Cloning and characterization of thermotolerant xylitol dehydrogenases from yeast Pichia angusta.
Pichia angusta (syn. Hansenula polymorpha) represents one of the rare yeast that can grow and ferment both xylose and glucose at higher temperature (50°C). However, little is known about the enzymes involved in xylose utilization from this species. Previous studies indicated the presence of one xylose reductase and two xylitol dehydrogenase genes in P. angusta. In this study, we have expressed both xylitol dehydrogenases (PaXdh1p and PaXdh2p) in Escherichia coli and purified them as 6X-Histidine-tagged proteins. Biochemical characterization of the recombinant proteins reveals that both PaXdh1p and PaXdh2p are thermotolerant enzymes. PaXdh2p contains a catalytic and a structural Zn atom. However, the structural Zn atom is not present in PaXdh1p. Both enzymes also differ in their affinity for the substrate as well as in the catalytic efficiency. Through mutagenesis and modeling approaches, we have also identified residues important for catalysis and substrate binding.
Biswas
Dipanwita
D
Institute of Microbial Technology, Council of Scientific and Industrial Research, Sector 39A, Chandigarh 160 036, India.
Datt
Manish
M
Ganesan
Kaliannan
K
Mondal
Alok K
AK
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D050542:D-Xylulose Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D015032:Zinc / Q000378:metabolism
D003067:Coenzymes; D004269:DNA, Bacterial; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D050542:D-Xylulose Reductase; D015032:Zinc