Identification of imine reductase‐specific sequence motifs

Abstract

Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database (www.IRED.BioCatNet.de) was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED-Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx(5 )[ATS]x(4) Gx(4) [VIL]WNR[TS]x(2) [KR] and the active site motif Gx[DE]x[GDA]x[APS]x(3){K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes.

Asymmetric Ketone Reduction by Imine Reductases

“…More than 30 IREDs have been characterizeda s catalysing intramolecular asymmetric reductions of various cyclic imines. [8,11] The question of how proteins in nature evolve new function is of fundamental significance. [5 -7] In the course of establishing the IRED database (Imine Reductase Engineering Database;h ttp://ired.biocatnet.de/), we observed that most imine reductases are annotated as dehydrogenases.…”

“…[5 -7] In the course of establishing the IRED database (Imine Reductase Engineering Database;h ttp://ired.biocatnet.de/), we observed that most imine reductases are annotated as dehydrogenases. [8,11] The question of how proteins in nature evolve new function is of fundamental significance. [8] We and others have recently demonstratedt hat IREDs appear to be incapableo fr educing C=Ob onds.…”

“…However,e xamination of the supposed dehydrogenasea ctivity of IREDs from Streptosporangium roseum, Streptomyces turgidiscabies and Paenibacillus elgii with glycerol-3-phosphateo r6phosphogluconate as substrate demonstrated no dehydrogenase activity. [8,11] The question of how proteins in nature evolve new function is of fundamental significance. [9,10] Conversely, no asymmetricr eduction of iminesb yd ehydrogenases or carbonyl/keto reductases (KREDs) has been reported.…”

“…[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.…”