Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase.

backgroundCreating designer molecules using a combination of select domains from polyketide synthases and/or nonribosomal peptide synthetases (NRPS) continues to be a synthetic goal. However, an incomplete understanding of how protein-protein interactions and dynamics affect each of the domain functions stands as a major obstacle in the field. Of particular interest is understanding the basis for a class of methyltransferase domains (MT) that are found embedded within the adenylation domain (A) of fungal NRPS systems instead of in an end-to-end architecture.

resultsThe MT domain from bassianolide synthetase (BSLS) was removed and the truncated enzyme BSLS-ΔMT was recombinantly expressed. The biosynthesis of bassianolide was abolished and

conclusionsThe embedding of MT into the A2 domain of BSLS is not required for the product assembly, but is critical for the overall yields of the final products. The substrate specificity of MT is significantly affected by the protein context within which it is present. While A domains are known to be responsible for selecting and activating the biosynthetic precursors for NRPS systems, our results suggest that embedding the MT acts as a secondary gatekeeper for the assembly line. This work thus provides new insights into the embedded MT domain in NRPSs, which will facilitate further engineering of this type of biosynthetic machinery to create structural diversity in natural products.