Site-directed mutation of the Escherichia coli ada gene: Effects of substitution of methyl acceptor cysteine-321 by histidine in Ada protein

Journal Article · Wed Mar 01 00:00:00 EST 1989 · Journal of Bacteriology; (USA)

OSTI ID:7266296

Tano, K; Bhattacharyya, D; Foote, R S ^[1]; Mural, R J; Mitra, S ^[2]

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1. Univ. of Tennessee Graduate, Oak Ridge (USA)
2. Protein Engineering and Molecular Mutagenesis Program, Oak Ridge, TN (USA)

Oligodeoxynucleotide-mediated mutagenesis of the ada gene of Escherichia coli was used to produce two mutant Ada proteins. In mutant I the methyl acceptor Cys-321 for O{sup 6} -methylguanine was replaced by histidine; and in mutant II the positions of Cys-321 and His-322 of the wild-type protein were inverted. Neither mutant protein had O{sup 6}-methylguanine-DNA methyltransferase activity, but both retained the phosphotriester-DNA methyltransferase activity involving methyl group transfer to Cys-69. Under the control of the endogenous promoter, synthesis of mutant I protein was undetectable before of after adaptation treatment with N-methyl-N{prime}-nitro-N-nitrosoguanidine. This appeared to be due to both inhibition of transcription of the mutant gene and degradation of the synthesized protein. On the other hand, mutant II protein was inducible by N-methyl-N{prime}-nitro-N-nitrosoguanidine, although to a smaller extent than the wild-type protein was, and the phosphotriester-DNA methyltransferase activity appeared to reside in 24- to 30-kilodalton cleavage products. Mutant I protein could be produced under lac promoter control, and its cleavage products, unlike those of mutant II protein, tended to aggregate. These result indicate that (i) Cys-321 cannot be replaced or transposed with the nucleophilic amino acid histidine for O{sup 6}-methylguanine-DNA methyltransferase function, (ii) single amino acid replacement or transposition at the O{sup 6}-methylguanine methyl acceptor site can have a profound effect on the in vivo stability and regulatory function of the Ada protein, and (iii) the integrity of the protein may not be absolutely needed for its transcription-activation function.

• DOE Contract Number: AC05-84OR21400
• OSTI ID: 7266296
• Journal Information: Journal of Bacteriology; (USA), Vol. 41:3; ISSN 0021-9193
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ESCHERICHIA COLI
MUTATIONS
METHYL TRANSFERASES
ENZYME ACTIVITY
NITROSO COMPOUNDS
BIOLOGICAL EFFECTS
PROTEINS
BIOLOGICAL FUNCTIONS
CHEMICAL PROPERTIES
CYSTEINE
GENES
HISTIDINE
TRANSCRIPTION
AMINO ACIDS
AZOLES
BACTERIA
CARBOXYLIC ACIDS
ENZYMES
FUNCTIONS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
IMIDAZOLES
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
THIOLS
TRANSFERASES
560300* - Chemicals Metabolism & Toxicology