CARBOXYPEPTIDASE TAQ, A THERMOSTABLE ZINC ENZYME, FROM THERMUS-AQUATICUS YT-1 - MOLECULAR-CLONING, SEQUENCING, AND EXPRESSION OF THE ENCODING GENE IN ESCHERICHIA-COLI

The gene for carboxypeptidase Tag, a thermostable metallo-carboxypeptidase from Thermus aquaticus YT-1, was cloned and sequenced. The gene comprised an open reading frame of 1,536 base pairs with a GTG initiation codon and a TGA termination codon, which encodes a protein of 56,210 Da consisting of 511 amino acid residues. The GTG initiation codon of the gene was replaced with ATG by site-directed mutagenesis, and then the gene was expressed in Escherichia coli. The enzyme purified from E. coli cells showed the same properties as those of carboxypeptidase Tag prepared from T. aquaticus cells. Analysis for metal ions bound to the enzyme found that one molecule of the enzyme contains one tightly bound zinc ion. Comparison of the entire sequence showed that the enzyme has no obvious sequence similarity to any other metallo-peptidases, However, a His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent endopeptidases and aminopeptidases, was found at positions 276 to 280 of the enzyme. These findings suggest that carboxypeptidase Tag is a novel type of zinc-dependent metallocarboxypeptidase.