5HPF

Crystal Structure of the Double Mutant of PobR Transcription Factor Inducer Binding Domain from Acinetobacter

5HPF の概要

• エントリーDOI: 10.2210/pdb5hpf/pdb
• 関連するPDBエントリー: 5HPI
• 分子名称: p-hydroxybenzoate hydroxylase transcriptional activator, PHOSPHATE ION, GLYCEROL, ... (5 entities in total)
• 機能のキーワード: transcription factor, ligand binding domain, transcription
• 由来する生物種: Acinetobacter baylyi str. ADP1
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 60837.28

構造登録者

Kim, Y.,Tesar, C.,Jedrejczak, R.,Jha, R.,Strauss, C.E.M.,Joachimiak, A. (登録日: 2016-01-20, 公開日: 2016-09-07, 最終更新日: 2024-12-25)

主引用文献

Jha, R.K.,Kern, T.L.,Kim, Y.,Tesar, C.,Jedrzejczak, R.,Joachimiak, A.,Strauss, C.E.
A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor.
Nucleic Acids Res., 44:8490-8500, 2016

PubMed Abstract: A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to 'sense' a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10(7) variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (∼2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.

PubMed: 27536006
DOI: 10.1093/nar/gkw687

実験手法

X-RAY DIFFRACTION (2.309 Å)