1887

Abstract

The thermotolerant Gram-positive methylotroph 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 by two putative genes, the chromosomally located and 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 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 from the plasmid encoded FBA, since FBA showed higher affinity towards fructose 1,6-bisphosphate ( 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 mM) than FBA. However, FBA exhibited a higher catalytic efficiency in gluconeogenesis (50.4 as compared to 1.4 s mM with dihydroxyacetone phosphate and 4 as compared to 0.4 s mM with glyceraldehyde 3-phosphate as limiting substrate). The aldolase-negative mutant Δ could be complemented with both FBA genes from . Based on the kinetic data, we propose that FBA acts as major aldolase in glycolysis, whereas FBA acts as major aldolase in gluconeogenesis in .

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2013-08-01
2021-10-21
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