Genetic engineering, production and characterisation of monomeric variants of the dimeric Serratia marcescens endonuclease.
The Serratia nuclease is a non-specific endonuclease which cleaves single- and double-stranded RNA and DNA. It is a member of a large family of related endonucleases, most of which are dimers of identical subunits, with the notable exception of the Anabaena nuclease which is a monomer. In order to find out whether the dimer state of the Serratia nuclease is essential for its function we have produced variants of this nuclease which based on the crystal structure (Miller, M.D. and Krause, K.L. (1996), Protein Science 5, 24-33) were expected to be unable to dimerise. We demonstrate here that these variants, H184A, H184N, H184T and H184R, are monomers and have the same secondary structure, stability towards chemical denaturation and activity as the wild-type enzyme. This allows to conclude that the dimeric state is not essential for the catalytic function of the Serratia nuclease. In contrast, the S179C variant which is also a monomer shows little activity, presumably because this amino acid substitution changes the structure of the enzyme.
Franke
I
I
Institut für Biochemie, Fachbereich Biologie, Justus-Liebig Universität, Giessen, Germany.
Meiss
G
G
Blecher
D
D
Gimadutdinow
O
O
Urbanke
C
C
Pingoud
A
A
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D005818:Genetic Engineering; D019791:Guanidine / Q000494:pharmacology; D006860:Hydrogen Bonding; D007700:Kinetics; D008970:Molecular Weight; D016296:Mutagenesis / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism; D012705:Serratia / Q000201:enzymology*
D001426:Bacterial Proteins; D012313:RNA; D004247:DNA; D004720:Endonucleases; D019791:Guanidine