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 fbaC and fbaP 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 FBAC from the plasmid encoded FBAP, since FBAC showed higher affinity towards fructose 1,6-bisphosphate (Km 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 FBAP. However, FBAP exhibited a higher catalytic efficiency in gluconeogenesis (50.4 as compared to 1.4 s−1 mM−1 with dihydroxyacetone phosphate and 4 as compared to 0.4 s−1 mM−1 with glyceraldehyde 3-phosphate as limiting substrate). The aldolase-negative Corynebacterium glutamicum mutant Δfda could be complemented with both FBA genes from B. methanolicus. Based on the kinetic data, we propose that FBAC acts as major aldolase in glycolysis, whereas FBAP acts as major aldolase in gluconeogenesis in B. methanolicus.
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