Covalent joining of the subunits of a homodimeric type II restriction endonuclease: single-chain PvuII endonuclease.
The PvuII restriction endonuclease has been converted from its natural homodimeric form into a single polypeptide chain by tandemly linking the two subunits through a short peptide linker. The arrangement of the single-chain PvuII (sc PvuII) is (2-157)-GlySerGlyGly-(2-157), where (2-157) represents the amino acid residues of the enzyme subunit and GlySerGlyGly is the peptide linker. By introducing the corresponding tandem gene into Escherichia coli, PvuII endonuclease activity could be detected in functional in vivo assays. The sc enzyme was expressed at high level as a soluble protein. The purified enzyme was shown to have the molecular mass expected for the designed sc protein. Based on the DNA cleavage patterns obtained with different substrates, the cleavage specificity of the sc PvuII is indistinguishable from that of the wild-type (wt) enzyme. The sc enzyme binds specifically to the cognate DNA site under non-catalytic conditions, in the presence of Ca2+, with the expected 1:1 stoichiometry. Under standard catalytic conditions, the sc enzyme cleaves simultaneously the two DNA strands in a concerted manner. Steady-state kinetic parameters of DNA cleavage by the sc and wt PvuII showed that the sc enzyme is a potent, but somewhat less efficient catalyst; the k(cat)/K(M) values are 1.11 x 10(9) and 3.50 x 10(9) min(-1) M(-1) for the sc and wt enzyme, respectively. The activity decrease is due to the lower turnover number and to the lower substrate affinity. The sc arrangement provides a facile route to obtain asymmetrically modified heterodimeric enzymes.
Simoncsits
A
A
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy. simoncs@icgeb.trieste.it
Tjörnhammar
M L
ML
Raskó
T
T
Kiss
A
A
Pongor
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011514:Proteus vulgaris / Q000201:enzymology* / Q000235:genetics; D012995:Solubility; D013379:Substrate Specificity; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D021122:Protein Subunits; D004247:DNA; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D002118:Calcium