Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis

Abstract

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.

Tuning an Imine Reductase for the Asymmetric Synthesis of Azacycloalkylamines by Concise Structure‐Guided Engineering

“…Those interactions could form a bottleneck that may hinder the entering and coupling of the bulky substrates. To interrupt such interactions, we superimposed NADP(H)‐complex structures of six IREDs [5a–d,q,r] with that of IR‐G36 (Supporting Information Figures S5 and S6) to seek the promising site on the loop 252–261 for mutation. In those structures, NADP(H) can form hydrogen bonds either with the top loop 252–261 or with the bottom loop 119–123 (Figure 1B, Supporting Information Figures S5 and S6), and accordingly generate two dissimilar types of IRED‐cavities, which we termed “narrow‐type” and “wide‐type” (Supporting Information Figures S5 and S6).…”

Actinomycetes-derived imine reductases with a preference towards bulky amine substrates

“…Except for the NADPH binding site, the pocket cavity of IR-G02 showed a strong negative electrostatic surface potential (Fig. 5a), which may favor the entry of positively charged amine donors and imine intermediate 27 , and contribute to its high substrate promiscuity.…”

Inverting the Stereoselectivity of an NADH‐Dependent Imine‐Reductase Variant

“…However, this approximate classification cannot be applied to all IREDs, as different stereoselectivities have been described depending on the substrate [12,18] . This is illustrated by recently characterized IREDs that were found to be ( S )‐selective despite having aspartic acid as a pendant residue in the active site [19,20] . In addition, single‐point variants of the IRED from Amycolatopsis orientalis displayed inverted stereoselectivity compared to the wild‐type [21] .…”

“…[ 12 , 18 ] This is illustrated by recently characterized IREDs that were found to be ( S )‐selective despite having aspartic acid as a pendant residue in the active site. [ 19 , 20 ] In addition, single‐point variants of the IRED from Amycolatopsis orientalis displayed inverted stereoselectivity compared to the wild‐type. [21] Despite extensive studies of IREDs,[ 12 , 16 , 18 , 22 , 23 , 24 , 25 , 26 ] the molecular mechanism of asymmetric imine reduction and, if relevant, imine protonation, remains unclear.…”