Partial purification and characterization of mannitol: mannose 1-oxidoreductase from celeriac (Apium graveolens var. rapaceum) roots.
A mannitol:mannose 1-oxidoreductase was isolated from celeriac (Apium graveolens var. rapaceum) root tips by fractionation with (NH4)2SO4, followed by chromatography on a Fractogel DEAE column and then concentration with (NH4)2SO4. This newly discovered mannitol dehydrogenase catalyzes the NAD-dependent oxidation of mannitol to mannose, not mannitol to fructose. The sugar product of the enzyme reaction was identified by three independent HPLC systems and by an enzymatically linked system as being mannose and not fructose or glucose. Normal Michaelis--Menten kinetics were exhibited for both mannitol and NAD with Km values of 72 and 0.26 mM, respectively, at pH 9.0. The Vmax was 40.14 mumol/h/mg protein for mannitol synthesis and 0.8 mumol/h/mg protein for mannose synthesis at pH 9.0. In the polyol oxidizing reaction, the enzyme was very specific for mannitol with a low rate of oxidation of sorbitol. In the reverse reaction, the enzyme was specific for mannose. The enzyme was strongly inhibited by NADH and sensitive to alterations of NAD/NADH ratio. The enzyme is of physiological importance in that it is mainly localized in root tips (sink tissue) where it functions to convert mannitol into hexoses which are utilized to support root growth. Product determination and kinetic characterization were carried out on an enzyme preparation with a specific activity (SA) of 30.44 mumol/h/mg protein. Subsequently, the enzyme was further purified to a SA of 201 mumol/h/mg protein using an NAD affinity column. This paper apparently represents the first evidence of the existence of a mannitol:mannose 1-oxidoreductase and also the first evidence of the presence of a mannitol dehydrogenase in vascular plants.
Stoop
J M
JM
Department of Horticultural Science, North Carolina State University, Raleigh 27695-7609.
Pharr
D M
DM
D002852:Chromatography, Ion Exchange; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D008353:Mannitol / Q000378:metabolism; D008354:Mannitol Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D013401:Sugar Alcohol Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*
D009243:NAD; D005632:Fructose; D008353:Mannitol; D013395:Sucrose; D008354:Mannitol Dehydrogenases; D013401:Sugar Alcohol Dehydrogenases; C077257:mannitol-mannose 1-oxidoreductase; D005947:Glucose