Transaldolase of Methanocaldococcus jannaschii

Abstract

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.

Figure 1.

Comparison of the probable reaction mechanisms of fructose-6-phosphate aldolase and transaldolase. Aldolase cleaves the fructose-6-phosphate substrate directly into glyceraldehyde-3-phosphate and dihydroxyacetone, whereas transaldolase requires an aldose substrate (such as erythrose-4-phosphate) to which it transfers the dihydroxyacetone group. Abbreviations: F6P = fructose-6-phosphate; GAP = glyceraldehyde-3-phosphate; E4P = erythrose-4-phosphate; S7P = sedoheptulose-7-phosphate; and DHA = dihydroxyacetone.

Figure 2.

SDS-PAGE analysis of purified M. jannaschii transaldolase. Lane 1: 10 kDa protein molecular mass marker. Lane 2: Crude extract. Lane 3: Crude extract after heat treatment. Lane 4: DE52-purified transaldolase.

Figure 3.

Requirement for an acceptor substrate for enzyme turnover. Each value represents the mean of triplicate assays. Erythrose-4-phosphate was added at the 5 min mark. See Materials and methods for details.

Figure 4.

Alignment of M. jannaschii transaldolase (MJ0960) and E. coli FSA (Ec FSA) sequences. Conserved residues among 40 transaldolase and six FSA sequences are in bold, with conservative substitutions in parentheses above or below the sequence. The regions corresponding to the eight β sheets of the FSA α/β barrel structure (Thorell et al. 2002) are underlined. Arrows indicate conserved residues that are unique to the transaldolase sequences and that are in or near the probable active site of the enzyme.