Purification and characterization of a NAD+-dependent sorbitol dehydrogenase from Japanese pear fruit.
NAD+-dependent sorbitol dehydrogenase NAD-SDH, EC 1.1.1.14) from Japanese pear fruit was purified to apparent homogeneity (single band by SDS-PAGE with silver staining), and had a specific activity of 916.7 nKatal/mg protein. The molecular of the native enzyme was calculated to be 160 kDa by gel filtration, whereas SDS-PAGE gave a subunit size of 40 kDa, indicating that the native enzyme is a homotetramer. The protein immunologically reacted with an antibody raised in rabbit against the fusion protein expressed in E. coli harboring an apple NAD-SDH cDNA. The Km, values for sorbitol and fructose were 96.4+/-8.60 and 4239+/-33.5 mM, respectively, and optimum pH for sorbitol oxidation was 9.0 and 7.0 for fructose reduction. Pear NAD-SDH had a very narrow substrate specificity, that is, sorbitol, L-iditol, xylitol and L-threitol were oxidized but not any of the other alcohols tested. These data suggest the structural importance of an S configuration at C-2 and an R configuration at C-4 in the substrate(s). Its enzymatic activity was strongly inhibited both by heavy metal ions such as mercury, and by thiol compounds, such as L-cysteine. However, the addition of zinc ion reversed the enzyme inactivation caused by addition of L-cysteine.
Oura
Y
Y
Laboratory of Horticultural Science, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Japan.
Yamada
K
K
Shiratake
K
K
Yamaki
S
S
D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005632:Fructose / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D019685:Rosales / Q000737:chemistry / Q000201:enzymology*; D013012:Sorbitol / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013379:Substrate Specificity
D005632:Fructose; D013012:Sorbitol; D007064:L-Iditol 2-Dehydrogenase