Using Chemical Knowledge to Uncover New Biological Function: Discovery of the Cylindrocyclophane Biosynthetic Pathway

Abstract

An understanding of organic chemistry can play a central role in uncovering enzymes with new biochemical functions. We have recently identified the enzymes involved in the biosynthesis of the cylindrocyclophanes, a structurally unique family of natural products, and found that this pathway employs a remarkable macrocyclization event that requires functionalization of an unactivated carbon atom. This work illustrates the potential of using chemically guided approaches for enzyme discovery.

Keywords: Biosynthesis; cylindrocyclophanes; enzymes; genomics; natural products.

Scheme 1

Chemically guided approaches for discovering and understanding biological function in microbial genome sequencing data.

Scheme 2

Isolation and total syntheses of the cylindrocyclophanes. A. Structures of the cylindrocyclophanes isolated from C. licheniforme. B. Strategies employed in previous cylindrocyclophane total syntheses.

Scheme 3

Previous studies of cylindrocyclophane biosynthesis by Bobzin and Moore. A. Feeding experiments with isotopically labeled sodium acetate reveal the polyketide origin of the natural products. B. Biosynthetic hypothesis for macrocycle formation.

Scheme 4

Identification of the cylindrocyclophane biosynthetic pathway A. HMG-CoA synthase homologs install β-methyl substituents onto polyketide scaffolds. B. Strategy for identifying the cylindrocyclophane (cyl) biosynthetic gene cluster in C. licheniforme genome sequencing data.

Scheme 5

Biosynthetic hypothesis for cylindrocyclophane assembly.

Scheme 6

The in vitro activities of the cyl enzymes support their involvement in cylindrocyclophane production A. Biochemical characterizations of fatty acid activating enzymes CylA and CylB. B. Biochemical characterization of type III PKS Cyll. C. Proposed mechanism for Cyll-catalyzed formation of resorcinol 9.

Scheme 7.

Feeding d₁₉-decanoic acid to C. licheniforme ATCC 29412 confirms its role as a biosynthetic precursor.

Scheme 8

Understanding the macrocyclization event in cylindrocyclophane biosynthesis. A. Possible biosynthetic logic employed in macrocyclization. B. Potentially related direct oxidative C–C bond formations.