Role of Allosteric Switch Residue Histidine 195 in Maintaining Active-Site Asymmetry in Presynaptic Filaments of Bacteriophage T4 UvsX Recombinase |
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Authors: | Joshua N Farb |
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Institution: | Departments of Biochemistry, and Microbiology and Molecular Genetics, and Vermont Cancer Center, University of Vermont College of Medicine, Burlington, VT 05405, USA |
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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 kcat of 16.5 s? 1 and a kcat/KM 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. |
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Keywords: | ssDNA single-stranded DNA EcRecA Escherichia coli RecA ScRad51 Saccharomyces cerevisiae Rad51 dsDNA double-stranded DNA RFIII replicative fragment III TLC thin-layer chromatography PPi inorganic pyrophosphate Pi inorganic phosphate EDTA ethylenediaminetetraacetic acid NCBI National Center for Biotechnology Information PDB Protein Data Bank |
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