Cho, Kye Woon
(Department of Life Science and Biochemical Engineering, Sun Moon University)
,
Kim, Tae-Su
(Department of Life Science and Biochemical Engineering, Sun Moon University)
,
Le, Tuoi Thi
(Department of Life Science and Biochemical Engineering, Sun Moon University)
,
Nguyen, Hue Thi
(Department of Life Science and Biochemical Engineering, Sun Moon University)
,
Oh, So Yeong
(Department of Pharmaceutical Engineering and Biotechnology, Sun Moon University Chungnam)
,
Pandey, Ramesh Prasad
(Department of Life Science and Biochemical Engineering, Sun Moon University)
,
Sohng, Jae Kyung
(Department of Life Science and Biochemical Engineering, Sun Moon University)
The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters ...
The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters were compared between YjiC and its variants with several acceptor substrates such as 7-hydroxyflavone (7-HF), 4',7-dihydroxyisoflavone, 7,8-dihydroxyflavone and curcumin. Molecular docking of TDP-glucose and 7-HF with YjiC model showed pi-alkyl interaction with Arg-282 and His-14, and pi-pi interaction with $His^{14}$ and thymine ring. YjiC (H14A) variant lost its glucosylation activity with TDP-glucose validating significance of His-14 in binding of TDP-sugars.
The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters were compared between YjiC and its variants with several acceptor substrates such as 7-hydroxyflavone (7-HF), 4',7-dihydroxyisoflavone, 7,8-dihydroxyflavone and curcumin. Molecular docking of TDP-glucose and 7-HF with YjiC model showed pi-alkyl interaction with Arg-282 and His-14, and pi-pi interaction with $His^{14}$ and thymine ring. YjiC (H14A) variant lost its glucosylation activity with TDP-glucose validating significance of His-14 in binding of TDP-sugars.
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문제 정의
Two residues (Arg 282 and His14) identified by in silico study and characterized by mutagenesis studies present evidence for promiscuous activity of YjiC with diverse donor substrates. The study also provides information for further engineering GT by saturated mutagenesis for target NDP-sugar specificity in the near future.
In conclusion, we tuned donor substrate specificity of YjiC from preferred UDP-glucose to TDP-glucose by sitedirected mutagenesis. This study provides evidence for better understanding of YjiC catalytic activity and donor substrate specificity. Two residues (Arg 282 and His14) identified by in silico study and characterized by mutagenesis studies present evidence for promiscuous activity of YjiC with diverse donor substrates.
제안 방법
In this study, we generated mutated variants of YjiC to explore the TDP-sugar specificity of the enzyme. By homology modeling and amino acid sequence alignment, two amino acids, His-14 and Arg-282, were identified as potential candidate amino acids for TDP-sugar specificities.
성능/효과
The predicted YjiC model displayed the GT-B fold typical of several GT families including GT-1, consisting of two Rossmann-like β/α/β domains that are not tightly associated.
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