The role of the C-terminal region of cyanophycin synthetase from Nostoc ellipsosporum NE1 in its enzymatic activity and thermostability: A key function of Glu856

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, CphANE1, was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphANE1 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 CphANE1 as a vector that contained the ochre codon TAA. CphANE1ΔC45, 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, Glu856, resulted in complete inactivation of CphANE1ΔC46. Replacement of Glu856 by valine or alanine confirmed the importance of this residue for the activity of CphANE1, as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphANE1 after removal of the region from Leu867 to Leu870. The gel filtration analysis showed that CphANE1Δ46C still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu856 is critical for CphANE1 catalytic activity and that the predicted α-helical region that ranges from Val858 to Leu870 is important for the thermostability of the enzyme.