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).
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(NE1DeltaC45), 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(NE1DeltaC46). 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(NE1Delta46C) 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 alpha-helical region that ranges from Val(858) to Leu(870) is important for the thermostability of the enzyme.
Hai
Tran
T
Institute of Bioscience and Biotechnology, Myongji University, Gyeonggi-Do 449-728, Republic of Korea.
Lee
Jin-Sook
JS
Kim
Tae-Jong
TJ
Suh
Joo-Won
JW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018388:Codon, Terminator; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D046937:Nostoc / Q000201:enzymology*; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014633:Valine / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D018388:Codon, Terminator; D018698:Glutamic Acid; D010453:Peptide Synthases; C113257:cyanophycin synthase, bacteria; D007930:Leucine; D014633:Valine