A Solanum torvum GH3 ß-glucosidase expressed in Pichia pastoris catalyzes the hydrolysis of furostanol glycoside.

Plant ß-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a ß-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26-O-ß-glucosides, torvosides A and H. Furostanol glycoside 26-O-ß-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 ß-glucosidase, but could not hydrolyze ß-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum ß-glucosidase (SBgl3) was isolated by the rapid amplification of cDNA ends method. The 1887bp ORF encoded 629 amino acids and showed high homology to other plant GH3 ß-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 ß-glucosidase that hydrolyzes furostanol 26-O-ß-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes.

Shrimp ATP synthase genes complement yeast null mutants for ATP hydrolysis but not synthesis activity.

The aim of this study was to examine the feasibility of employing a yeast functional complementation assay for shrimp genes by using the shrimp mitochondrial F(1)F(0)-ATP synthase enzyme complex as a model. Yeast mutants defective in this complex are typically respiratory-deficient and cannot grow on non-fermentable carbon sources such as glycerol, allowing easy verification of functional complementation by yeast growth on media with them as the only carbon source. We cloned the previous published sequence of ATP2 (coding for ATP synthase ß subunit) from the Pacific white shrimp Penaeus vannamei (Pv) and also successfully amplified a novel PvATP3 (coding for the ATP synthase γ subunit). Analysis of the putative amino acid sequence of PvATP3 revealed a significant homology with the ATP synthase γ subunit of crustaceans and insects. Complementation assays were performed using full-length ATP2 and ATP3 as well as a chimeric form of ATP2 containing a leader peptide sequence from yeast and a mature sequence from shrimp. However, the shrimp genes were unable to complement the growth of respective yeast mutants on glycerol medium, even though transcriptional expression of the shrimp genes from plasmid-borne constructs in the transformed yeast cells was confirmed by RT-PCR. Interestingly, both PvATP2 and PvATP3 suppressed the lethality of the yeast F(1) mutants after the elimination of mitochondrial DNA, which suggests the assembly of a functional F(1) complex necessary for the maintenance of membrane potential in the ρ(0) state. These data suggest an incompatibility of the shrimp/yeast chimeric F(1)-ATPase with the stalk and probably also the F(0) sectors of the ATP synthase, which is essential for coupled energy transduction and ATP synthesis.