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.

Keywords: 2-ethylhexanol; biocatalysis; crystal structure; enzyme; imine reductase; substrate binding; substrate soaking.

Figure 1

Overall structure of S-IRED-Ms. S-IRED-Ms, depicted in cartoon representation, shows the canonical dimeric domain-swapped structure. Helices of monomer A of the dimer are colored and labeled in blue, β-sheets in red, loops and turns in light pink. Monomer B is colored in light orange. The cofactor NADPH is depicted as stick model in gray.

Figure 2

NADPH-binding site of S-IRED-Ms. NADPH, shown as stick model, resides in a highly conserved binding site. Carbon atoms are depicted in gray, oxygen in red, nitrogen in dark blue and phosphorus in orange. (a) Apart from hydrogen bonds to two serines in the C-terminal domain (light blue) NADPH mainly interacts with adjacent residues of the N-terminal Rossmann-fold domain (light orange) and interactions are indicated by dotted yellow lines. (b) Visualization of the electrostatic surface potential of the NADPH-binding site displays a positive potential (blue surface) around the adenine moiety of NADPH and its 2′-phosphorylated ribose, while a strongly negative potential (red surface) can be observed in the rest of the channel leading to the substrate binding site. The surface of the C-terminal domain, normally partially covering the NADPH binding site, was omitted from the depiction in order to provide a better view.

Figure 3

Second entrance and active site of S-IRED-Ms. (a) The opening to the active site cavity shows a negative electrostatic potential. This is largely due to D125, E218 and possibly P214 and S232 outlining the entrance. (b) 2-ethylhexanol (2-EH) was modelled into the active site. 2-EH is sandwiched by hydrophobic residues like P123, L174, F177, and W178 and also forms two hydrogen bonds to nearby waters which are part of a wider network inside the active site, further holding 2-EH in place. Amino acids within 5 Å of 2-EH as well as D170 in the proposed protic position are depicted as sticks, with the two chains forming the dimer colored in light orange and marine blue. NADPH is colored in light gray. The 2Fo-Fc electron density of NADPH, contoured at 1 σ, is shown in dark gray. The hydrogen bonds of the 2-EH hydroxyl to nearby water molecules (red spheres) are indicated as pink dashed lines.