TY - JOUR
T1 - Reversible control of enantioselectivity by the length of ketone substituent in biocatalytic reduction
AU - Koesoema, Afifa Ayu
AU - Sugiyama, Yosuke
AU - Sriwong, Kotchakorn T.
AU - Xu, Zichang
AU - Verina, Samantha
AU - Standley, Daron M.
AU - Senda, Miki
AU - Senda, Toshiya
AU - Matsuda, Tomoko
N1 - Funding Information:
This study was funded by Japan Society for the Promotion of Science under grant number JP16K05864 to Tomoko Matsuda, and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant numbers 19am0101108j0003 to Daron M. Standley and 19am0101071 to Toshiya Senda.
Publisher Copyright:
© 2019, Springer-Verlag GmbH Germany, part of Springer Nature.
PY - 2019/12/1
Y1 - 2019/12/1
N2 - Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.
AB - Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.
KW - Alcohol dehydrogenase
KW - Asymmetric reduction
KW - Docking simulation
KW - Enantioselectivity inversion
KW - Enzyme engineering
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U2 - 10.1007/s00253-019-10206-5
DO - 10.1007/s00253-019-10206-5
M3 - Article
C2 - 31720775
AN - SCOPUS:85075193691
VL - 103
SP - 9529
EP - 9541
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
SN - 0175-7598
IS - 23-24
ER -