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The cloning and DNA sequence of the gene for the glutathione-regulated potassium-efflux system KefC of Escherichia coli (1991)

Munro, A. W. ; Ritchie, G. Y. ; Lamb, A. J. ; [et al.]

Oxford, UK : Blackwell Publishing Ltd

Molecular microbiology 5 (1991), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The kefC gene of Escherichia coli encodes a potassium-efflux system that is regulated by glutathione metabolites. The close proximity of the E. coli kefC gene to the folA gene, encoding dihydrofolate reductase, has been utilized to clone the structural gene for the system from a Clarke-Carbon plasmid. The cloned gene has been refined to a region of DNA approximately 2.1 kb in length using exonuclease III-generated deletions and random Muc/M1734 (IacZ) insertions. The direction of transcription has been deduced from the orientation of the Mu insertions in the cloned DNA. A hybrid protein consisting of approximately two thirds of the KefC protein fused to β-galactosidase has been shown to be membrane-located. The DNA sequence of the gene has been determined and an open reading frame of 1.86kb has been located which could encode a protein of 620 amino acids (7901 0Da). Using the T7 expression system a membrane protein, of apparent molecular mass 55–60 kDa, has been shown to be encoded by the kefC gene. The predicted protein sequence shows a highly hydrophobic amino-terminus and a strongly hydrophilic carboxy-terminus, Comparison of the amino acid sequence of the kefC gene product with those of two glutathione-utilizing enzymes, glyoxalase and dehalogenase, has revealed some similarities.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.1991.tb00731.x

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Analysis of the structure of the flavin-binding sites of flavocytochrome P450 BM3 using surface enhanced resonance Raman scattering (1999)

Macdonald, I. D. G. ; Smith, W. E. ; Munro, A. W.

Springer

European biophysics journal 28 (1999), S. 437-445

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ISSN: 1432-1017

Keywords: Key words Raman ; Surface enhanced resonance Raman scattering spectroscopy ; Flavins ; Flavocytochrome P450 BM3

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Physics

Notes: Abstract Flavocytochrome P450 BM3, an FMN-deficient mutant (G570 D), the component reductase and an FAD-containing domain were studied using surface enhanced resonance Raman scattering (SERRS). They were compared to spectra obtained from the free flavins FAD and FMN. For the holoenzyme and reductase domain, FMN is displaced during SERRS analysis. However, studies with the G570 D mutant indicate that FAD is retained in its active site. Analysis of SERRS frequencies and intensities provides information on the nature of the flavin binding site and the planarity of the ring, and enables an interpretation of the hydrogen bonding environment around ring III of the isoalloxazine moiety. Hydrogen bonding is strong at N3–H, C2=O and C4=O, but weak at N5. Structural alteration of the FAD domain of P450 BM3 is caused by removal of the FMN-binding domain. Further, the hydrogen bond at N3–H is lost and that at C2=O is weakened and the isoalloxazine ring system in the FAD domain appears to adopt a more planar arrangement. Alterations in the environment of the FAD in its isolated domain are likely to relate to changes in the redox properties and suggest a close structural interplay of FAD with the FMN-binding domain in intact flavocytochrome P450 BM3.