Structural basis for an unprecedented enzymatic alkylation in cylindrocyclophane biosynthesis
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Curated by eLife
Evaluation Summary:
The de novo crystal structure of the cyanobacterial enzyme CylK, which assembles cylindrocyclophane natural products, is reported. The substrate-binding site and critical catalytic residues were identified through a combination of anion soaking, mutagenesis, molecular dynamic simulations. The insights from this work are relevant in understanding biological Friedel-Crafts alkylation and also in enzyme engineering and catalyst designs. This is a very comprehensive study that provides new mechanistic insights for this enzyme and it will be of interest to all who are involved in enzyme engineering, catalyst design, and natural product discovery.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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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 Ca 2+ -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.
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Evaluation Summary:
The de novo crystal structure of the cyanobacterial enzyme CylK, which assembles cylindrocyclophane natural products, is reported. The substrate-binding site and critical catalytic residues were identified through a combination of anion soaking, mutagenesis, molecular dynamic simulations. The insights from this work are relevant in understanding biological Friedel-Crafts alkylation and also in enzyme engineering and catalyst designs. This is a very comprehensive study that provides new mechanistic insights for this enzyme and it will be of interest to all who are involved in enzyme engineering, catalyst design, and natural product discovery.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the …
Evaluation Summary:
The de novo crystal structure of the cyanobacterial enzyme CylK, which assembles cylindrocyclophane natural products, is reported. The substrate-binding site and critical catalytic residues were identified through a combination of anion soaking, mutagenesis, molecular dynamic simulations. The insights from this work are relevant in understanding biological Friedel-Crafts alkylation and also in enzyme engineering and catalyst designs. This is a very comprehensive study that provides new mechanistic insights for this enzyme and it will be of interest to all who are involved in enzyme engineering, catalyst design, and natural product discovery.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors present the structure characterization of the unusual enzymatic Friedel-Crafts alkylation catalyzed by CylK in cylindrocyclophane biosynthesis. Based on the mutagenic screening, anomalous diffraction datasets with Bromide ions and molecular dynamic simulation, they identified the key residues for catalysis, and proposed an activation mechanism. The bioinformatics analysis of cylk homologues from other cyanobacteria indicates the preservation of key catalytic amino acids. The study is infomative for enzyme engineering, catalyst design, and natural product discovery.
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Reviewer #2 (Public Review):
CylK is an enzyme with unusual Friedel-Crafts alkylation activity. Its 3D structure was determined by MR-SAD using the terbium(III) chloride soaking method and revealed a two-domain architecture. Although the substrate-bound structure was not feasible, the authors did not stop there and tried to locate the substrate chloride and hydroxyl group binding positions in a mechanistic sense by NaBr soaking. This effort resulted in identifying a putative substrate-binding site, which was further supported by mutant activity screens and the molecular dynamics simulation. Placing the active site at its N- and C-terminal domains interface is convincing and inspiring. Moreover, the CylK structure with mutagenesis study also identified the key residues Asp440 and Arg105 for the catalytic activity at the interface between …
Reviewer #2 (Public Review):
CylK is an enzyme with unusual Friedel-Crafts alkylation activity. Its 3D structure was determined by MR-SAD using the terbium(III) chloride soaking method and revealed a two-domain architecture. Although the substrate-bound structure was not feasible, the authors did not stop there and tried to locate the substrate chloride and hydroxyl group binding positions in a mechanistic sense by NaBr soaking. This effort resulted in identifying a putative substrate-binding site, which was further supported by mutant activity screens and the molecular dynamics simulation. Placing the active site at its N- and C-terminal domains interface is convincing and inspiring. Moreover, the CylK structure with mutagenesis study also identified the key residues Asp440 and Arg105 for the catalytic activity at the interface between Ca(II)-binding and beta-propeller domains.
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