Triose-phosphate isomerase of Leishmania mexicana mexicana. Cloning and characterization of the gene, overexpression in Escherichia coli and analysis of the protein.
The gene of triose-phosphate isomerase in Leishmania mexicana has been cloned and characterized. The gene encodes a polypeptide of 251 amino acids, with a calculated molecular mass of 27,561 Da and a net charge of +2. Only one gene could be detected, although the enzyme is present in two different compartments of the cell, in microbody-like organelles called glycosomes and in the cytosol. The primary structure of the enzyme has many features in common with that of triose-phosphate isomerase in the related organism Trypanosoma brucei. Their sequences are 68% identical. The residues constituting the subunit interface are highly conserved between the enzyme of L. mexicana and T. brucei, but are mostly different from those in the enzyme of other organisms. One major substitution was detected in the interface region of the L. mexicana protein: a glutamate was found at position 66, instead of glutamine in all other available 20 sequences. The glutamine is thought to be important for the stability of the dimeric enzyme. L. mexicana triose-phosphate isomerase has been overexpressed in Escherichia coli. Growth conditions were established to obtain high levels of soluble and active protein. The enzyme has been purified to near homogeneity. It appears a stable dimeric protein with a specific activity of 5500 units/mg protein, a subunit mass of 28 kDa and an isoelectric point of 9.0. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. Some kinetic properties of the recombinant protein have been compared with those of the promastigote enzyme and with the values previously reported for the T. brucei enzyme. The kinetics of the different enzyme preparations were very similar. For the recombinant enzyme the following values were measured: with glyceraldehyde 3-phosphate as substrate Km = 0.30 +/- 0.05 mM and kcat = 2.5 x 10(5) min-1; with dihydroxyacetone phosphate as substrate Km = 1.3 +/- 0.3 mM and kcat = 2.8 x 10(4) min-1.
Kohl
L
L
Research Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Callens
M
M
Wierenga
R K
RK
Opperdoes
F R
FR
Michels
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology* / Q000235:genetics*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014305:Triose-Phosphate Isomerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D016054:DNA, Protozoan; D046911:Macromolecular Substances; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014305:Triose-Phosphate Isomerase