DIHYDROXYACETONE SYNTHASE FROM A METHANOL-UTILIZING CARBOXYDOBACTERIUM, ACINETOBACTER SP. STRAIN JC1-DSM-3803

Actinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyaceton e kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyr uvate reductase and very low hexulose 6-phosphate synthase, activities , The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase, The dihydroxyacetone synth ase was purified 19-fold in eight steps to homogeneity, with a yield o f 9%, The final specific activity of the purified enzyme was 1.12 mu m ol of NADH oxidized per min per mg of protein, The molecular weight of the native enzyme was determined to be 140,000, Sodium dodecyl sulfat e-gel electrophoresis revealed a subunit of molecular weight 73,000, T he optimum temperature and pH were 30 degrees C and 7.0, respectively, The enzyme was inactivated very rapidly at 70 degrees C, The enzyme r equired Mg2+ and thiamine pyrophosphate for maximal activity, Xylulose 5-phosphate was found to be the best substrate when formaldehyde was used as a glycoaldehyde acceptor, Erythrose 4-phosphate, glycolaldehyd e, and formaldehyde were found to act as excellent substrates when xyl ulose 5-phosphate was used as a glycoaldehyde donor, The K(m)s for for maldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 mu M, respect ively, The enzyme produced dihydroxyacetone from formaldehyde and xylu lose 5-phosphate. The enzyme was found to be expressed only in cells g rown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.