Gene cloning and catalysis features of a new mannitol-1-phosphate dehydrogenase (BbMPD) from Beauveria bassiana.
A long-chain mannitol-1-phosphate dehydrogenase (MPD) was characterized for the first time from fungal entomopathogen Beauveria bassiana by gene cloning, heterogeneous expression and activity analysis. The cloned gene BbMPD consisted of a 1334-bp open reading frame (ORF) with a 158-bp intron and the 935-bp upstream and 780-bp downstream regions. The ORF-encoded 391-aa protein (42kDa) showed less than 75% sequence identity to 17 fungal MPDs documented and shared two conserved domains with the fungal MPD family at the N- and C-terminus, respectively. The new enzyme was expressed well in the Luria-Bertani culture of engineered Escherichia coli BL21 by 16-h induction of 0.5 mM isopropyl 1-thio-beta-d-galactopyranoside at 20 degrees C after 5-h growth at 37 degrees C. The purified BbMPD exhibited a high catalytic efficiency (k(cat)/K(m)) of 1.31 x 10(4) mM(-1)s(-1) in the reduction of the highly specific substrate d-fructose-6-phosphate to d-mannitol-1-phosphate. Its activity was maximal at the reaction regime of 37 degrees C and pH 7.0 and was much more sensitive to Cu(2+) and Zn(2+) than to Li(+) and Mn(2+). The results indicate a crucial role of BbMPD in the mannitol biosynthesis of B. bassiana.
Wang
Zheng-Liang
ZL
Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, People's Republic of China.
Ying
Sheng-Hua
SH
Feng
Ming-Guang
MG
D000595:Amino Acid Sequence; D001483:Base Sequence; D052982:Beauveria / Q000235:genetics*; D055162:Biocatalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005636:Fructosephosphates / Q000378:metabolism; D007202:Indicators and Reagents / Q000494:pharmacology; D008355:Mannitol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D005636:Fructosephosphates; D007202:Indicators and Reagents; D008355:Mannitol Phosphates; C030070:mannitol-1-phosphate; C027618:fructose-6-phosphate; D013401:Sugar Alcohol Dehydrogenases; C021544:mannitol-1-phosphate dehydrogenase