Structure-guided design of Serratia marcescens short-chain dehydrogenase/reductase for stereoselective synthesis of (R)-phenylephrine

Abstract

Bioconversion is useful to produce optically pure enantiomers in the pharmaceutical industry, thereby avoiding problems with side reactions during organic synthesis processes. A short-chain dehydrogenase/reductase from Serratia marcescens BCRC 10948 (SmSDR) can stereoselectively convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-phenylephrine [(R)-PE], which is marketed medically as a nasal decongestant agent. The whole-cell conversion process for the synthesis of (R)-PE using SmSDR was reported to have an unexpectedly low conversion rate. We reported the crystal structure of the SmSDR and designed profitable variants to improve the enzymatic activity by structure-guided approach. Several important residues in the structure were observed to form hydrophobic clusters that stabilize the mobile loops surrounding the pocket. Of these, Phe98 and Phe202 face toward each other and connect the upper curvature from the two arms (i.e., the α7 helix and loopβ4-α4). The mutant structure of the double substitutions (F98YF202Y) exhibited a hydrogen bond between the curvatures that stabilizes the flexible arms. Site-directed mutagenesis characterization revealed that the mutations (F98Y, F98YF202Y, and F98YF202L) of the flexible loops that stabilize the region exhibited a higher transformation activity toward HPMAE. Together, our results suggest a robust structure-guided approach that can be used to generate a valuable engineered variant for pharmaceutical applications.

Figure 1

Structure of SmSDR. (A) Structure of the apo form of SmSDR. A presumed NADPH-binding cleft is surrounded by β2–α2, β4–α4, β3–α4, α5–α6, β6–α7, and an α7 helix. The α-helix, β-sheet, and loop are colored cyan, magenta, and brown, respectively. Phe98 and Phe202 are depicted as green stick models. The residues of the hydrophobic cluster are colored as purple stick models. The carbon, nitrogen, and oxygen atoms are shown as green, blue, and red, respectively. (B) Close-up view of the hydrophobic core on the presumed binding pocket. Phe98 (green) from the β4–α4 loop and Phe202 (green) from the α7 helix face toward each other and contact with the L100, Y158, N196, and I206 residues (purple). (C) Superposition of subunit A and B reveals flexible side chain of the Phe98.

Figure 2

Structure-based alignment of the homologous structures of SDRs. Secondary structural elements are presented above the sequence. The β-strands (β1–7) and α-helices (α1–8) are numbered from the N terminus. TT, β-turns; η (η1, η2), 3₁₀ helix. The conserved catalytic tetrad residues are indicated as green circles. (B) Close-up view of the superimposed conserved catalytic tetrad. The tetrad residues in SmSDR are drawn as thick stick model.

Figure 3

Proposed catalytic mechanism of SmSDR for the conversion of HPMAE into (R)-PE. The hydrogen atom of the nicotinamide approaches the C3 atom of HPMAE, initiating a hydride transfer to the atom. Subsequent protonation of Tyr158 by a water molecule then reduces the carbonyl group to ethanol.

Figure 4

Comparison of the hydrophobic surface in the F98-F202 region. (A) Superposition of four SmSDRs [apo form of SmSDR (green), F98L·F202L·NADPH (purple), F98Y·F202Y SmSDR (yellow), and F98A·F202L (magenta),]. NADPH is depicted as a stick model, and the nitrogen, oxygen, and phosphorus atoms are depicted as purple, red, and orange, respectively. (B) Apo form of SmSDR, (C) F98L·F202L·NADPH·SmSDR, (D) F98Y·F202Y·SmSDR, and (E) F98A·F202L·SmSDR. The hydrophobic surface was generated according to the Kyte‐Doolittle hydrophobic scale and is colored gray. The α-helices and loops are colored cyan and orange. The residues are depicted as thick stick models. Dashed lines indicate the distance between the depicted atoms.

Figure 5

Differential scanning fluorimetry (DSF) analysis of SmSDR variants. Tm is denoted as the midpoint of the unfolding transition of each protein. Tm and ΔTm values per variant are shown in the bottom table.