Transaldolase of Methanocaldococcus jannaschii

• Published in: Archaea 2004, Vol.2004 (4), p.255-262
• Main Authors: Soderberg, Tim, Alver, Robert C
• Format: Article
• Language: English
• Subjects: Aldehyde-Lyases - metabolism; Amino Acid Sequence; Cloning, Molecular; Enzyme Stability; Escherichia coli - genetics; Escherichia coli Proteins - metabolism; Fructosephosphates - metabolism; Molecular Sequence Data; Molecular Weight; Pentose Phosphate Pathway - physiology; Protein Subunits; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Ribosemonophosphates - metabolism; Sequence Alignment; Substrate Specificity; Sugar Phosphates - metabolism; Temperature; Transaldolase - chemistry; Transaldolase - genetics; Transaldolase - isolation & purification; Transaldolase - metabolism
• ISSN: 1472-3646; 1472-3654
• DOI: 10.1155/2004/608428

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 retained full activity for 4 h at 80 °C, and for 3 weeks at 25 °C. Methanocaldococcus jannaschii transaldolase has a maximal velocity (V_(max)) of 1.0 ± 0.2 µmol min^(-1) mg^(-1) at 25 °C, whereas V_(max) = 12.0 ± 0.5 µmol min^(-1) mg^(-1) at 50 °C. Apparent Michaelis constants at 50 °C were K_m = 0.65 ± 0.09 mM for fructose-6-phosphate and K_m = 27.8 ± 4.3 µM for erythrose-4-phosphate. When ribose-5-phosphate replaced erythrose-4-phosphate as an aldose acceptor, V_(max) decreased twofold, whereas the K_m 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.