D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions

Abstract

When Absidia glauca was grown in minimal media with D-mannitol as the only source of carbon, an NAD+ specific D-mannitol dehydrogenase (EC 1.1.1.67) was induced. The crude extract also gave evidence of mannitol kinase, mannitol-1-phosphate dehydrogenase, phosphofructokinase, and L-iditol dehydrogenase activity. The heat labile purified preparation was judged enzymically homogeneous based on evidence derived from substrate specificity studies and activity staining, following disc gel electrophoresis. The enzymic monomer, with a weight of about 67000 daltons, slowly polymerizes when stored at -20 degrees C, giving a multiplicity of protein bands on electrophoresis distributed predominantly across a spectrum from dimer to pentamer, with enzymic activity resident predominantly in even multiples of the monomer. Depolymerization occurred rapidly (hours) when a frozen preparation was brought to and held between 4 and 20 degrees C. Aggregate fragmentation with sodium dodecyl sulfate showed a time-temperature dependence, terminating in a subunit component of 13000 daltons. pH optimum for polyol oxidation occurs at 9.6 (NaOH-glycine buffer) while ketose reduction proceeded most rapidly at pH 7.0-7.2 (phosphate buffer). A regulatory role is suggested for this enzyme based on dead-end inhibition by mannitol 1-phosphate, multiple enzyme forms, and its locus at the initiation site for mannitol utilization. The physiological relevance of low-temperature aggregation to regulatory control remains to be established.

Mannitol metabolism in the phytopathogenic fungus Alternaria alternata

“…The ability of this mutant to readily utilize mannitol is not consistent with a hypothesis of MtDH as the primary catabolic enzyme. We hypothesize that the mannitol is phosphorylated and then utilized through the enzyme MPDH, as enzymes with the ability to phosphorylate mannitol have been documented in fungi (Lee, 1967;Ueng et al, 1976). It has been shown that growth of Aspergillus species and Sphaerosporella brunnea on mannitol causes a decrease in both MtDH and mannitol 1-phosphatase activity, and an increase in MPDH activity (Lee, 1967;Strandberg, 1969;Ramstedt et al, 1987), consistent with MPDH serving as a degradative enzyme in vivo, in addition to its biosynthetic role.…”

D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate

“…In fact, no evidence of substrate inhibition was observed up to the limit of solubility for mannitol. The enzyme is, however, uniquely susceptible to metabolic control by mannitol 1-phosphate acting as a deadend inhibitor (see Figure 1 of Ueng et al, 1976). This polyol phosphate binds twice and nonpreferentially to the E and/or EA forms of the enzyme when the levels of mannitol and NAD+ are relatively low, but multiply (Le., more than twice) and predominantly to one (or both) of the E Q conformers when the level of mannitol is sufficiently high to drive the reaction rapidly through the central complexes.…”

Effect of Different Carbon Sources on Relative Growth Rate, Internal Carbohydrates, and Mannitol 1-Oxidoreductase Activity in Celery Suspension Cultures

“…The M6PR functions primarily in a synthetic manner, with only very low rates of mannitol-1-P oxidation observed . M6PR is distinctly different from mannitol-metabolizing enzymes in lower organisms, a11 of which can be characterized as 2-oxidoreductases, catalyzing the interconversion of Fru or Fru-6-P to mannitol or mannitol-1-P (Martinez et al, 1963;Ueng et al, 1976;Niehaus and Dilts, 1982;Morton et al, 1985;Teschner et al, 1990). Many of the mannitol dehydrogenases in lower organisms can function in both directions, e.g.…”