Systematic Evaluation of Imine-Reducing Enzymes: Common Principles in Imine Reductases, β-Hydroxy Acid Dehydrogenases, and Short-Chain Dehydrogenases/ Reductases

Abstract

The enzymatic, asymmetric reduction of imines is catalyzed by imine reductases (IREDs), members of the short-chain dehydrogenase/reductase (SDR) family, and β-hydroxy acid dehydrogenase (βHAD) variants. Systematic evaluation of the structures and substrate-binding sites of the three enzyme families has revealed four common principles for imine reduction: structurally conserved cofactor-binding domains; tyrosine, aspartate, or glutamate as proton donor; at least four characteristic flanking residues that adapt the donor's pK_a and polarize the substrate; and a negative electrostatic potential in the substrate-binding site to stabilize the transition state. As additional catalytically relevant positions, we propose alternative proton donors in IREDs and βHADs as well as proton relays in IREDs, βHADs, and SDRs. The functional role of flanking residues was experimentally confirmed by alanine scanning of the imine-reducing SDR from Zephyranthes treatiae. Mutating the "gatekeeping" phenylalanine at standard position 200 resulted in a tenfold increase in imine-reducing activity.

Keywords: beta-hydroxy acid dehydrogenases; flanking residues; imine reductases; intrinsic disorder; short-chain dehydrogenases/reductases.

Figure 1

Structural scheme of A) IREDs, B) “short‐chain” βHADs, C) “long‐chain” βHADs, and D) classical SDRs. The black box marks the Rossmann‐like NADPH‐binding domains which were superimposed; the red boxes mark the secondary structures involved in cofactor binding. Blue coloring indicates the structures that are involved in multimerization, and the green areas indicate substrate‐interacting regions. In (D), secondary structures of the Rossmann‐like domain that were not superimposable with those of IREDs/βHADs are colored in black, and terminal helices that only appear in the tetrameric SDR are labeled (tN and tC).

Figure 2

A) General scheme of imine‐reducing catalytic sites. Dashed coloring shows flanking residues that are not strictly present in all considered enzymes. The proton donor (green) is always flanked by one putatively imine‐polarizing amino acid (orange). Except for the conventional βHADs, all proton donors are flanked by at least one nonpolar amino acid (pink). One or more donor‐polarizing amino acids (blue) are present in all considered enzymes except R‐IRED‐Sr. The characteristic flanking methionine (red) was not observed in the imine‐reducing SDRs. Optionally, proton‐mediating residues flanking the imine‐polarizing residue can be present (gray). The proposed conventional and catalytic sites are schematically shown for the considered IREDs (B) and βHADs (C), where the dashed lines mark the respective affiliation of the flanking residues and dashed green coloring represents a non‐proton‐donating residue at the proposed alternative proton donor position. D) The considered imine‐reducing classical SDRs did not present the characteristic flanking methionine or an alternative catalytic site.

Figure 3

Biotransformation results for wild‐type, alanine variants, and C150S+C150D variants of SDR‐Zt. The specific activity (dark gray) on TMI and its conversion after reaction for 3 h (light gray) are shown. Due to the significantly increased activity of variant F202A, a scaling interruption has been introduced for visualization purposes.