The Structural Basis of [beta]-Peptide-Specific Cleavage by the Serine Protease Cyanophycinase

Journal Article · Fri Oct 01 04:00:00 EDT 2010 · J. Mol. Biol.

DOI:https://doi.org/10.1016/j.jmb.2009.07.001· OSTI ID:1006149

Law, Adrienne M; Lai, Sandy W.S.; Tavares, John; Kimber, Matthew S

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-{angstrom} 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{sub cat} of 16.5 s{sup -1} and a k{sub cat}/K{sub M} of 7.5 x 10{sup -6} M{sup -1} s{sup -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.

• Research Organization: Argonne National Laboratory (ANL)
• Sponsoring Organization: USDOE
• OSTI ID: 1006149
• Journal Information: J. Mol. Biol., Journal Name: J. Mol. Biol. Journal Issue: (2) ; 01, 2009 Vol. 392; ISSN JMOBAK; ISSN 0022-2836
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
CATALYSIS
CLEAVAGE
CYANOBACTERIA
ENZYMES
KINETICS
MUTAGENESIS
NITROGEN
PEPTIDES
POLYMERS
RESIDUES
RESOLUTION
SERINE
SIMULATION
STORAGE
SUBSTRATES
SYNTHESIS