Characterization of salt-regulated mannitol-1-phosphate dehydrogenase in the red alga Caloglossa continua.
Mannitol-1-phosphate (M1P) dehydrogenase (M1PDH; EC 1.1.1.17), an enzyme catalyzing the reduction of Fru-6-phosphate (F6P) to M1P in algal mannitol biosynthesis, was purified to homogeneity from a cell homogenate of the eulittoral red alga Caloglossa continua (Okamura) King et Puttock. The enzyme was a monomer with an apparent molecular mass of 53 kD, as determined by gel filtration and SDS-PAGE, and exhibited an pI of approximately 5.5. The substrate specificity was very high toward F6P and M1P for respective reductive and oxidative reactions. The enzyme was found to be a sulfhydryl-type, because its activity was inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and the inhibition by p-hydroxymercuribenzoate was rescued by 2-mercaptoethanol. Some unknown factors in the extract may also have inhibited the activity, because the total activity was greatly increased through the purification procedure. The optimum pH for F6P reduction was changed from 6.0 or lower to 7.2 by the addition of 200 mm NaCl. The reduction of F6P showed strong substrate inhibition above 0.5 mm. However, Km(F6P) of M1PDH was increased eight times by the addition of 200 mm NaCl, whereas Vmax was in a similar range with the avoidance of substrate inhibition by F6P. These results indicate that the enzyme was finely and directly regulated by the salt concentration without the requirement for gene expression. M1PDH can therefore be a key enzyme for regulating mannitol biosynthesis when the alga is stressed by a salinity change.
Iwamoto
Koji
K
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
Kawanobe
Hideaki
H
Ikawa
Tomoyoshi
T
Shiraiwa
Yoshihiro
Y
D007700:Kinetics; D008970:Molecular Weight; D009994:Osmolar Concentration; D000461:Rhodophyta / Q000201:enzymology*; D013401:Sugar Alcohol Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*
D013401:Sugar Alcohol Dehydrogenases; C021544:mannitol-1-phosphate dehydrogenase