Protein engineering of BamHI restriction endonuclease: replacement of Cys54 by Ala enhances catalytic activity.
Chemical modification studies of BamHI endonuclease indicated the importance of the cysteine residue in catalysis [Nath, K. (1981) Arch. Biochem. Biophys, 212, 611-617]. Of the three cysteine residues at positions 34, 54 and 64 in the BamHI endonuclease Cys54 and Cys64 are at the DNA-protein interface. The co-crystal structure of the BamHI-DNA complex, however, does not indicate any role of cysteines either in binding or catalysis. In the context of strong biochemical evidence, Cys54 in BamHI was changed to Ala54 to investigate its role in catalysis. The mutation was carried out by PCR overlap extension, the mutant gene was cloned and characterized by sequencing. The mutant BamHI was expressed and purified to homogeneity and the kinetic parameters (K(M) and kcat) of the wild type and the C54A mutant were determined. The mutation results in up to approximately 40% enhancement of kcat and some increase in K(M). These in vitro results were also supported by in vivo SOS induction assays: the C54A mutant gene under the T7 promoter caused complete lysis in JH139 in absence of T7 RNA polymerase whereas the wild-type gene gave deep blue colonies under the same conditions. The results suggest no direct role of Cys54 in catalysis, but it can influence the catalytic activity through Val57 backbone contact seen in the co-crystal structure.
Mukhopadhyay
P
P
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Roy
K B
KB
D000409:Alanine / Q000235:genetics*; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology; D010582:Bacteriophage lambda; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013014:SOS Response, Genetics; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D004247:DNA; D015245:Deoxyribonuclease BamHI; D003545:Cysteine; D000409:Alanine