The structural basis of beta-peptide-specific cleavage by the serine protease cyanophycinase.
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.
Law
Adrienne M
AM
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Lai
Sandy W S
SW
Tavares
John
J
Kimber
Matthew S
MS
D001426:Bacterial Proteins; D018360:Crystallography, X-Ray; D019281:Dimerization; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D046939:Synechocystis / Q000201:enzymology*
D001426:Bacterial Proteins; D010940:Plant Proteins; C024250:cyanophycin; D010447:Peptide Hydrolases; C120585:cyanophycinase