Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme.
The gene encoding tyrosine aminotransferase (TAT, EC 2.6.1.5) from the parasitic protozoan Trypanosoma cruzi was amplified from genomic DNA, cloned into the pET24a expression vector and functionally expressed as a C-terminally His-tagged protein in Escherichia coli BL21(DE3)pLysS. Purified recombinant TAT exhibited identical electrophoretic and enzymatic properties as the authentic enzyme from T. cruzi. Both recombinant and authentic T. cruzi TATs were highly resistant to limited tryptic cleavage and contained no disulfide bonds. Comprehensive analysis of its substrate specificity demonstrated TAT to be a broad substrate aminotransferase, with leucine, methionine as well as tyrosine, phenylalanine, tryptophan and alanine being utilized efficiently as amino donors. Valine, isoleucine and dicarboxylic amino acids served as poor substrates while polar aliphatic amino acids could not be transaminated. TAT also accepted several 2-oxoacids, including 2-oxoisocaproate and 2-oxomethiobutyrate, in addition to pyruvate, oxaloacetate and 2-oxoglutarate. The functionality of the expression system was confirmed by constructing two variants; one (Arg389) being a completely inactive enzyme; the other (Arg283) retaining its full activity, as predicted from the recently solved three-dimensional structure of T. cruzi TAT. Thus, only one of the two strictly conserved arginines which are essential for the enzymatic activity of subfamily Ialpha aspartate and aromatic aminotransferases is critical for T. cruzi's TAT activity.
Nowicki
C
C
Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina. cnowicki@criba.edu.ar
Hunter
G R
GR
Montemartini-Kalisz
M
M
Blankenfeldt
W
W
Hecht
H
H
Kalisz
H M
HM
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity / Q000502:physiology; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D014444:Tyrosine Transaminase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D000596:Amino Acids; D011994:Recombinant Proteins; D014444:Tyrosine Transaminase; D014357:Trypsin