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

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Author(s)

  • Gideon James Grogan
  • Godwin Aleku
  • Henry Man
  • Scott P. France
  • Friedemann Leipold
  • Shahed Hussain
  • Laura Toca-Gonzalez
  • Rebecca Marchington
  • Sam J. Hart
  • Johan P. Turkenburg
  • Nicholas J. Turner

Department/unit(s)

Publication details

JournalACS Catalysis
DateAccepted/In press - 10 May 2016
DateE-pub ahead of print (current) - 18 May 2016
Issue number6
Volume6
Number of pages10
Pages (from-to)3880–3889
Early online date18/05/16
Original languageEnglish

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 e.e.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-crystallisation 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 co-crystallization with NADPH and 1-methyl-1,2,3,4-tetrahydroisoquinoline [(MTQ], and 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.

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© 2016, American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details

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