Probing diverse β-branching mechanisms in polyketide biosynthesis with in vitro state-of-the-art NMR assays Annabel Phillips, Christine L. Willis, Matthew P. Crump University of Bristol, UK
The incorporation of functionalised β-branches is a key route to structural complexity in polyketide biosynthesis. 1 This modification of a β-ketothioester intermediate is carried out by a set of discrete, trans -acting enzymes known collectively as a 3-hydroxy-3-methylglutaryl synthase (HMGS) cassette and necessitates finely-tuned recruitment of cis - and trans -acting components to prevent anomalous branching. 2 The variety of building blocks and additional tailoring domains that can be recruited in β-branching pathways has resulted in a diverse range of functionalities present in many bioactive polyketides. The molecular diversification introduced by an HMGS cassette provides an exciting opportunity for rational engineering to produce novel polyketides, which requires a greater understanding of β-branching mechanisms. 3 Many systems that install β-branches have unusual features that warrant detailed investigation, including modified HMGS cassettes, atypical acyl carrier proteins (ACPs) and intriguing tailoring enzymes. However, the multiple interactions between numerous in- cis and in- trans components requires advanced methods to analyse this complex interplay. An in vitro assay has therefore been developed that utilises state-of-the-art NMR techniques to monitor ACP-bound intermediates at atomic resolution in β-branching pathways. 4 Through the synthesis of substrate mimics with site-specific incorporation of a [ 13 C] label and subsequent ACP loading, protein-protein interactions and multi-enzyme cascades can be delineated by tracking a 1 H- 13 C cross-peak. The power of this technique has most recently been applied to analysing β-branch programming in the leinamycin biosynthetic pathway, in which the selectivity of unusual modular β-branching ACPs with HMGS constituents has been probed. Critically, this work demonstrates the extensive applications of this assay, particularly to elucidate the molecular features responsible for intricate β-branch incorporation, and could contribute to polyketide synthase reprogramming to produce desirable bioactive compounds.
References 1. P. D. Walker, A. N. M. Weir, C. L. Willis and M. P. Crump, Nat. Prod. Rep. , 2021, 38 , 723-756. 2. C. T. Calderone, W. E. Kowtoniuk, N. L. Kelleher, C. T. Walsh and P. C. Dorrestein, Proc. Natl. Acad. Sci. U. S. A. , 2006, 103 , 8977-8982. 3. M. Till and P. R. Race, Biotechnol. Lett. , 2014, 36 , 877-888. 4. P. D. Walker, C. Williams, A. N. M. Weir, L. Wang, J. Crosby, P. R. Race, T. J. Simpson, C. L. Willis and M. P. Crump, Angew. Chem., Int. Ed. , 2019, 58 , 12446-12450.
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