Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of <i>Escherichia coli</i>

Alefounder, Peter R.; Baldwin, S. A.; Perham, Richard N.; Short, Nicholas J.

doi:10.1042/bj2570529

Cited by 63 publications

(28 citation statements)

References 33 publications

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“…I). The E. coli enzyme contains six arginine residues (Alefounder et al, 1989), and this result suggests either that more than one of these residues are accessible for modification by phenylglyoxal and that their modification in some way affects the activity of the enzyme, or that more than one arginine residue is involved in the substrate recognition process. The number of arginines involved in substrate binding was therefore determined by carrying out the modification of the enzyme with [7-14C] phenylglyoxal (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…No crystal structure is yet available for a Class I1 FBP-aldolase, and so it remains unclear whether, and how, the mode of substrate binding is different in the two classes. As part of a study of the use of protein engineering to produce novel enzyme activities with use in biotechnology, the Class I1 FBPaldolase of Escherichia coli has been cloned (Alefounder et al, 1989) and a program of X-ray crystallography and characterization has been initiated (Naismith et al, 1992;Berry & Marshall, 1993;Kitagawa et al, 1995;Packman & Berry, 1995). In this paper, we extend this characterization to a study of the substrate binding site.…”

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confidence: 99%

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Identification of arginine 331 as an important active site residue in the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli

1996

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Treatment of the Class 11 fructose-1.6-bisphosphate aldolase of Escherichia coli with the arginine-specific a-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate. Modification with [7-"C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class I1 FBP-aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg-331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased the K,,, for fructose 1.6-bisphosphate. Comparatively small differences in the inhibitor constant K, for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (111) revealed that the K , for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate.

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Section: Resultsmentioning

confidence: 99%

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Identification of arginine 331 as an important active site residue in the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli

1996

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“…Recently, a class I aldolase (dhnA; see Ref. 7) has been described for E. coli in addition to the well known class II FBP aldolase of glycolysis (9).…”

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

Schürmann

Sprenger

2001

Journal of Biological Chemistry

187

171

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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.

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“…This mass was larger (+79.1 Da) than expected from thefdu gene sequence (39 016.1 Da) [23] allowing for post-translational removal of the N-terminal methionine [13,241. Although this extra mass was consistent with phosphorylation of the enzyme subunit (+SO Da), the mass of a control sample of the enzyme prepared in the absence of 2-mercaptoethanol was found to be 39013.4 2 2.1 Da (Table l), in excellent agreement with that of 39016.1 Da expected.…”

Section: Resultsmentioning

confidence: 85%

A Reactive, Surface Cysteine Residue of the Class‐II Fructose‐1,6‐Bisphosphate Aldolase of Escherichia coli Revealed by Electrospray Ionisation Mass Spectrometry

Packman

Berry

1995

European Journal of Biochemistry

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The state of post-translational modification of the class-I1 fructose-I ,6-bisphosphate aldolase (FBPaldolase) purified from Escherichia coli was examined by electrospray ionisation mass spectrometry (ESI-MS). The mass was larger than that expected from the known DNA sequence by approximately 80 ? 6 Da, suggesting the presence of a covalent modification on the protein. Phosphorylation (+80 Da), a known modification in an FBP-aldolase from Bacillus subtilis and a suspected modification in this E. coli aldolase, was ruled out as the extra mass was readily removed by treatment with dithiothreitol. Purification of aldolase by a protocol which omitted 2-mercaptoethanol from all buffers resulted in the purified protein having the expected mass (39016 Da). The extra mass was therefore established as a covalent adduct of the protein with 2-mercaptoethanol (+76 Da). Reduction and alkylation studies, followed by isolation of tryptic peptides, established that the site of attachment was Cys36. Although no significant effect of the modification on the activity of the protein was observed, the study underlines the ease with which a protein can be modified covalently by a simple and mild purification procedure; such labelling, which may not always be benign, would be undetectable without the routine use of mass spectrometric analysis.Keywords. Fructose-l,6-bisphosphate aldolase ; mass spectrometry ; 2-mercaptoethanol ; cysteine residue ; protein chemistry.Aldolases can be classified into two groups depending on their molecular properties and their catalytic mechanism [l, 21. The most widely studied are the class-I fmctose-I ,6-bisphosphate aldolases (FBP-aldolases). This enzyme catalyses the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The catalytic mechanism of the class-I aldolases proceeds via a carbanion intermediate stabilised by a protonated Schiff base formed between the substrate and an active-site lysine residue [3-51. In contrast, the class-I1 enzymes utilise a bound metal ion to fulfil a similar role 161.X-ray structures of a number of class-I aldolases, including the FBP-aldolases from rabbit 17, 81 and human tissues [9, 101 and from Drosophilu [ I l l have been determined. They are tetramers of molecular mass around 160kDa and are members of the a/p barrel family of enzymes. Comparatively little is known about the class-I1 aldolases. They are dimers of molecular mass around 80kDa and bind one zinc atodsubunit. The only detailed structural information available for a class-I1 aldolase is for the fuculose-I-phosphate aldolase of Escherichia coli [12]. Unlike the class-I enzymes, this enzyme does not adopt the alp barrel structure.In order to increase our understanding of the class-I1 aldolases, we have developed an expression system for the class- I1 FBP-aldolase from E. coli [13] which yields large amounts (approximately 1 g/l in crude extracts) of the enzyme, and a program of site-directed mutagenesis 1131 and X-ray crystallographic analysis has ...

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Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli

Cited by 63 publications

References 33 publications

Identification of arginine 331 as an important active site residue in the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli

Identification of arginine 331 as an important active site residue in the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli

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

A Reactive, Surface Cysteine Residue of the Class‐II Fructose‐1,6‐Bisphosphate Aldolase of Escherichia coli Revealed by Electrospray Ionisation Mass Spectrometry

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