Characterization of three novel enzymes with imine reductase activity
Characterization of three novel enzymes with imine reductase activity
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DOI：
10.1016/j.molcatb.2014.09.017
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发表时间：
2014-12-01
影响因子：
--
通讯作者：
Hohne, M.
中科院分区：
文献类型：
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作者：
Gand, M.;Muller, H.;Hohne, M.
亚胺还原酶（IRED）是合成光学纯仲环胺的理想催化剂。通过氨基酸序列或结构相似性搜索，从类芽孢杆菌（PaenibacilluseleveniB 69）、番茄链霉菌（Streptomycesipomoeae）91-03和假单胞菌（Pseudomonasputida）KT 2440中鉴定出3个新的IRED，克隆并在E.大肠杆菌中的表达及其底物范围进行了研究。除了接受环胺，也可以确定为所有IRED的底物的非环胺。对于来自恶臭假单胞菌的IRED，晶体结构（PDB-代码3L 6D）可在数据库中获得，但迄今为止尚未研究蛋白质的功能。对于本研究中研究的IRED的（R）-甲基吡咯烷，该酶显示出最高的表观E值，约为E-app=52。因此，在使用静息细胞的生物催化反应中，24小时后达到>99%的优异对映体纯度和97%的转化率。有趣的是，组氨酸残基可以通过诱变确认为催化残基，但与卡那霉素链霉菌IRED的催化Asp 187的正式已知位置相比，该残基被放置在一侧。(C)2014爱思唯尔有限公司版权所有。
Imine reductases (IRED) are promising catalysts for the synthesis of optically pure secondary cyclic amines. Three novel IREDs from Paenibacillus elgii B69, Streptomyces ipomoeae 91-03 and Pseudomonasputida KT2440 were identified by amino acid or structural similarity search, cloned and recombinantly expressed in E. coli and their substrate scope was investigated. Besides the acceptance of cyclic amines, also acyclic amines could be identified as substrates for all IREDs. For the IRED from P. putida, a crystal structure (PDB-code 3L6D) is available in the database, but the function of the protein was not investigated so far. This enzyme showed the highest apparent E-value of approximately E-app=52 for (R)-methylpyrrolidine of the IREDs investigated in this study. Thus, an excellent enantiomeric purity of >99% and 97% conversion was reached in a biocatalytic reaction using resting cells after 24 h. Interestingly, a histidine residue could be confirmed as a catalytic residue by mutagenesis, but the residue is placed one turn aside compared to the formally known position of the catalytic Asp187 of Streptomyces kanamyceticus IRED. (C) 2014 Elsevier B.V. All rights reserved.