Abstract
A putative prenyltransferase gene of the dimethylallyltryptophan synthase (DMATS) family, An13g01840, was identified in the genome sequence of Aspergillus niger. The deduced polypeptide CAK41583 consists of 465 amino acids with a calculated molecular mass of 52.7 kDa. To evaluate gene function, the coding sequence was cloned into pET28a and overexpressed in Escherichia coli. The soluble His6-fusion protein was purified to near homogeneity on Ni-NTA agarose and used for enzyme assays with diverse aromatic substrates in the presence of dimethylallyl diphosphate. HPLC analysis revealed product formation in the incubation mixtures with l-tyrosine and five derivatives thereof. Structure elucidation of the enzyme products by NMR and MS analyses confirmed O-prenylations and proved the identification of a tyrosine O-prenyltransferase (TyrPT). As in the case of SirD from Leptosphaeria maculans, TyrPT also accepted 4-amino-l-phenylalanine for an N-prenylation and l-tryptophan for a C7-prenylation. The K M values of TyrPT for l-tyrosine, l-tryptophan, and dimethylallyl diphosphate (DMAPP) were found to be 0.24, 0.19, and 0.71 mM, respectively. The k cat of l-tyrosine and l-tryptophan reactions were determined at 0.58 and 0.0053 s−1, respectively. The results presented in this study enhance the relationship of tyrosine O- and tryptophan C7-prenyltranferases and provide meanwhile a new enzyme for production of prenylated derivatives. In comparison to the known tyrosine prenyltransferase SirD, TyrPT showed significantly higher catalytic activity for several substrates, e.g., 4-amino-l-phenylalanine as well as 4- and 5-methyl-DL-tryptophan.
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Acknowledgments
We thank Prof. Michael Müller (Freiburg, Germany) for providing the A. niger strain, Lena Ludwig for synthesis of DMAPP, and Nina Zitzer and Stefan Newel for taking MS and NMR spectra, respectively. This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Li844/4-1 to S.-M. L.) and by a PPP program of Deutscher Akademischer Austauschdienst and China scholarship council (to S.-M. L. and H.X.). Aili Fan is a recipient of a scholarship from China scholarship council.
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Fan, A., Chen, H., Wu, R. et al. A new member of the DMATS superfamily from Aspergillus niger catalyzes prenylations of both tyrosine and tryptophan derivatives. Appl Microbiol Biotechnol 98, 10119–10129 (2014). https://doi.org/10.1007/s00253-014-5872-7
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DOI: https://doi.org/10.1007/s00253-014-5872-7