Chloro-methoxy-tryptophan biogenesis during NRP biosynthesis Michael Brigham , Daniel C.L. Van, Glyn R. Hemsworth, Ryan F. Seipke University of Leeds, UK Non-ribosomal peptides often possess modified amino acids and, when present, these moieties are often essential for compound activity. In most cases, the precursor biosynthetic pathways are co-encoded within the NRPS biosynthetic gene cluster (BGC) and overall are mechanistically relatively unexplored. Characterisation of such precursor pathways is essential for a holistic understanding of non-ribosomal peptide biology, which is required for achieving the field’s wider aim of end-to-end pathway engineering. Here we identified and characterised a cluster-encoded precursor biogenesis system (BifEFKI) that makes free chloro-methoxy- tryptophan. Detailed bioinformatics and de novo modelling defined the functions of BifEF as a haem-dependent tryptophan hydroxylase and SAM-dependent methylase, respectively, while BifK is a flavin-dependent tryptophan halogenase. BifI is a flavin-reductase responsible for co-factor regeneration for BifK. Overproduction of all four enzymes in a streptomycete host generated chloro-methoxy-tryptophan and a pathway intermediate, methoxy-tryptophan, which were characterised by LC-MSMS and NMR. Interestingly, not even trace quantities of chloro-tryptophan were detected in this experiment, suggesting that the BifK halogenase may act after BifEF have installed the methoxy moiety and possesses a tight substrate specificity for methoxy-tryptophan. Given the sequential action of BifE and BifF during the installation of the methoxy group, we hypothesised these two enzymes may form a complex. This hypothesis was supported by AlphaFold 2.0 models of each enzyme revealing BifE possesses a distinct, additional domain compared to other structurally characterised tryptophan dioxygenases. To assess this hypothetical BifE-BifF complex, an in vitro pulldown assay followed by analytical size exclusion chromatography and native-PAGE is currently in development. Preliminary results support formation of a heterodimeric complex. These data have established biological production of chloro-methoxy- tryptophan for the first time and is indicative of a unique biotransformation complex being formed for installation of the methoxy moiety. Future experiments are aimed at characterising the structural molecular biology of the system.
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© The Author(s), 2022
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