Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037, Marburg, Germany.
Appl Microbiol Biotechnol. 2014 Dec;98(24):10119-29. doi: 10.1007/s00253-014-5872-7. Epub 2014 Jun 28.
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.
在黑曲霉基因组序列中鉴定到一个假定的二甲基烯丙基色氨酸合酶（DMATS）家族的prenyltransferase 基因，An13g01840。推导的多肽 CAK41583 由 465 个氨基酸组成，计算分子量为 52.7 kDa。为了评估基因功能，将编码序列克隆到 pET28a 中，并在大肠杆菌中过表达。在二甲基烯丙基二磷酸存在的情况下，用 Ni-NTA 琼脂糖纯化可溶性 His6-融合蛋白，并用各种芳香族底物进行酶测定。HPLC 分析显示在与 L-酪氨酸及其五种衍生物的孵育混合物中形成产物。通过 NMR 和 MS 分析对酶产物的结构阐明证实了 O-prenylations，并证明了酪氨酸 O-prenyltransferase（TyrPT）的鉴定。与 Leptosphaeria maculans 中的 SirD 一样，TyrPT 也接受 4-氨基-L-苯丙氨酸进行 N-prenylation 和 L-色氨酸进行 C7-prenylation。TyrPT 对 L-酪氨酸、L-色氨酸和二甲基烯丙基二磷酸（DMAPP）的 K M 值分别为 0.24、0.19 和 0.71 mM。L-酪氨酸和 L-色氨酸反应的 k cat 分别为 0.58 和 0.0053 s(-1)。本研究中提出的结果增强了酪氨酸 O-和色氨酸 C7-prenyltranferases 的关系，同时为制备prenylated 衍生物提供了一种新的酶。与已知的酪氨酸 prenyltransferase SirD 相比，TyrPT 对几种底物（例如 4-氨基-L-苯丙氨酸以及 4-和 5-甲基-DL-色氨酸）的催化活性明显更高。
