Molecular characterization of glycosomal NAD(+)-dependent glycerol 3-phosphate dehydrogenase from Trypanosoma brucei rhodesiense.
The primary structure of a 38-kDa protein isolated from membrane preparations of African trypanosomes was determined by protein and DNA sequencing. Searching of the protein database with the trypanosome translated amino acid sequence identified glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) from various prokaryotic and eukaryotic organisms as the optimal scoring protein. Surprisingly, the eukaryotic trypanosome enzyme showed the highest degree of sequence identity with the corresponding enzyme from the prokaryote Escherichia coli. The trypanosome molecule was expressed in Escherichia coli and found to be enzymatically active, thus confirming the identity of the molecule as an NAD(+)-dependent glycerol 3-phosphate dehydrogenase. A monoclonal antibody specific for the 38-kDa protein was used to localize the enzyme to glycosomes. Immunoblotting showed that the monoclonal antibody bound to a 38-kDa protein in African trypanosomes but not in T. cruzi, Leishmania or Crithidia. The enzyme has a pI of 9.1, a net charge of +17 and contains the peroxisome-like targeting tripeptide SKM at its C-terminus, all characteristic of glycosomal enzymes. Amino acids predicted to be involved in the NAD(+)-dependent glycerol 3-phosphate dehydrogenase active site have diverged from those of the mammalian enzyme. Kinetic analyses of the trypanosome GPD and GPD from rabbit muscle showed that the Km values of the two enzymes are different. The data suggest that the trypanosome protein may be a candidate target for rational drug design.
Stebeck
C E
CE
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Frevert
U
U
Mommsen
T P
TP
Vassella
E
E
Roditi
I
I
Pearson
T W
TW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+); D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry*; D015151:Immunoblotting; D008565:Membrane Proteins / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017385:Sequence Homology; D016833:Trypanosoma brucei rhodesiense / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D009243:NAD; D005993:Glycerolphosphate Dehydrogenase; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+)