Chloramphenicol acetyl transferase

from Wikipedia, the free encyclopedia
Chloramphenicol acetyl transferase
Chloramphenicol acetyl transferase
other names

CAT

Existing structure data : 3CLA , 3U9F , 1PD5 , 1Q23

Mass / length primary structure 25663 Da , 219 amino acids
Secondary to quaternary structure Homotrimer
Identifier
External IDs
Enzyme classification
EC, category 2.3.1.28
Substrate Chloramphenicol, acetyl-CoA
Products Chloramphenicol-3-acetate

Chloramphenicol acetyltransferase (or CAT ) is a bacterial enzyme ( EC  2.3.1.28 ). It acetylates the antibiotic chloramphenicol using acetyl-CoA in two places. Since acetylated chloramphenicol no longer binds to ribosomes and can no longer inhibit translation , the gene conveys resistance to this antibiotic. The antibiotic effect of chloramphenicol is used to inhibit bacterial growth.

structure

The crystal structure of the chloramphenicol-bound type III enzyme from Escherichia coli was clarified as early as 1990. CAT is a trimer of identical subunits (monomer Mr 25,000) whose structure is stabilized by hydrogen bonds . Chloramphenicol binds a deep pocket of neighboring subunits so that most contacts are made with the residues of one subunit, while the catalytically essential histidine -195 of the neighboring subunit is ideally positioned for catalysis.

application

The gene for chloramphenicol acetyltransferase is in the microbiology and molecular biology as resistance gene used to transformed to select bacteria. The cat gene is also used as a reporter gene to e.g. B. Measure promoter strengths after transient transfection . In a typical experiment, the so-called CAT assay , a cell extract is usually produced 24-48 hours after the transfection, in which the enzyme activity is determined using radioactive chloramphenicol and acetyl-CoA . The chloramphenicol is extracted and separated by thin layer chromatography . The acetylated product of the enzyme reaction runs faster than the non-acetylated form. The substrate turnover can be measured by measuring the radioactivity in the scintillation counter or with a phosphoimager . The widespread method has been replaced with the advent of the simple luciferase assay , which can be carried out without radioactive substrates .

Individual evidence

  1. ^ William V. Shaw: Chloramphenicol Acetyltransferase: Enzymology and Molecular Biology . In: Critical Reviews in Biochemistry and Molecular Biology . 14, No. 1, 1983, ISSN  1040-9238 , pp. 1-46. doi : 10.3109 / 10409238309102789 .
  2. ^ Theodor Dingermann (Ed.), Rudolf Hänsel (Ed.) And Ilse Zündorf (Ed.): Pharmaceutical Biology: Molecular Basics and Clinical Applications . Springer Verlag Berlin; 1st edition 2002; ISBN 3-540-42844-5 ; P. 301.
  3. ^ AGW Leslie: Refined crystal structure of type III chloramphenicol acetyltransferase at 1 · 75 Å resolution . In: Journal of Molecular Biology . 213, No. 1, 1990, ISSN  0022-2836 , pp. 167-186. doi : 10.1016 / S0022-2836 (05) 80129-9 .
  4. ^ CM Gorman, LF Moffat, BH Howard: Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. . In: Molecular and Cellular Biology . 2, No. 9, 1982, ISSN  0270-7306 , pp. 1044-1051. doi : 10.1128 / MCB.2.9.1044 .
  5. ↑ Steven R. Kain, Subinay Ganguly: Overview of Genetic Reporter Systems. . In: Current Protocols in Molecular Biology . 9, 2001. doi : 10.1002 / 0471142727.mb0906s36 .