Open AccessN-terminal amino acid sequence of the novel type IIIb trimethoprim-resistant plasmid-encoded dihydrofolate reductase from Shigella sonnei
Author(s) -
C. J. Thomson,
Neil Barg,
S. G. B. Amyes
Publication year - 1990
Publication title -
journal of general microbiology/journal of general microbiology
Language(s) - English
Resource type - Journals
eISSN - 2059-9323
pISSN - 0022-1287
DOI - 10.1099/00221287-136-4-673
Subject(s) - dihydrofolate reductase , enzyme , biochemistry , plasmid , trimethoprim , shigella sonnei , biology , microbiology and biotechnology , amino acid , peptide sequence , affinity chromatography , chemistry , escherichia coli , shigella , dna , gene , antibiotics
The type IIIb dihydrofolate reductase, a novel plasmid-encoded enzyme recently identified in Shigella sonnei, has been shown to have some similar biochemical properties to the type IIIa dihydrofolate reductase which was first identified in New Zealand in 1979. However, the type IIIb enzyme has a Ki for trimethoprim of 0.4 microM, and a pI of 5.35 (as compared to 19 nM and 6.1 for the type IIIa); both these results suggest that it is a different enzyme from the prototype type IIIa. The type IIIb dihydrofolate reductase was purified by methotrexate agarose affinity chromatography, yielding a pure protein as determined by HPLC. Automatic amino acid analysis of the purified enzyme showed it to be distinct from all other known plasmid-encoded dihydrofolate reductases and quite different from the type IIIa enzyme. The purified enzyme was examined by SDS-PAGE, which revealed that the type IIIb dihydrofolate reductase was a monomeric protein of Mr 17,200.