Recombinant expression of twelve evolutionarily diverse subfamily Ialpha aminotransferases.
Aminotransferases are essential enzymes involved in the central metabolism of all organisms. The Ialpha subfamily of aspartate and tyrosine aminotransferases (AATases and TATases) is the best-characterized grouping, but only eight enzymes from this subfamily, representing relatively little sequence diversity, have been experimentally characterized for substrate specificity (i.e., AATase vs. TATase). Genome annotation, based on this limited dataset, provides tentative assignments for all sequenced members of this subfamily. This procedure is, however, subject to error, particularly when the experimental basis set is limited. To address this problem we cloned twelve additional subfamily Ialpha enzymes from an evolutionarily divergent set of organisms. Nine were purified to homogeneity after heterologous expression in Escherichia coli in native, intein-tagged or His(6)-tagged forms. The two Saccharomyces cerevisiae isoforms were recombinantly produced in yeast. The effects of the C-terminal tags on expression, purification and enzyme activity are discussed.
Muratore
Kathryn E
KE
University of California, Berkeley, Department of Molecular and Cell Biology, Berkeley, CA 94720-3206, USA.
Srouji
John R
JR
Chow
Margaret A
MA
Kirsch
Jack F
JF
D000345:Affinity Labels; D017360:Arabidopsis / Q000201:enzymology; D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000201:enzymology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D014644:Genetic Variation; D005822:Genetic Vectors; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D000637:Transaminases / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014734:Vibrio cholerae / Q000201:enzymology
D000345:Affinity Labels; D007527:Isoenzymes; D011994:Recombinant Proteins; D006639:Histidine; D000637:Transaminases