Cloning, Expression and Purification of Yeast Aminotransferase

Abstract

Lysine is synthesized in Saccharomyces cerevisiae via the α‐aminoadipate pathway.α‐aminoadipate is made from α‐ ketoadipate via the action of a PLP‐dependent aminotransferase that has not been identified. Sequence alignments and a homology search for conserved amino acid residues were used to identify six aminotransferase genes in S. cerevisiae. Based upon the homology, the identified genes code for two aromatic aminotransferases, two aspartate aminotransferases, one kynurenine aminotransferas and one putative aminotransferase of unkown function. The cloned genes were obtained from the Harvard Institute of Proteomics Plasmid Repository. The genes were subcloned into expression vector pET16b. The expressed his‐tagged proteins have been purified using a Ni‐NTA affinity column. The aromatic aminotransferase coded by the Aro 8 gene has Km values for L‐tyrosine and L‐phenylalanine of 0.5 and 1 mM respectively. The Kcat values are about 10 s‐1 for both substrates. The uv/vis spectra of the aromatic aminotransferases have been determined as a function of pH. Substrate titrations and initial rate studies have been done to determine the substrate specificity of these enzymes.