Characterization of two isotypes of l-threonine dehydratase from Entamoeba histolytica.
The genome sequence of the enteric protozoan parasite Entamoeba histolytica suggests that amino acid catabolism plays an important role in energy metabolism. In the present study, we described kinetic and regulatory properties of catabolic l-threonine and l-serine dehydratase (TD) from E. histolytica. TD catalyses the pyridoxal phosphate-dependent dehydrative deamination of l-threonine and l-serine to ammonia and keto acids (2-oxobutyrate and pyruvate, respectively). E. histolytica possesses two TD isotypes (EhTD1-2) showing 38% mutual identity, a calculated molecular mass of 45.0 or 46.5kDa, and an isoelectric point of 6.68 or 5.88, respectively. Only EhTD1 showed l-threonine and l-serine dehydrative deaminating activities whereas EhTD2, in which the amino acid residues involved in the substrate and cofactor binding were not conserved, was devoid of these activities. The k(cat)/K(m) value of EhTD1 was >3 fold higher for l-threonine than l-serine. EhTD1 was inhibited by l-cysteine in a competitive manner with the K(i) values of 1.1mM and 2.2mM for l-serine and l-threonine, respectively. EhTD1 was insensitive to the allosteric activation by AMP or CMP. Three major substitutions of EhTD1 likely attribute to the insensitivity. EhTD1 was also inhibited about 50% by 20mM 2-oxobutyrate, pyruvate, and glyoxylate; the inhibition was not, however, reversed by AMP. Together, these data showed that EhTD1 possesses unique regulatory properties distinct from other organisms and may play an important role in energy metabolism via amino acid degradation in E. histolytica.
Husain
Afzal
A
Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Jeelani
Ghulam
G
Sato
Dan
D
Ali
Vahab
V
Nozaki
Tomoyoshi
T
D000249:Adenosine Monophosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D002264:Carboxylic Acids / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology; D003568:Cytidine Monophosphate / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011730:Pyridoxal / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*; D013913:Threonine Dehydratase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D002264:Carboxylic Acids; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D015800:Protozoan Proteins; D013912:Threonine; D011730:Pyridoxal; D000249:Adenosine Monophosphate; D012694:Serine; D000641:Ammonia; D013913:Threonine Dehydratase; D003568:Cytidine Monophosphate; D003545:Cysteine