Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.
Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate 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 synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. The final specific activity of the purified enzyme was 1.12 micromol of NADH oxidized per min per mg of protein. The molecular weight of the native enzyme was determined to be 140,000. Sodium dodecyl sulfate-gel electrophoresis revealed a subunit of molecular weight 73,000. The optimum temperature and pH were 30 degrees C and 7.0, respectively. The enzyme was inactivated very rapidly at 70 degrees C. The enzyme required 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, glycolaldehyde, and formaldehyde were found to act as excellent substrates when xylulose 5-phosphate was used as a glycoaldehyde donor. The Kms for formaldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 microM, respectively. The enzyme produced dihydroxyacetone from formaldehyde and xylulose 5-phosphate. The enzyme was found to be expressed only in cells grown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.
Ro
Y T
YT
Department of Biology, Yonsei University, Seoul, Korea.
Eom
C Y
CY
Song
T
T
Cho
J W
JW
Kim
Y M
YM
D000150:Acinetobacter / Q000201:enzymology* / Q000254:growth & development; D019880:Aldehyde-Ketone Transferases; D004098:Dihydroxyacetone / Q000096:biosynthesis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007700:Kinetics; D000432:Methanol / Q000378:metabolism*; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D014166:Transferases / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D014166:Transferases; D019880:Aldehyde-Ketone Transferases; C031222:formaldehyde transketolase; D004098:Dihydroxyacetone; D000432:Methanol