Escherichia coli active site mutations

Induced mutagenesis in Escherichia coli is an active process involving proteins with DNA replication, repair, and recombination functions. The available evidence suggests that mutations are generated at sites where DNA has been damaged and that they arise via an error-prone repair activity. In an attempt to understand what specific contributions to mutagenesis are made by DNA lesions, we have studied the mutational specificity of some carcinogens, such as benzo[a]pyrene and aflatoxin, whose chemical reactions with DNA are... 

Murray, J. M., Wilmot, C. M., Saysell, C. G., Jaeger, J., Knowles, P. F., Phillips, S. E. V., and McPherson, M. J., 1999, The active site base controls cofactor reactivity in Escherichia coli amine oxidase X-ray crystallographic studies with mutational variants. Biochemistry, 38 8217n8227. 

The structure of the bacterial ArsC from Escherichia coli plasmid R773 has been solved at 1.65 A resolution, and revealed that arsenate reductase (ArsC) has only one cysteine residue (Cys-12) in the active site, surrounded by an arginine triad composed of Arg-60, Arg-94, and Arg-107 (Mukhopadhyay et al, 2002). However, the arsenate reductase from Staphylococcus aureus has three cysteine residues (Cys-10, Cys-82, and Cys-89). The biochemical and mutational studies established that the arsenate binds to the triad of arginine (Arg-60, Arg-94, and Arg-107) residues and forms a covalent bond with the cysteine (Cys-12) residue near the N-terminus at the active site of arsenate reductase and/or participates in catalysis (Rosen, 2002a Silver and Phung, 2005 Shi etal, 2003 Martin et al, 2001). 

Ex ample 1 The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary alcohol dehydrogenase was overexpressed in Escherichia coli to form more than 10% to total protein11361. The recombinant enzyme was purified by heat treatment and precipitation with aqueous (NH4)2S04 and isolated in 67% yield. Enzymes with mutation(s) around the active site residues were also created to examine the catalytically important zinc binding motif in the proteins. 

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