Recombinant expression of twelve evolutionarily diverse subfamily Iα aminotransferases

Abstract

Aminotransferases are essential enzymes involved in the central metabolism of all organisms. The Iα subfamily of aspartate and tyrosine aminotransferases (AATases and TATases) is the bestcharacterized 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 Iα enzymes from an evolutionarily divergent set of organisms. Nine were purified to homogeneity after heterologous expression in E. coli in native, intein-tagged or His 6 -tagged forms and the two S. cerevisiae isoforms were recombinantly produced in yeast. The effects of the Cterminal tags on expression, purification and enzyme activity are discussed.

Molecular function prediction for a family exhibiting evolutionary tendencies toward substrate specificity swapping: Recurrence of tyrosine aminotransferase activity in the Iα subfamily

“…There is significant variance in the data presented here relative to those reported in previous work . The kinetic data presented in Table are for enzymes with C‐terminal histidine tags . No saturation of aspartate up to 40 m M was observed here for C‐terminal His 6 ‐tagged TbmAT, while Berger et al .…”

“…Malate dehydrogenase (MDH) and hydroxyisocaproate dehydrogenase (HO‐HxoDH) were prepared as described previously, except that HO‐HxoDH was expressed in Rosetta(DE3)pLysS cells (EMD, San Diego, CA) from the plasmid pHicHis described below. The cloning, expression, and purification of aminotransferases are described elsewhere …”

“…Using the D&V scores, we selected 10 distantly related aminotransferases that were previously uncharacterized to subject to kinetic analysis. As reported previously, attempts to obtain pure Arabidopsis thaliana cytosolic aminotransferase (AtcAT) were unsuccessful and the enzyme could not be characterized, but is included in the phylogenetic analyses here. The yeast cytosolic aminotransferase was also characterized because, while its crystal structure was solved, there are no reports in the literature of its kinetic activity with aromatic substrates .…”

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

“…9 Moreover, A293D enhances the specificities of HEX 9 and SRHEPT 12 ; it makes them more TATase like. SRHEPT þ A293D is a very efficient enzyme whose substrate specificity is within the range of those observed for natural TATases 13 (Kathryn Muratore, PhD thesis, UC Berkeley), and it is very similar to that of eTATase. 12 The accumulated knowledge on aminotransferase specificity was used here to design an enzyme in which one active site would behave as an AATase and the other as a TATase.…”

“…expect the 1:2:1 ratio between OCT3/WT5:OCT/ WT:OCT5/WT3, because each contains several mutations in regions that are crucial for stability, that is, the dimer interface and the N-terminal tail. [13][14][15] The diethylaminoethyl (DEAE) column elution profile in Figure 1(A) shows the successful resolution of OCT/WT from its homodimeric parents. Dimer purity was confirmed by native gel electrophoresis Eq.…”