Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1 ′ subsite from pancreatic carboxypeptidase B

A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1 ′ subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1 ′ subsite was crystallized and its three-dimensional structure was determined at 1.29   Å resolution by X-ray crystallography. A comparison of the three-dimensional structures of CPT, the G215S/A251G/T257A/D260G/T262D CPT mutant and CPB showed that the S1 ′ subsite of CPT has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1 ′ subsite. The CPB-like mutant differs from CPB in substrate selectivity owing to differences between the two enzymes outside the S1 ′ subsite. Moreover, the difference in substrate specificity between the enzymes was shown to be affected by residues other than those that directly contact the substrate.

Source: Acta Crystallographica Section F - September 19, 2018 Category: Biochemistry Authors: Akparov, V.K. Timofeev, V.I. Kuranova, I.P. Rakitina, T.V. Tags: metallocarboxypeptidase T Thermoactinomyces vulgaris metallocarboxypeptidase B S1 ′ subsite substrate selectivity X-ray crystallography research communications Source Type: research