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

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 2 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-1,2,3,4tetrahydroisoquinoline [(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 structureinformed mutation of the active site, resulting in mutants including Y179A, Y179F and N241A, of altered activity and stereoselectivity.

Asymmetric Ketone Reduction by Imine Reductases

“…[23] Unlike the situation for carbonyl-reducing enzymes, the catalytic residues and the reduction mechanism of IREDs are still under debate. [25] The recent investigations by Tawfik and co-workers on the evolution of the Rossmann-fold motif [26] and the structurals imilarity of IREDs to hydroxyisobutyratea nd b-hydroxyacidd ehydrogenases [8,11] strongly suggest ac ommona ncestry.D espite these common elements, IREDs and KREDs differ in their ability to reduce imines. [24] NADPH is considered the hydride donor,a lthough the proton-donating residue remains elusive.…”

“…Theoretically calculated activation energies encourage the speculation that imines are protonated prior to hydride transfer from NADPH. [10,20] Regarding speculations of the roles of amino acid residues for C=Np rotonation in IREDs, [11,25] there is some supporting evidenceb ased on mutations of putative catalytically important aspartic acid residues displaying reduced catalytic efficiencies over wild-type enzyme. This is in accordance with the work of Mayr and co-workers,w ho described the electrophilicity of benzaldehyde-derived iminium ions and their higherr eactivities compared to substituted imines.…”

Synthesis of 2,5‐Disubstituted Pyrrolidine Alkaloids via A One‐Pot Cascade Using Transaminase and Reductive Aminase Biocatalysts

“…GF3587, S ‐IRED from Streptomyces sp . GF3546 and Ao IRED from Amycolatopsis orientalis . Biotransformations were initially performed in a whole‐cell system as it promotes in vivo cofactor regeneration by only supplementing the reaction medium with glucose.…”

Biocatalytic Potential of Enzymes Involved in the Biosynthesis of Isoprenoid Quinones

“…Recently, Turner and co‐workers described the unexpected stereoselective patterns of an imine reductase from A. orientalis (AoIRED). In their case, AoIRED was shown to exhibit selectivity patterns that defy the Cahn‐Ingold‐Prelog priority rule . As enzymes displaying unusual selectivity properties emerge, further studies are required to uncover the mechanism for such unique behaviour.…”