MS09-P05 Crystal structures of Serratia marcescens reveal a homotetramer and insight into a flexible catalytic cleft Wen-Ching Wang (Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan)email: wcwang@mx.nthu.edu.tw 
The short-chain dehydrogenase/reductase (SDR) from erratia marcescens BCRC 10948 (SmSDR) that catalyzes an asymmetric reduction of alkyl ketones to the corresponding chiral alcohols is capable of converting 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-phenylephrine, which is marketed medically as a nasal decongestant agent. Here, we report the crystal structure of the apo-form SmSDR solved to 1.47 Å. The SmSDR structure shows a homotetramer of which each subunit consists a nucleotide-binding Rossmann domain and a presumed binding pocket surrounded by five loops and an helix (α7). Phe98 and Phe202 stand on the α7 helix and loop b4-a4) and form hydrophobic contacts with nearby residues. Site-directed mutagenesis characterization (WT, F98Y, F98YF202Y, and F98YF202L) revealed that F98YF202L exhibited a higher transformation activity toward HPMAE. Crystal structures of F98AF202L SmSDR, F98LF202L·NADPH, and F98YF202Y variants show an overall homologous structure. Substitution of F98 with alanine leads to the loss of the hydrophobic contacts between two arms, whereas F98YF202Y creates a strong H bond. In addition, the whole-cell conversion of F202A had an increased yield (1.9-fold) than that of WT. Together, our results suggest a robust structure-guided approach to stabilize the binding pocket that can be used to generate a valuable SmSDR variant for pharmaceutical applications.
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Keywords: short-chain dehydrogenase/reductase), Serratia marcescens BCRC 10948, (R)-phenylephrine