Molecular and structural characterization of NADPH-dependent d-glycerate dehydrogenase from the enteric parasitic protist Entamoeba histolytica.
Putative NADPH-dependent GDH (L-glycerate dehydrogenase) of the protozoan parasite Entamoeba histolytica (EhGDH) has been characterized. The EhGDH gene encodes a protein of 318 amino acids with a calculated isoelectric point of 6.29 and a molecular mass of 35.8 kDa. EhGDH showed highest identities with GDH from epsilon-proteobacteria. This close kinship was also supported by phylogenetic analyses, suggesting possible lateral transfer of the gene from epsilon-proteobacteria to E. histolytica. In contrast with the implications from protein alignment and phylogenetic analysis, kinetic studies revealed that the amoebic GDH showed biochemical properties similar to those of mammalian GDH, i.e. a preference for NADPH as cofactor and higher affinities towards NADPH and beta-hydroxypyruvate than towards NADP+ and L-glycerate. Whereas the amino acids involved in nucleotide binding and catalysis are totally conserved in EhGDH, substitution of a negatively charged amino acid with a non-charged hydroxy-group-containing amino acid is probably responsible for the observed high affinity of EhGDH for NADP+/NADPH. In addition, the amoebic GDH, dissimilar to the bacterial and mammalian GDHs, lacks glyoxylate reductase activity. Native and recombinant EhGDH showed comparable subunit structure, kinetic parameters and elution profiles on anion-exchange chromatography. We propose that the GDH enzyme is likely to be involved in regulation of the intracellular concentration of serine, and, thus, also in controlling cysteine biosynthesis located downstream of serine metabolic pathways in this protist.
Ali
Vahab
V
Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Shigeta
Yasuo
Y
Nozaki
Tomoyoshi
T
D000595:Amino Acid Sequence; D000818:Animals; D002237:Carbohydrate Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004748:Entamoeba histolytica / Q000201:enzymology / Q000235:genetics*; D005988:Glyceric Acids / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D010802:Phylogeny; D011773:Pyruvates / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D016054:DNA, Protozoan; D005988:Glyceric Acids; D011773:Pyruvates; D011994:Recombinant Proteins; D009249:NADP; C042971:glyceric acid; C012375:hydroxypyruvic acid; D002237:Carbohydrate Dehydrogenases; C020669:Glycerate dehydrogenase