Cloning and characterization of a gene encoding trehalose phosphorylase (TP) from Pleurotus sajor-caju.
Complementary DNA for a gene encoding trehalose phosphorylase (TP) that reversibly catalyzes trehalose synthesis and degradation from alpha-glucose-1-phosphate (alpha-Glc-1-P) and glucose was cloned from Pleurotus sajor-caju. The cDNA of P. sajor-caju TP (designated PsTP, GenBank Accession No. AF149777) encodes a polypeptide of 751 amino acids with a deduced molecular mass of 83.7 kDa. The PsTP gene is expressed in mycelia, pilei, and stipes of fruiting bodies. Trehalose phosphorylase PsTP was purified from PsTP-transformed Escherichia coli. The enzyme catalyzes both the phosphorolysis of trehalose to produce alpha-Glc-1-P and glucose, and the synthesis of trehalose. The apparent K(m) values for trehalose and Pi in phosphorolytic reaction at pH 7.0 were 74.8 and 5.4 mM, respectively. The PsTP gene complemented Saccharomyces cerevisiae Deltatps1, Deltatps2 double-mutant cells, allowing their growth on glucose medium. Furthermore, yeast transformed with PsTP produced 2-2.5-fold more trehalose than non-transformants or cells transformed with empty vector only.
Han
Sang-Eun
SE
Molecular Physiology Division, National Institute of Agricultural Biotechnology, 441-707 Suwon, Republic of Korea.
Kwon
Hawk-Bin
HB
Lee
Seung-Bum
SB
Yi
Bu-Young
BY
Murayama
Ikuo
I
Kitamoto
Yutaka
Y
Byun
Myung-Ok
MO
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D013696:Temperature; D014199:Trehalose / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D014199:Trehalose; D005964:Glucosyltransferases; C023181:alpha,alpha-trehalose phosphorylase