Dataset Information

An L213A variant of ?-glycosidase from Sulfolobus solfataricus with increased ?-L-arabinofuranosidase activity converts ginsenoside Rc to compound K.

ABSTRACT: Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. ?-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. ?-Glycosidase from S. solfataricus can hydrolyze ?-d-glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not ?-l-arabinofuranoside in these ginsenosides. To determine candidate residues involved in ?-l-arabinofuranosidase activity, compound Mc (C-Mc) was docking to ?-glycosidase from S. solfataricus in homology model and sequence was aligned with ?-glycosidase from Pyrococcus furiosus that has ?-l-arabinofuranosidase activity. A L213A variant ?-glycosidase with increased ?-l-arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased ?-l-arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant ?-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing ?-l-arabinofuranoside linked to Rc, whereas the wild-type ?-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides.

SUBMITTER: Choi JH

PROVIDER: S-EPMC5764348 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

An L213A variant of β-glycosidase from Sulfolobus solfataricus with increased α-L-arabinofuranosidase activity converts ginsenoside Rc to compound K.

Choi Ji-Hyeon JH Shin Kyung-Chul KC Oh Deok-Kun DK

PloS one 20180111 1

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β-d-glucopyranoside in ginsenosides Rc, C-M ...[more]

PMID: 29324789

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Project description:The local and global dynamics of the Sulfolobus solfataricus beta-glycosidase were studied by electron spin resonance and time-resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine -SH groups, at position 344 and at position 101, where Ser-101 was changed into a cysteine by site-directed mutagenesis. The greater reactivity of exposed Cys-101 suggested the exclusive modification of this amino acid compared with Cys-344. The labeled proteins underwent temperature perturbation in the range 290-335 K and the values of the spin-label rotation correlation frequencies (nu(c)) ranged from 6 x 10(7) to 2 x 10(8) sec(-1) for the protein labeled at position C344 and from 5.62 x 10(7) to 1.10 x 10(8) sec(-1) for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (nu(c)) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long-lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short-lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long-lived and short-lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.

Dataset Information

An L213A variant of ?-glycosidase from Sulfolobus solfataricus with increased ?-L-arabinofuranosidase activity converts ginsenoside Rc to compound K.

Publications

An L213A variant of β-glycosidase from Sulfolobus solfataricus with increased α-L-arabinofuranosidase activity converts ginsenoside Rc to compound K.

Similar Datasets

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