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Molecular genetic analysis of enoyl-acyl carrier protein reductase inhibition by diazaborine (1999)

De Boer, Gert-Jan ; Pielage, Gerlof J. A. ; Nijkamp, H. John J. ; [et al.]

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 31 (1999), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Diazaborine and isoniazid are, at first sight, unrelated anti-bacterial agents that inhibit the enoyl-ACP reductase (ENR) of Escherichia coli and Mycobacterium tuberculosis respectively. The crystal structures of these enzymes including that of the diazaborine-inhibited E. coli ENR have been obtained at high resolution. Site-directed mutagenesis was used to study the importance of amino acid residues in diazaborine susceptibility and enzyme function. The results show that drug binding and inhibition require the presence of a glycine residue at position 93 of E. coli ENR or at the structurally equivalent position in the plant homologue, which is naturally resistant to the drug. The data confirm the hypothesis that any amino acid side-chain other than hydrogen at this position within the three-dimensional structure of these enzymes will affect diazaborine resistance by encroaching into the drug binding site. Substitutions of Gly-93 by amino acids with small side-chains, such as serine, alanine, cysteine and valine, hardly affected the catalytic parameters and rendered the bacterial host resistant to the drug. Larger amino acid side-chains, such as that of arginine, histidine, lysine and glutamine, completely inactivated the activity of the enzyme.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01182.x

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Sequences surrounding the transcription initiation site of the Arabidopsis enoyl-acyl carrier protein reductase gene control seed expression in transgenic tobacco (1999)

de Boer, Gert-Jan ; Testerink, Christa ; Pielage, Gerlof ; [et al.]

Springer

Plant molecular biology 39 (1999), S. 1197-1207

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ISSN: 1573-5028

Keywords: Arabidopsis thaliana ; enoyl-ACP reductase ; fatty acid synthesis ; GUS ; 5′-flanking region ; transgenic tobacco

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract The NADH-specific enoyl-acyl carrier protein (ACP) reductase, which catalyses the last reducing step during the fatty acid biosynthesis cycle, is encoded in Arabidopsis thaliana encoded by a single housekeeping gene (ENR-A) which is differentially expressed during plant development. To identify elements involved in its tissue-specific transcriptional control, a fragment comprising the 1470 bp region directly upstream of the ATG start codon of the ENR-A gene was fused to the uidA (GUS) reporter gene and analysed in transgenic Nicotiana tabacum plants. GUS activity found during development of the transgenic plants was similar to endogenous ENR protein levels found in both tobacco and Arabidopsis plants, except for developing flowers. In floral tissue the promoter fragment showed very little activity in contrast to the relatively high level of endogenous ENR expression. Successive deletions from the 5′ and 3′ regions of the promoter fragment revealed the presence of at least three elements which control GUS expression in different stages of development in the transgenic tobacco plants. First, expression in young developing leaves required both the presence of sequences between −329 to −201 relative to the transcription start and part of the untranslated leader comprising the first intron. Second, root-specific GUS expression was still observed after deletion of the 5′-upstream sequences up to 19 bp of the transcription initiation site. Further, the additional removal of the intron from the untranslated leader increased root-specific expression by ca. 4- to 5-fold. Third, high expression in seeds was still observed with the minimal upstream promoter segment of 19 bp. This seed expression level was found to be independent of the presence or absence of the intron in the untranslated leader. Finally, 3′ deletion of the leader sequence up to 17 bp of the transcription start greatly impaired GUS activity during all stages of plant development, suggesting that the deleted sequence of the leader either functions as an enhancer for transcription initiation or stabilizes the mRNA.