title: 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.
authors: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors: Silvia
authors: S
authors: Fademrecht
authors: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors: Philipp N
authors: PN
authors: Scheller
authors: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors: Bettina M
authors: BM
authors: Nestl
authors: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors: Bernhard
authors: B
authors: Hauer
authors: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors: Jürgen
authors: J
authors: Pleiss
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D003067:Coenzymes; D019295:Computational Biology; D030562:Databases, Protein; D007097:Imines / Q000737:chemistry* / Q000378:metabolism*; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D013237:Stereoisomerism
chemical_list: D003067:Coenzymes; D007097:Imines; D009249:NADP; D010088:Oxidoreductases
keywords: IRED; biocatalysis; conservation; database analysis; sequence motif; sequence-function-relationship