The Structural Basis of β-Peptide-Specific Cleavage by the Serine Protease Cyanophycinase

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-Å 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 7.5 × 10 − 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 β-Asp–Arg dipeptide residue immediately N-terminal to the scissile bond in an extended conformation in this pocket, primarily recognizing this penultimate β-Asp–Arg residue of the polymeric chain. Because binding and catalysis depend on substrate features unique to β-linked aspartyl peptides, cyanophycinase is able to act within the cytosol without non-specific cleavage events disrupting essential cellular processes.