Cloning, expression and biochemical characterization of UDP-glucose 6-dehydrogenase, a key enzyme in the biosynthesis of an anti-tumor polysaccharide from the marine fungus Phoma herbarum YS4108
2011
Zhou, Tian | Song, Yong C. | Feng, Ming Q. | Tan, Ren X.
There is an urgent requirement for unraveling the pathway for biosynthesis of the polysaccharide YCP from the marine fungus Phoma herbarum YS4108 in order to exploit its potential as an anti-tumor agent. Here we present cloning and characterization of UDP-glucose 6-dehydrogenase (UDP-GlcDHase), a key enzyme for the biosynthesis of this glycan. A full-length cDNA encoding UDP-GlcDHase was obtained by PCR using degenerate primers and the RACE strategy. The cDNA was 1846bp in length with an open reading frame of 1560bp encoding a protein of 520 amino acids. The deduced amino acid sequence showed about 50% overall identity to its homologs and a high degree of conservation in the nucleotide binding and catalytic domains. The cDNA was cloned in the Pichia pastoris GS115 on the plasmid vector, pPIC3.5k, to allow inducible expression of the protein with an N-terminal histidine-tag. Recombinant UDP-GlcDHase was affinity purified from crude, cytosolic extracts of the host cells, and characterized in terms of the its substrate affinity and the optimum temperature and pH for its activity. The biochemical properties of the purified recombinant enzyme were comparable with those of its homologs. The present investigation provides a promising start for manipulating YCP biosynthesis in P. herbarum.
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