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J. Biol. Chem., Vol. 276, Issue 14, 11055-11061, April 6, 2001
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From the Institut für Biotechnologie 1, Forschungszentrum
Jülich GmbH, P. O. Box 1913, D-52425 Jülich, Germany
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 Vmax of 7 units
mg
Fructose-6-phosphate Aldolase Is a Novel Class I Aldolase
from Escherichia coli and Is Related to a Novel Group
of Bacterial Transaldolases*
1 of protein for fructose 6-phosphate
cleavage (at 30 °C, pH 8.5 in 50 mM glycylglycine
buffer). For the aldolization reaction a Vmax
of 45 units mg1 of protein was found;
Km 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.
*
This work was supported by Grant SFB380/B21 of the Deutsche
Forschungsgemeinschaft.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-2461-616205;
Fax: 49-2461-612710; E-mail: g.sprenger@fz-juelich.de.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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