Fructose-6-phosphate Aldolase Is a Novel Class I Aldolase from Escherichia coli and Is Related to a Novel Group of Bacterial Transaldolases

Abstract

We have cloned an open reading frame from the Escherichia coli K-12 chromosome that had been assumed earlier to be a transaldolase or a transaldolase-related protein, termed MipB. Here we show that instead a novel enzyme activity, fructose-6-phosphate aldolase, is encoded by this open reading frame, which is the first report of an enzyme that catalyzes an aldol cleavage of fructose 6-phosphate from any organism. We propose the name FSA (for fructose-six phosphate aldolase; gene name fsa). The recombinant protein was purified to apparent homogeneity by anion exchange and gel permeation chromatography with a yield of 40 mg of protein from 1 liter of culture. By using electrospray tandem mass spectroscopy, a molecular weight of 22,998 per subunit was determined. From gel filtration a size of 257,000 (؎ 20,000) was calculated. The enzyme most likely forms either a decamer or dodecamer of identical subunits. The purified enzyme displayed a V max of 7 units mg ؊1 of protein for fructose 6-phosphate cleavage (at 30°C, pH 8.5 in 50 mM glycylglycine buffer). For the aldolization reaction a V max of 45 units mg ؊1 of protein was found; K m values for the substrates were 9 mM for fructose 6-phosphate, 35 mM for dihydroxyacetone, and 0.8 mM for glyceraldehyde 3-phosphate. FSA did not utilize fructose, fructose 1-phosphate, fructose 1,6-bisphosphate, or dihydroxyacetone phosphate. FSA is not inhibited by EDTA which points to a metal-independent mode of action. The lysine 85 residue is essential for its action as its exchange to arginine (K85R) resulted in complete loss of activity in line with the assumption that the reaction mechanism involves a Schiff base formation through this lysine residue (class I aldolase). Another fsa-related gene, talC of Escherichia coli, was shown to also encode fructose-6-phosphate aldolase activity and not a transaldolase as proposed earlier.

Regulation ofLactobacillus caseiSorbitol Utilization Genes Requires DNA-Binding Transcriptional Activator GutR and the Conserved Protein GutM

“…Two putative rho-independent terminators were identified downstream from gutA, one between gutA and the following gene (fsa) (⌬G, Ϫ19.4 kcal/mol) and another located in the fsa 3Ј region (⌬G, Ϫ12.3 kcal/mol). The deduced amino acid sequence of Fsa shows significant homology to transaldolase-like fructose-6-phosphate aldolases (24). Genes encoding transaldolase-like proteins have been found associated with catabolic operons in other organisms, for example, the gut operon from C. beijerinckii (27) and the sorbose operon from L. casei (33).…”

Building carbon–carbon bonds using a biocatalytic methanol condensation cycle

“…The formaldehyde assimilation is catalyzed by hexulose-6-phosphate synthase (Hps) and phosphohexulose isomerase (Phi), respectively. A similar conversion could be achieved using dihydroxyacetone synthase (12) and fructose-6-phosphate aldolase (16) as shown in Fig. S2.…”

A mechanism of covalent substrate binding in the x-ray structure of subunit K of the Escherichia coli dihydroxyacetone kinase

“…In methylotrophic yeast, Dha is the primary product of methanol assimilation (1). In bacteria, Dha is formed by oxidation of glycerol or aldol cleavage of fructose-6-phosphate (2,3). In animal cells, Dha is a gluconeogenic precursor (4)(5)(6).…”