Broadening substrate specificity of a chain-extending ketosynthase through a single active-site mutation
Royal Society of Chemistry
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Murphy, A., Hong, H., Vance, S., Broadhurst, B., & Leadlay, P. (2016). Broadening substrate specificity of a chain-extending ketosynthase through a single active-site mutation. Chemical Communications, 52 8373-8376. https://doi.org/10.1039/c6cc03501a
Type I modular polyketide synthase multienzymes, which catalyze the assembly-line biosynthesis of an impressive array of bioactive natural products, use a different $\beta$-ketoacyl-acyl carrier protein synthase domain for each cycle of chain elongation. Despite intensive study, the extent to which the KS domains determine the selectivity of chain extension remains poorly understood. The KS domains of the PKS for biosynthesis of the toxin mycolactone produced by Mycobacterium ulcerans appear uniquely permissive because they link diverse substrates yet they have essentially identical amino acid sequences. Using an in vitro model system based on the KS domain of module 3 of the erythromycin PKS, we have examined the effect of altering individual active site residues to their counterparts in the mycolactone PKS. While other mutations had little effect on the native selectivity of carbon-carbon bond formation, alteration of a specific alanine residue to tryptophan led to an emphatic increase in the turnover of a range of substrates. This residue lies at the boundary between the buried active site and a surface loop whose sequence is notably variable among PKS KS domains, and which has previously been implicated in the recognition of the acyl carrier protein substrates. These results encourage the view that the efficiency of engineered PKSs can be improved by targeted mutagenesis.
Wellcome Trust (094252/Z/10/Z)
External DOI: https://doi.org/10.1039/c6cc03501a
This record's URL: https://www.repository.cam.ac.uk/handle/1810/256351