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

DOI: 10.1021/acscatal.6b00782 DOI Help

Authors: G. A. Aleku (University of Manchester) , H. Man (University of York) , S. P. France (University of Manchester) , F. Leipold (University of Manchester) , S. Hussain (University of Manchester) , L. Toca- Gonzalez (University of Manchester) , R. Marchington (University of Manchester) , S. Hart (University of York) , J. Turkenburg (University of York) , G. Grogan (University of York) , N. J. Turner (University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: May 2016
Diamond Proposal Number(s): 9948

Abstract: 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.

Journal Keywords: biocatalysis; chiral amine; imine reductase; oxidoreductase; NADPH

Subject Areas: Biology and Bio-materials


Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 22/05/2016 19:25

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