Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichiacoli and is related to a novel group of bacterial transaldolases

We have cloned an open reading frame from the Escherichia coli K-12 chromos ome that had been assumed earlier to be a transaldolase or a transaldolase- related protein, termed MipB, Here we show that instead a novel enzyme acti vity, 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 fruct ose-six phosphate aldolase; gene name fsa). The recombinant protein was pur ified to apparent homogeneity by anion exchange and gel permeation chromato graphy with a yield of 40 mg of protein from 1 liter of culture. By using e lectrospray tandem mass spectroscopy, a molecular weight of 22,998 per subu nit 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 i dentical subunits, The purified enzyme displayed a V-max of 7 units mg(-1) of protein for fructose B-phosphate cleavage (at 30 degreesC, 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 f ructose 6-phosphate, 35 mM for dihydroxyacetone, and 0.8 mM for glyceraldeh yde 3-phosphate. FSA did not utilize fructose, fructose l-phosphate, fructo se 1,6-bisphosphate, or dihydroxyacetone phosphate. FSA is not inhibited by EDTA which points to a metal-independent mode of action. The lysine 85 res idue is essential for its action as its exchange to arginine (K85R) resulte d in complete loss of activity in line with the assumption that the reactio n mechanism involves a Schiff base formation through this lysine residue (c lass I aldolase). Another fsa-related gene, talC of Escherichia coli, was s hown to also encode fructose-6-phosphate aldolase activity and not a transa ldolase as proposed earlier.