opportunity to study alkyl halide utilization in biological systems In this organism, the putative diiron carboxylate halogenase CylC generates an alkyl chloride intermediate which is further elaborated to produce resorcinol-containing alkyl chloride 1. Uncharacterized enzymes with homology to CylK are found in numerous cyanobacteria, one of which (BrtB) was recently revealed to catalyze carbon-oxygen (C–O) bond formation between the carboxylate groups of fatty acids and bartoloside A, an alkyl chloride-containing natural product (Fig. 1B) [9] This suggests that the mechanism by which CylK binds and activates alkyl chlorides might be shared with this and other homologs that use diverse nucleophilic substrates. Our structural information and mechanistic model will guide future enzyme engineering efforts, inform the design of non-enzymatic catalysts, and enable genome mining to uncover new natural products constructed by related biosynthetic strategies
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