Investigation of the release and cyclisation steps from the PKS- NRPS hybrid during the biosynthesis of cytochalasans Henrike Heinemann University of Hanover, Germany Pyrichalasin H belongs to the cytochalasans, a broad family of fungal secondary metabolites with a wide range of biological activities. The early construction steps during the biosynthesis of pyrichalasin H, involving methyl tyrosine production and the action of a PKS-NRPS hybrid have been revealed by our group. Similarly, the later tailoring steps have been shown to involve parallel oxidative transformations. However, three central steps, involving reductive release, Knoevenagel cyclisation and Diels Alder cyclisation are not yet elucidated at a detailed molecular level. An N-terminal reductive release domain of the PKS-NRPS hybrid, marks the first step in this process. The proposed function of the reductase domain is to release an aldehyde from a thiolester intermediate by a two- electron reduction. However, this hypothesis is not yet confirmed in vitro . To answer this question, we investigated the thiolation (T) and reductase (R) domains of the PKS-NRPS hybrid in vitro . In chemical and chemo-enzymatic approaches, synthetic thiolesters were generated and used as substrate mimics for in vitro assays. These assays demonstrated that the R-domain is only able to process substrates that are tethered to the T-domain, but it cannot accept free substrates (SNAC and (phospho-)pantetheine derivates). In addition, the experiments reveal an intrinsic substrate specificity of the R-domain. The NADPH dependent release from the thiolation domain occurs without interplay of other enzymes of the pathway and forms an aldehyde intermediate which quickly undergoes Knoevenagle cyclisation spontaneously. These results give detailed insights into the reductive release during the biosynthesis of cytochalasans for the first time and confirm the aldehyde as a pathway intermediate in vitro . In future, this builts a platform for investigations of the following cyclization steps. References 1. Wang, C., Becker, K., Pfu, S., Kuhnert, E., Stadler, M., Cox, R. J., & Skellam, E. (2019). Investigating the Function of Cryptic Cytochalasan Cytochrome P450 Monooxygenases Using Combinatorial Biosynthesis . https://doi.org/10.1021/acs. orglett.9b03372 2. Wang, C., Hantke, V., Cox, R. J., & Skellam, E. (2019). Targeted Gene Inactivations Expose Silent Cytochalasans in Magnaporthe grisea NI980. Organic Letters . https://doi.org/10.1021/acs.orglett.9b01344 3. Hantke, V., Skellam, E. J., & Cox, R. J. (2020). Evidence for enzyme catalysed intramolecular [4+2] Diels-Alder cyclization during the biosynthesis of pyrichalasin H. Chemical Communications (Cambridge, England) , 56 (19), 2925–2928. https://doi. org/10.1039/c9cc09590j 4. Zhang, H., Hantke, V., Bruhnke, P., Skellam, E. J., & Cox, R. J. (2021). Chemical and Genetic Studies on the Formation of Pyrrolones During the Biosynthesis of Cytochalasans. Chemistry - A European Journal , 27 (9), 3106–3113. https://doi. org/10.1002/chem.202004444 5. Zhang, J. M., Liu, X., Wei, Q., Ma, C., Li, D., & Zou, Y. (2022). Berberine bridge enzyme-like oxidase-catalysed double bond isomerization acts as the pathway switch in cytochalasin synthesis. Nature Communications , 13 (1). https://doi.org/10.1038/ s41467-021-27931-z
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