Abstract
A putative brevianamide F reverse prenyltransferase gene brePT was amplified from Aspergillus versicolor NRRL573 by using primers deduced from its orthologue notF in Aspergillus sp. MF297-2 and overexpressed in Escherichia coli. The soluble His-tagged protein BrePT was purified to near homogeneity and assayed with tryptophan-containing cyclic dipeptides in the presence of dimethylallyl diphosphate. BrePT showed much higher flexibility towards its aromatic substrates than NotF and accepted all of the 14 tested tryptophan-containing cyclic dipeptides. Structure elucidation of the enzyme products by NMR and MS analyses proved unequivocally the highly regiospecific reverse prenylation at C2 of the indole nucleus. K M values of BrePT were determined for its putative substrates brevianamide F and DMAPP at 32 and 98 μM, respectively. Average turnover number (k cat) at 0.4 s−1 was calculated from kinetic data of brevianamide F and DMAPP. K M values in the range of 0.082–2.9 mM and k cat values from 0.003 to 0.15 s−1 were determined for other 11 cyclic dipeptides. Similar to known fungal indole prenyltransferases, BrePT did not accept geranyl or farnesyl diphosphate as prenyl donor for its prenylation.
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Acknowledgments
This work was financially supported in part by grants from the Deutsche Forschungsgemeinschaft (Li844/1-3 to S.-M. Li) and China Natural Science Foundation (31070067 to X.-Q. Liu). Xia Yu is a recipient of a fellowship from China Scholarship Council.
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Suqin Yin and Xia Yu contributed equally to this work.
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Yin, S., Yu, X., Wang, Q. et al. Identification of a brevianamide F reverse prenyltransferase BrePT from Aspergillus versicolor with a broad substrate specificity towards tryptophan-containing cyclic dipeptides. Appl Microbiol Biotechnol 97, 1649–1660 (2013). https://doi.org/10.1007/s00253-012-4130-0
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DOI: https://doi.org/10.1007/s00253-012-4130-0