Hibi Makoto, Mori Ryosuke, Miyake Ryoma, Kawabata Hiroshi, Kozono Shoko, Takahashi Satomi, Ogawa Jun
Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Appl Environ Microbiol. 2016 Jan 22;82(7):2070-2077. doi: 10.1128/AEM.03764-15.
Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward L-pipecolic acid (L-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme L-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of L-Pip, and produces optically pure trans-4-hydroxy-L-pipecolic acid (trans-4-L-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had L-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the L and D forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-L-HyPip was successfully developed. Thus, we report a fungal family of L-Pip hydroxylases and the enzymatic preparation of trans-4-L-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.
羟基哌啶酸是广泛分布于自然界的生物活性化合物，是药物有机合成中有价值的构建模块。我们在丝状真菌尖孢镰刀菌c8D中发现了一种对L-哌啶酸（L-Pip）具有活性的新型羟化酶。尖孢镰刀菌的L-Pip反式-4-羟化酶（Pip4H）（FoPip4H）属于依赖Fe（II）/α-酮戊二酸的双加氧酶超家族，催化L-Pip的区域和立体选择性羟化反应，并产生光学纯的反式-4-羟基-L-哌啶酸（反式-4-L-HyPip）。氨基酸序列分析揭示了几种与FoPip4H同源的真菌酶，其中五种也具有L-Pip反式-4-羟化活性。特别是，源自构巢曲霉FGSC A4（AnPip4H）的同源Pip4H酶比其他同源物具有更广泛的底物特异性谱，并且能与各种环状和脂肪族氨基酸的L型和D型发生反应。以FoPip4H作为生物催化剂，成功开发了一种用于制备规模生产手性反式-4-L-HyPip的系统。因此，我们报道了一个L-Pip羟化酶的真菌家族以及反式-4-L-HyPip的酶促制备方法，反式-4-L-HyPip是一种生物活性化合物，也是具有有用生理活性的次生代谢产物的组成成分。
