Stereoselectivity and Structural Characterization of an Imine Reductase (IRED) from Amycolatopsis orientalis

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• 作者：Godwin A. Aleku ; Henry Man ; Scott P. France ; Friedemann Leipold ; Shahed Hussain ; Laura Toca-Gonzalez ; Rebecca Marchington ; Sam Hart ; Johan P. Turkenburg ; Gideon Grogan ; Nicholas J. Turner
• 刊名：ACS Catalysis
• 出版年：2016
• 出版时间：June 3, 2016
• 年：2016
• 卷：6
• 期：6
• 页码：3880-3889
• 全文大小：447K
• 年卷期：0
• ISSN：2155-5435

文摘

The imine reductase AoIRED from Amycolatopsis orientalis (Uniprot R4SNK4) catalyzes the NADPH-dependent reduction of a wide range of prochiral imines and iminium ions, predominantly with (S)-selectivity and with ee’s of up to >99%. AoIRED displays up to 100-fold greater catalytic efficiency for 2-methyl-1-pyrroline (2MPN) compared to other IREDs, such as the enzyme from Streptomyces sp. GF3546, which also exhibits (S)-selectivity, and thus, AoIRED is an interesting candidate for preparative synthesis. AoIRED exhibits unusual catalytic properties, with inversion of stereoselectivity observed between structurally similar substrates, and also, in the case of 1-methyl-3,4-dihydroisoquinoline, for the same substrate, dependent on the age of the enzyme after purification. The structure of AoIRED has been determined in an “open” apo-form, revealing a canonical dimeric IRED fold in which the active site is formed between the N- and C-terminal domains of participating monomers. Co-crystallization with NADPH gave a “closed” form in complex with the cofactor, in which a relative closure of domains, and associated loop movements, has resulted in a much smaller active site. A ternary complex was also obtained by cocrystallization with NADPH and 1-methyl-1,2,3,4-tetrahydroisoquinoline [(MTQ], and it reveals a binding site for the (R)-amine product, which places the chiral carbon within 4 Å of the putative location of the C4 atom of NADPH that delivers hydride to the C═N bond of the substrate. The ternary complex has permitted structure-informed mutation of the active site, resulting in mutants including Y179A, Y179F, and N241A, of altered activity and stereoselectivity.