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.

Applications of Phosphorene and Black Phosphorus in Energy Conversion and Storage Devices

“…Further cracking of P 2 at 2000 °C yields the atomic monomer in the vapor phase . The other important sources for elemental phosphorus are adenosine triphosphate, phosphoric acid (H 3 PO 4 ), the fertilizer salt monopotassium phosphate, KH 2 PO 4 , and phosphane (PH 3 ) …”

Structural analysis of an anthrol reductase inspires enantioselective synthesis of enantiopure hydroxycycloketones and β-halohydrins

“…2a, b ), in which continuous β-sheets and seven α-helices constitute the standard Rossmann-fold motif responsible for NADPH binding and the C-terminal domain constitutes a substrate binding pocket (Fig. 2a ) 32 , 33 . Based on structure alignment, although the root mean square difference (RMSD) of superposition for the Cα atoms of crystal structures of CbAR-NADP + and CbAR-NADP + -Emodin is 0.25Å, the regions from residue 209 to 216 are clearly different (Fig.…”

Flexibility of Short-chain dehydrogenase is interconnected to its promiscuity for the reduction of multiple ketone intermediates

“…was engineered to mimic the flexibility of another native SDR, the PhaB, for increasing the production of Polyhydroxy butyrate. Conversely, the flexibility was reduced in Serratia marcescens SDR for the production of (R)-phenylephrine (37)(38). Contextually, it is possible to engineer SDRs to obtain a specific product of interest; however, for multi-substrate catalysis, the overall architecture, flexibility, and an estimate of catalytic activity need to be considered for establishing a viable candidate.…”