Fructose diphosphate aldolase from Mycobacterium smegmatis

Investigation of aldolase 1, the class-I D-fructose 1,6-bisphosphate aldolase (EC4.1.2.13) from Escherichia coli (Crookes' strain), showed it to have unusual kinetic and structural properties. The enzyme appeared to be larger than was previously supposed and may be a decamer with a mol. wt. of approx. 340000. Its fructose 1,6-bisphosphate-cleavage activity was unaffected by these compounds. The enhancement exhibited a strong dependence on pH. These novel kinetic properties do not seem to be shared by any other fructose 1,6-bisphosphate aldolase, but recall the activation by polycarboxylic acids of the deoxyribose 3-phosphate aldolases from some other organisms. In view of its unusual properties, it is unlikely that aldolase 1 from E. coli is closely related to the class-1 aldolases that have been detected in several other prokaryotes, or to the typical class-1 enzymes from eukaryotes.

Section: Discussionmentioning

confidence: 99%

Novel kinetic and structural properties of the class-I D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes' strain)

Baldwin¹,

Perham²

1978

“…Excellent crystal structures are available for several eukaryotic Class I FBP aldolases [3][4][5], revealing that these proteins are members of the α\β-barrel family of proteins, of which triosephosphate isomerase is the archetype. Class I FBP aldolases in prokaryotes have been discovered in Escherichia coli [6,7], Micrococcus aerogenes [8,9], Lactobacillus casei [10], Mycobacterium smegmatis [11,12], Staphylococcus aureus [13], Staphylococcus carnosus [14], Synechocystis sp. strain PCC6803 [15] and Haloarcula allismortis [16].…”

Section: Introductionmentioning

confidence: 99%

The dhnA gene of Escherichia coli encodes a Class I fructose bisphosphate aldolase

Thomson

Howlett

Ashcroft

et al. 1998

The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBanktrade mark database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies.

“…In contrast with this wealth of information about the Class I aldolases, comparatively little is known about the Class II enzymes. The aldolase of yeast is metaldependent, as are the aldolases of most prokaryotes studied (Rutter, 1964), but at least some prokaryotes, such as Escherichia coli (Stribling & Perham, 1973;Baldwin & Perham, 1978), Lactobacillus casei (London, 1974) and Mycobacterium smegmatis (Bai et al, 1975), have been shown also to contain Class I enzymes. The Class II aldolases of Saccharomyces cerevisiae (Rutter, 1964;Jack & Harris, 1971) and E. coli (Baldwin et al, 1978) are perhaps the best characterized.…”

Section: Introductionmentioning

confidence: 99%

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

Alefounder

Baldwin

Perham

et al. 1989

Nucleotide sequence analysis of the Escherichia coli chromosomal DNA inserted in the plasmid pLC33-5 of the Clarke and Carbon library [Clarke & Carbon (1976) Cell 9, 91-99] revealed the existence of the gene, fda, encoding the Class II (metal-dependent) fructose 1,6-bisphosphate aldolase of E. coli. The primary structure of the polypeptide chain inferred from the DNA sequence of the fda gene comprises 359 amino acids, including the initiating methionine residue, from which an Mr of 39,146 could be calculated. This value is in good agreement with that of 40,000 estimated from sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified dimeric enzyme. The amino acid sequence of the Class II aldolase from E. coli showed no homology with the known amino acid sequences of Class I (imine-forming) fructose 1,6-bisphosphate aldolases from a wide variety of sources. On the other hand, there was obvious homology with the N-terminal sequence of 40 residues already established for the Class II fructose 1,6-bisphosphate aldolase of Saccharomyces cerevisiae. These Class II aldolases, one from a prokaryote and one from a eukaryote, evidently are structurally and evolutionarily related. A 1029 bp-fragment of DNA incorporating the fda gene was excised from plasmid pLC33-5 by digestion with restriction endonuclease HaeIII and subcloned into the expression plasmid pKK223-3, where the gene came under the control of the tac promoter. When grown in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside, E. coli JM101 cells transformed with this recombinant expression plasmid generated the Class II fructose 1,6-bisphosphate aldolase as approx. 70% of their soluble protein. This unusually high expression of an E. coli gene should greatly facilitate purification of the enzyme for any future structural or mechanistic studies.