Purification, characterization, cloning and expression of pyruvate decarboxylase from Torulopsis glabrata IFO005.
In the production of pyruvate and optically active alpha-hydroxy ketones by Torulopsis glabrata, pyruvate decarboxylase (PDC, EC 4.1.1.1) plays an important role in pyruvate metabolism and in catalyzing the biotransformation of aromatic amino acid precursors to alpha-hydroxy ketones. In this paper, we have purified and characterized PDC from T. glabrata IFO005 and cloned the corresponding gene. A simple, rapid and efficient purification protocol was developed that provided PDC with high specific activity. Unlike other yeast or higher plant enzymes, known as homotetramers (alpha(4) or beta(4)) or heterotetramers (alpha(2)beta(2)), two active isoforms of PDC purified from T. glabrata IFO005 were homodimeric proteins with subunits of 58.7 kDa. We isolated the T. glabrata PDC gene encoding 563 amino acid residues and succeeded in overproducing the recombinant PDC protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Recombinant PDC from E. coli was purified as a homotetramer. Targeted gene disruption of PDC confirmed that T. glabrata has only one gene of PDC. This PDC gene showed about 80% homology with the genes of other yeasts, and amino acid residues involved in the allosteric site for pyruvate in other yeast PDCs were conserved in T. glabrata PDC. Both native PDC and recombinant PDC were activated by pyruvate and exhibited sigmoidal kinetics similar to those of Saccharomyces cerevisiae and higher plants. They also exhibited the similar catalytic properties: low thermostability, similar pH stability and optimal pH, and complete inhibition by glyoxylate.
Wang
Qinhong
Q
Center for Microbial Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, P.R. China.
He
Peng
P
Lu
Dajun
D
Shen
An
A
Jiang
Ning
N
D000495:Allosteric Site; D000595:Amino Acid Sequence; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D041221:Candida glabrata / Q000201:enzymology*; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006038:Glyoxylates / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D006038:Glyoxylates; D007659:Ketones; D020033:Protein Isoforms; D011994:Recombinant Proteins; C020927:phenyl-sepharose; D012685:Sepharose; D011767:Pyruvate Decarboxylase