Cloning of NAD-SDH cDNA from plum fruit and its expression and characterization.
A full-length cDNA consisting of 1444 bp for NAD dependent sorbitol dehydrogenase (NAD-SDH) was cloned from fruit of plum (Prunus salicina var. cordata cv. Younai) by means of RT-PCR and RACE. The cDNA containing an open reading frame (ORF) of 1101 bp encoded a polypeptide of 367 amino acid residues. The maltose binding protein fusion SDH (MBP-SDH) was expressed and partially purified from Escherichia coli cells, and biochemical properties of MBP-SDH and SDH cleaved from the fusion protein by factor Xa were characterized. The MBP-SDH had the specific affinity for NAD and was able to oxidize sorbitol, xylitol, l-ribitol and mannitol but not ethyl alcohol, arabitol and other polyols. The optimum pH for the oxidation of sorbitol and the reduction of fructose was 9.0 and 7.0, respectively; the maximum reaction rate occurred when temperature increased up to 50 °C in the presence of sorbitol. The MBP-SDH with a subunit of 80 kDa appears to be a hexamer. Its molecular weight was 478.6 kDa estimated by gel filtration and 493.2 kDa estimated using native linear gradient PAGE. The K(m) values for sorbitol, NAD, fructose and NADH were 95.86 mM, 0.31 mM, 1.04 M and 0.038 mM, respectively. However, when MBP was cleaved from the fusion enzyme, the SDH exists as a homotetramer with the native molecular weight of 164.8 kDa estimated by gel filtration. The K(m) values were 111.8 mM, 0.35 mM, 1.25 M and 0.048 mM for sorbitol, NAD, fructose and NADH, respectively. The MBP-SDH and the SDH were similar with respect to their kinetic characteristics despite their difference in quaternary structures.
Institute of Postharvest Science and Technology of Horticultural Products, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, PR China. gzhhs@163.com
Zhi-Xiong
ZX
Guo
Teng-Fei
TF
Pan
Kai-Tuo
KT
Li
Feng-Lin
FL
Zhong
Lin
L
Lin
Dong-Ming
DM
Pan
Liu-Xin
LX
Lu
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005638:Fruit / Q000201:enzymology* / Q000378:metabolism; D027861:Prunus / Q000201:enzymology*; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D013401:Sugar Alcohol Dehydrogenases