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The structural basis of beta-peptide-specific cleavage by the serine protease cyanophycinase.
- Source :
-
Journal of molecular biology [J Mol Biol] 2009 Sep 18; Vol. 392 (2), pp. 393-404. Date of Electronic Publication: 2009 Jul 08. - Publication Year :
- 2009
-
Abstract
- Cyanophycin, or poly-L-Asp-multi-L-Arg, is a non-ribosomally synthesized peptidic polymer that is used for nitrogen storage by cyanobacteria and other select eubacteria. Upon synthesis, it self-associates to form insoluble granules, the degradation of which is uniquely catalyzed by a carboxy-terminal-specific protease, cyanophycinase. We have determined the structure of cyanophycinase from the freshwater cyanobacterium Synechocystis sp. PCC6803 at 1.5-A resolution, showing that the structure is dimeric, with individual protomers resembling aspartyl dipeptidase. Kinetic characterization of the enzyme demonstrates that the enzyme displays Michaelis-Menten kinetics with a k(cat) of 16.5 s(-1) and a k(cat)/K(M) of 7.5x10(-6) M(-1) s(-1). Site-directed mutagenesis experiments confirm that cyanophycinase is a serine protease and that Gln101, Asp172, Gln173, Arg178, Arg180 and Arg183, which form a conserved pocket adjacent to the catalytic Ser132, are functionally critical residues. Modeling indicates that cyanophycinase binds the beta-Asp-Arg dipeptide residue immediately N-terminal to the scissile bond in an extended conformation in this pocket, primarily recognizing this penultimate beta-Asp-Arg residue of the polymeric chain. Because binding and catalysis depend on substrate features unique to beta-linked aspartyl peptides, cyanophycinase is able to act within the cytosol without non-specific cleavage events disrupting essential cellular processes.
- Subjects :
- Bacterial Proteins
Crystallography, X-Ray
Dimerization
Kinetics
Models, Biological
Models, Molecular
Mutagenesis, Site-Directed
Peptide Hydrolases genetics
Protein Structure, Quaternary
Peptide Hydrolases chemistry
Peptide Hydrolases metabolism
Plant Proteins metabolism
Synechocystis enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 1089-8638
- Volume :
- 392
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 19591842
- Full Text :
- https://doi.org/10.1016/j.jmb.2009.07.001