A Solanum torvum GH3 β-glucosidase expressed in Pichia pastoris catalyzes the hydrolysis of furostanol glycoside. (July 2016)

• Record Type: Journal Article
• Authors: Suthangkornkul, Rungarun; Sriworanun, Pornpisut; Nakai, Hiroyuki; Okuyama, Masayuki; Svasti, Jisnuson; Kimura, Atsuo; Senapin, Saengchan; Arthan, Dumrongkiet
• Is Part Of: Phytochemistry. Volume 127(2016:Jul.)
• Page Start: 4
• Page End: 11
• Publication Date: 2016-07
• Subjects: β-Glucosidase -- Family 3 -- Torvoside A -- Pichia pastoris -- Solanum torvum -- Solanaceae; Botanical chemistry -- Periodicals; Biochemistry -- Periodicals; Botany -- Periodicals; Chimie végétale -- Périodiques; 572.2
• DOI: 10.1016/j.phytochem.2016.03.015
• Languages: English
• ISSNs: 0031-9422

Abstract

Graphical abstract: Recombinant Solanum torvum GH3 β-glucosidase showed the same unique substrate specificity as the natural enzyme, namely hydrolyzing furostanol glycoside, torvoside A. Highlights: The polyhistidine-tagged β-glucosidase, SBgl3 was expressed in Pichia pastoris . The recombinant SBgl3 showed similar kinetic properties to the natural SBgl3. The recombinant SBgl3 showed similar substrate specificity to the natural SBgl3. The substrate specificity of SBgl3 differs from other family 3 β-glucosidases. Structural elements in active site of SBgl3 differ from family 3 β-glucosidases. Abstract: Plant β-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a β-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26- O -β-glucosides, torvosides A and H. Furostanol glycoside 26- O -β-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 β-glucosidase, but could not hydrolyze β-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum β-glucosidase (SBgl3) was isolated by the rapid amplification of cDNA ends method. The 1887 bp ORF encoded 629 amino acids and showed high homology to other plant GH3 β-glucosidases. Internal peptide sequences of purified native Sbgl3 determined by LC–MS/MS matched the deduced amino acid sequence of the Sbgl3 cDNA, suggesting that it encoded the natural enzyme. Recombinant SBgl3 with a polyhistidine tag (SBgl3His) was successfully expressed in Pichia pastoris . The purified SBgl3His showed the same substrate specificity as natural SBgl3, hydrolyzing torvoside A with much higher catalytic efficiency than other substrates. It also had similar biochemical properties and kinetic parameters to the natural enzyme, with slight differences, possibly attributable to post-translational glycosylation. Quantitative real-time PCR (qRT-PCR) showed that SBgl3 was highly expressed in leaves and germinated seeds, suggesting a role in leaf and seedling development. To our knowledge, a recombinant GH3 β-glucosidase that hydrolyzes furostanol 26- O -β-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes.