Transaldolase of Methanocaldococcus jannaschii.
The Methanocaldococcus jannaschii genome contains putative genes for all four nonoxidative pentose phosphate pathway enzymes. Open reading frame (ORF) MJ0960 is a member of the mipB/talC family of 'transaldolase-like' genes, so named because of their similarity to the well-characterized transaldolase B gene family. However, recently, it has been reported that both the mipB and the talC genes from Escherichia coli encode novel enzymes with fructose-6-phosphate aldolase activity, not transaldolase activity (Schürmann and Sprenger 2001). The same study reports that other members of the mipB/talC family appear to encode transaldolases. To confirm the function of MJ0960 and to clarify the presence of a nonoxidative pentose phosphate pathway in M. jannaschii, we have cloned ORF MJ0960 from M. jannaschii genomic DNA and purified the recombinant protein. MJ0960 encodes a transaldolase and displays no fructose-6-phosphate aldolase activity. It etained full activity for 4 h at 80 degrees C, and for 3 weeks at 25 degrees C. Methanocaldococcus jannaschii transaldolase has a maximal velocity (Vmax) of 1.0 +/- 0.2 micromol min(-1) mg(-1) at 25 degrees C, whereas Vmax = 12.0 +/- 0.5 micromol min(-1) mg(-1) at 50 degrees C. Apparent Michaelis constants at 50 degrees C were Km = 0.65 +/- 0.09 mM for fructose-6-phosphate and Km = 27.8 +/- 4.3 microM for erythrose-4-phosphate. When ribose-5-phosphate replaced erythrose-4-phosphate as an aldose acceptor, Vmax decreased twofold, whereas the Km was 150-fold higher. The molecular mass of the active enzyme is 271 +/- 27 kDa as estimated by gel filtration, whereas the predicted monomer size is 23.96 kDa, suggesting that the native form of the protein is probably a decamer. A readily available source of thermophilic pentose phosphate pathway enzymes including transaldolase may have direct application in enzymatic biohydrogen production.
Soderberg
Tim
T
Division of Science and Mathematics, University of Minnesota, Morris, 600 E. 4th Street, Morris, MN 56267, USA. soderbt@mrs.umn.edu
Alver
Robert C
RC
D000446:Aldehyde-Lyases / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010427:Pentose Phosphate Pathway / Q000502:physiology; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism; D013696:Temperature; D014153:Transaldolase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D029968:Escherichia coli Proteins; D005636:Fructosephosphates; D021122:Protein Subunits; D011994:Recombinant Proteins; D012267:Ribosemonophosphates; D013403:Sugar Phosphates; C031626:ribose-5-phosphate; C026959:erythrose 4-phosphate; C027618:fructose-6-phosphate; D014153:Transaldolase; D000446:Aldehyde-Lyases; C426024:fsaA protein, E coli