The methylotrophic Bacillus methanolicus MGA3 possesses two distinct fructose 1,6-bisphosphate aldolases

Abstract

The thermotolerant Gram-positive methylotroph Bacillus methanolicus is able to grow with methanol, glucose or mannitol as a sole carbon and energy source. Fructose 1,6-bisphosphate aldolase (FBA), a key enzyme of glycolysis and gluconeogenesis, is encoded in the genome of B. methanolicus by two putative fba genes, the chromosomally located fba C and fba P on the naturally occurring plasmid pBM19. Their amino acid sequences share 75 % identity and suggest a classification as class II aldolases. Both enzymes were purified from recombinant Escherichia coli and were found to be active as homotetramers. Both enzymes were activated by either manganese or cobalt ions, and inhibited by ADP, ATP and EDTA. The kinetic parameters allowed us to distinguish the chromosomally encoded FBA C from the plasmid encoded FBA P , since FBA C showed higher affinity towards fructose 1,6-bisphosphate (K m of 0.16±0.01 mM as compared to 2±0.08 mM) as well as higher glycolytic catalytic efficiency (31.3 as compared to 0.8 s "1 mM "1 ) than FBA P

Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

“…Their similar thermal stabilities and temperature optima correlate well with the physiology of thermophilic B. methanolicus, which is able to grow at temperatures of between 35°C and 60°C (1). It is peculiar that the optimal temperature, especially for GlpX C , in the in vitro activity assay was higher than the temperature at which the enzyme is stable, but this is not unprecedented and has also been described for both FBAs of B. methanolicus (10) and for the FBAs of Bacillus stearothermophilus (53) and Anoxybacillus gonensis (54). However, a diverse stability of the GlpX enzymes in vitro cannot be excluded.…”

“…While it was shown previously that both enzymes are active in the cleavage of FBP as well as in the reverse aldol condensation reaction leading to FBP, the catalytic efficiencies allowed distinguishing them. FBA C is the major glycolytic enzyme and FBA P is the major gluconeogenic one from B. methanolicus (10). As E4-P is no longer commercially available, kinetic parameters could not be determined, and it remains to be shown whether one of the enzymes shows a higher catalytic efficiency for the aldol condensation of E4-P and DHAP to SBP.…”

“…E4-P and xylulose 5-phosphate (XU5-P) were generated from F-6P and GAP by transketolase from S. cerevisiae. Aldol condensation of E-4P with DHAP to yield SBP was tested for by using purified FBA C or FBA P from B. methanolicus (10). Subsequently, hydrolysis of SBP to S7-P was assayed by using purified GlpX C or GlpX P from B. methanolicus.…”

“…Fructose 1,6-bisphosphate (FBP) is cleaved to glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) by fructose 1,6-bisphosphate aldolase (FBA). B. methanolicus possesses a chromosomally encoded FBA (FBA P ) and a plasmid-encoded FBA (FBA C ) (10). FBA P is the major gluconeogenic FBA since it shows a Ͼ10-fold-higher catalytic efficiency for aldol condensation than FBA C .…”

“…FBA P is the major gluconeogenic FBA since it shows a Ͼ10-fold-higher catalytic efficiency for aldol condensation than FBA C . FBA C is the major glycolytic FBA in this bacterium, since it shows a Ͼ30-fold-higher catalytic efficiency for FBP cleavage than FBA P (10). Two different variants of the regeneration part of the RuMP pathway are known for the conversion of triosephosphates and F6-P to Ru5-P: the TA (transaldolase) variant and the SBPase (sedoheptulose 1,7-bisphosphatase) variant.…”