Summary: Recombinant expression of twelve evolutionarily diverse
subfamily Ia aminotransferases
Kathryn E. Muratore 1
, John R. Srouji, Margaret A. Chow 2
, Jack F. Kirsch *
University of California, Berkeley, Department of Molecular and Cell Biology, Berkeley, CA 94720-3206, USA
Received 9 July 2007, and in revised form 2 September 2007
Available online 14 September 2007
Aminotransferases are essential enzymes involved in the central metabolism of all organisms. The Ia subfamily of aspartate and tyro-
sine aminotransferases (AATases and TATases) is the best-characterized grouping, but only eight enzymes from this subfamily, repre-
senting 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 pro-
cedure is, however, subject to error, particularly when the experimental basis set is limited. To address this problem we cloned twelve
additional subfamily Ia enzymes from an evolutionarily divergent set of organisms. Nine were purified to homogeneity after heterologous
expression in Escherichia coli in native, intein-tagged or His6-tagged forms. The two Saccharomyces cerevisiae isoforms were recombi-
nantly produced in yeast. The effects of the C-terminal tags on expression, purification and enzyme activity are discussed.
Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Aminotransferase; Pyridoxal phosphate; Intein; Heterologous expression; Substrate specificity; Annotation
Aminotransferases are pivotal enzymes in cellular