FRUCTOSE-6-PHOSPHATE ALDOLASE IS A NOVEL CLASS I ALDOLASE FROM ESCHERICHIA COLI AND IS RELATED TO A NOVEL GROUP OF BACTERIAL TRANSALDOLASES

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 degrees 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.


INTRODUCTION
Aldolases are lyases which typically catalyze a stereoselective addition of a keto donor on an aldehyde acceptor molecule (1). Aldol condensation and cleavage reactions play crucial roles in the central sugar metabolic pathways of all organisms. For instance in glycolysis, fructose-1,6-bisphosphate is reversibly cleaved into the trioses dihydroxyacetone-phosphate and glyceraldehyde 3-phosphate, whereas in gluconeogenesis, the bisphosphate is formed through action of aldolase (fructose-1,6-bisphosphate or FBP aldolase, EC 4.1.2.13). FBP aldolases and other aldolases can be broadly divided into two groups according to their reaction mechanisms.
Class I aldolases are characterized by a covalent intermediate, which is a protonated Schiff base formed between a lysine residue and the carbonyl carbon of the substrate (2)(3)(4). Class II aldolases have an absolute requirement for a divalent metal ion which stabilizes the reaction intermediates by polarization of the substrate carbonyl (5). Class I and II aldolases vary in other criteria such as subunit structure, pH profile, and substrate affinity. They share little if any sequence homology and are apparently of different evolutionary origins (2). Class II aldolases prevail in bacteria, in fungi and algae (4). Class I FBP aldolases are mainly distributed in higher eukaryotes including animals, plants, protozoa and algae; they generally are tetramers (4). Bacterial class I FBP aldolases are known from Staphylococcus carnosus (6), Escherichia coli (7), or from the archaeon Halobacterium vallismortis (4,8). They either form monomers (S. carnosus; 6), or homodecamers (H. vallismortis; 8). Recently, a class I aldolase (dhnA; 7) has been described for Escherichia coli in addition to the well-known class II FBP aldolase of glycolysis (9).
Transaldolases (EC 2.2.1.2) are class I aldolases which serve in transfer reactions in the pentose phosphate cycle. Transaldolases use fructose-6-P as donor and transfer a dihydroxyacetone group to acceptor compounds as erythrose-4-P or glyceraldehyde-3-P (3,(15)(16)(17)(18). As a side reaction, formation of fructose-6-P from dihydroxyacetone and glyceraldehyde-3-P is known but the corresponding aldol cleavage reaction has not been documented (3). Recently, a group of gene sequences presumably encoding transaldolase-like proteins (19) have been reported as outcome of total genome analyses of various Eu-and Archaebacteria. We have cloned two of these sequences (mipB, talC) from the genome of Escherichia coli K-12. During the course of characterization of the gene products, however, we noticed that the corresponding proteins did not act as transaldolases. Instead, they perform a novel reaction, cleavage or formation of fructose-6-phosphate: were purchased from Biomol (Hamburg,Germany). Bacterial media were from Difco.

Bacterial strains and growth conditions
The bacterial strains and plasmids used in this study are listed in Table 1. The strains were grown under aeration at 37 o C in LB medium (20) with appropriate antibiotics added. Ampicillin was used in a concentration of 100 mg/l.

DNA techniques
Chromosomal DNA of E. coli strain MC4100 (21) was prepared and used as template for oligonucleotide-directed DNA amplification (22). Standard techniques for cloning (20) and transformation (23)   proteins, the subunit mass of the FSA was calculated from a plot of the log of the molecular mass versus the relative mobility on SDS-polyacrylamide gels. Purified FSA was blotted onto PVDF membranes (Immobilon-P from Millipore) in a semi-dry blot apparatus and stained with amidoblack. The protein band was cut out and subjected to N-terminal sequenation. Electrospray tandem mass spectroscopy was carried out as described (27) using a Q-TOF (Micromass, Manchester England).

Aldolase Assays
Two different assays for fructose-6-phosphate aldolase activitiy were used (all at 30°C in a Shimadzu UV160A -spectrophotometer with a thermostatted cuvette holder at a wavelength of 340 nm): I) Cleavage of fructose-6-phosphate (Fru-6-P, 50 mM) was followed using the auxiliary enzymes triosephosphate isomerase and glycerol-3-phosphate dehydrogenase to detect formation of Dglyceraldehyde 3-phosphate. The oxidation of NADH (0.5 mM) was monitored and 1 µmole of NADH oxidized was set equivalent to 1 µmole of Fru-6-P cleaved. Enzyme activities are given in U (µmol/min). The standard buffer was glycylglycine (50 mM, pH 8.5) including 1 mM DTT in a total volume of 1 ml.
II) Using the same buffer system as in I), the formation of Fru-6-P from glyceraldehyde 3phosphate and dihydroxyacetone (3 and 50mM, respectively ) was monitored by the combined enzymes phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. The reduction of NADP (0.5 mM) was followed. A prereaction of glyceraldehyde 3-phosphate with the auxiliary enzymes and NADP was run until no further NADPH formation occurred. Influence of possible 9 inhibitors of aldolase activity was measured by aldolase assays I and II. Glycerol was added at different concentrations up to 230 mM; inorganic phosphate was added up to 5 mM and EDTA was added at 10mM. Transaldolase activity was determined as described earlier (16). A dyebinding method (28) was used to estimate the concentration of protein in solution.

Cloning of the fsa (mipB) gene and expression of the plasmid-encoded aldolase
During a databank search for transaldolase-like proteins in the genome of E. coli K-12 strain MG1655 (24; accession number U00096) we found two open reading frames (ORFs) which showed a degree of identical amino acid residues in the range of 25% to the derived peptide sequence of talB (Tab. 2; 16). One of the putative ORFs ("talC") had been classified earlier by the group of Saier as a transaldolase, albeit without experimental evidence (19). The other (mipB) was originally proposed as a transaldolase-like protein (Footnote 1; 29).
In our efforts to understand the transaldolase activities of E. coli (16,17,30), we amplified the mipB-containing region with a PCR method (22)   To verify that the novel enzyme activity was the true product of the fsa (mipB) gene, the purified protein was subjected to SDS-PAGE, blotted onto a PVDF membrane and stained with amidoblack. The first 10 amino acid residues were determined by an automated Edman degradation and analysed by reversed phase high performance liquid chromatography. The sequence was determined as: The formyl-methionine was cleaved off in a portion of the sample. The N-terminal amino acid sequence was in full agreement with the sequence submitted by Isomura and co-workers (EMBL entry ECD188; SwissProt entry P78,055).

Properties of the novel aldolase
Examination of the comparative SDS gel electrophoretic mobility of the novel E. coli recombinant aldolase with a number of known reference proteins indicated a subunit mass for the purified protein of 24,000 + 1,000 (Fig. 2). Using a Q-TOF electrospray tandem mass spectrometer the molecular mass of FSA was determined to 22, 998 (Fig 3). This was in excellent agreement with the mass calculated from the deduced protein sequence (including the initial f-Met) of 22,997 Da. (SwissProt entry P78,055). The molecular mass of native E. coli recombinant aldolase was judged by gel filtration with reference proteins of known molecular 12 masses ranging from 12 to 400 kDa. Active aldolase was eluted at a volume of 152 ml buffer. In a logarithmic plot of elution volume versus molecular mass an average mass of 257,000 +/-20, 000 Da was calculated. This points to either a decameric or dodecameric structure of E. coli Fru-6-P aldolase, consisting of ten or twelve identical subunits, respectively.
The influence of different buffer substances, pH and temperature on the activity of the enzyme as well as the storage stability were analysed using enzyme assay I (see Experimental Procedures).
The auxiliary enzymes were first checked for activity under the different reaction conditions and were added to the reaction mixture in excess. As buffer substances, Tris, glycylglycine, Hepes, Imidazole, CHAS, or phosphate were used. Of these, glycylglycine (50mM) was the best buffer compound. Optimal activity was found around pH 8.5, with a broad range of activity in buffers from pH 6.0 to 12.0 . FSA was inhibited by glycerol, inorganic phosphate, and arabinose-5-phosphate, but not by EDTA (at 10mM). Rapid loss of activity was seen if kept in contact with glycerol (see Fig.   4a). After 10 min of incubation in the presence of 20% glycerol, a decrease of more than 70% of enzyme activity was found. This inhibition was fully reversible (by dilution or removal through ultrafiltration) and appeared to be of the uncompetitive type. Inorganic phosphate was a 13 competitive inhibitor with an apparent K i value of 0.22 mM (see Fig. 4b). Arabinose-5phosphate was a competitive inhibitor (K i of 0.07 mM; data not shown).

Kinetic studies on aldolase substrates
The kinetic constants K m and V max were determined in 50mM glycylglycine buffer, at Aldol forming activity of FSA (dihydroxyacetone as donor, glyceraldehyde-3-P as standard acceptor) was followed by measuring NADPH formation in the presence of phosphoglucoseisomerase and glucose-6-phosphate dehydrogenase (assay II). Aldol formation took place at a faster rate than the cleavage reaction (V max was calculated to be at 45 U/mg). Using HPLC measurements we checked whether other donor compounds are used by FSA. Dihydroxyacetone served as standard donor compound for comparison. Hydroxyacetone (acetol) served as donor but at reduced rates, erythrose and glycolaldehyde were weak acceptors (data not shown). DHAP did not serve as donor compound, nor was D-glyceraldehyde used as acceptor (i.e. no fructose was formed).

Occurrence of FSA homologs in other organisms
Databank searches with total genome sequences from various eu-and archaebacterial microorganisms revealed sequences with apparent homology to FSA. Databank searches were 14 done using the NCBI Blast server (25). Preliminary sequence data were obtained, a.o., from The Institute for Genomic Research website at http://www.tigr.org. In E. coli, another sequence is present (talC, see above) which shared 68% identical (79% similar) residues with FSA. fsarelated genes with prominent similarity were only found in prokaryotic genomes such as in To test whether this conserved lysyl-residue indeed fulfills a function in enzyme activity, we changed the Lys-85 residue to an arginine residue by site-directed mutagenesis (see Fig.1 and Experimental procedures for details). The K85R mutein was expressed at good quantity and was purified through the same procedure as wild-type FSA. The K85R mutein nearly lacked enzyme activity (less than 0.03 U/mg of protein), both for cleavage of fructose-6-phosphate or its formation. We propose that FSA is therefore likely to be a class I aldolase with a reactive lysine residue (Lys-85).
As the talC gene from E. coli showed striking similarity to the fsa gene, we tested whether it also encoded an aldolase activity. Recombinant strains of E. coli carrying a high-copy number plasmid with the PCR-amplified talC-gene, indeed showed fructose-6-phosphate aldolase activity in the crude extracts. The purified protein lacked transaldolase activity and is thus the second example of a fructose-6-phosphate aldolase (although with reduced specific activities when compared with FSA; data not shown). In order to find whether other related proteins included in Fig. 5 display transaldolase or the novel fructose 6-P aldolase activities, we 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; this could be best interpreted if the reaction mechanism involves a Schiff base formation through this lysine residue. This we take for evidence that FSA is a class I aldolase.
To our knowledge, this is the first report on a genuine fructose-6-phosphate aldolase from any source. As we show here, the gene talC of E. coli, also encodes a fructose-6-phosphate aldolase and not a transaldolase as proposed earlier (19 We were not able to determine the reaction equilibrium constants due to the rapid chemical degradation of one of the cleavage products, glyceraldehyde 3-phosphate (data not shown). However, we estimated a standard free energy change of reaction ∆G 0´o f + 32 kJ mole -1 , which is about 10 kJ mole -1 more endergonic than the fructose-bisphosphate cleavage reaction (33). If the subsequent reactions cannot compensate for this strongly endergonic reaction it is not likely that