The role of the C-terminal region of cyanophycin synthetase from Nostoc ellipsosporum NE1 in its enzymatic activity and thermostability: A key function of Glu 856

Abstract

The biosynthesis of cyanophycin granule polypeptides is catalyzed by cyanophycin synthetase, CphA. In this study, the role of the C-terminal region of CphA from Nostoc ellipsosporum NE1, CphA NE1, was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphA NE1 in E. coli depended on the stop codons that were used. The expression vector that had the amber stop codon TAG produced more than twice amount of CphA NE1 as a vector that contained the ochre codon TAA. CphA NE1ΔC45, which was truncated up to 45 amino acids at its C-terminus, retained full enzymatic activity and produced polymers. However, the removal of one additional amino acid, Glu 856, resulted in complete inactivation of CphA NE1ΔC46. Replacement of Glu 856 by valine or alanine confirmed the importance of this residue for the activity of CphA NE1, as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphA NE1 after removal of the region from Leu 867 to Leu 870. The gel filtration analysis showed that CphA NE1Δ46C still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu 856 is critical for CphA NE1 catalytic activity and that the predicted α-helical region that ranges from Val 858 to Leu 870 is important for the thermostability of the enzyme.