+データを開く
-基本情報
登録情報 | データベース: SASBDB / ID: SASDDJ7 |
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試料 | Citrate-binding PAS domain from the sensor histidine kinase, CitA, fused to lipase EstA
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引用 | ジャーナル: Sci Rep / 年: 2017 タイトル: A combination of mutational and computational scanning guides the design of an artificial ligand-binding controlled lipase. 著者: Marco Kaschner / Oliver Schillinger / Timo Fettweiss / Christina Nutschel / Frank Krause / Alexander Fulton / Birgit Strodel / Andreas Stadler / Karl-Erich Jaeger / Ulrich Krauss / 要旨: Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme's active site, represents a mechanism essential for sustaining life. The rational design of ...Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme's active site, represents a mechanism essential for sustaining life. The rational design of allostery is a non-trivial task but can be achieved by fusion of a sensory domain, which responds to environmental stimuli with a change in its structure. Hereby, the site of domain fusion is difficult to predict. We here explore the possibility to rationally engineer allostery into the naturally not allosterically regulated Bacillus subtilis lipase A, by fusion of the citrate-binding sensor-domain of the CitA sensory-kinase of Klebsiella pneumoniae. The site of domain fusion was rationally determined based on whole-protein site-saturation mutagenesis data, complemented by computational evolutionary-coupling analyses. Functional assays, combined with biochemical and biophysical studies suggest a mechanism for control, similar but distinct to the one of the parent CitA protein, with citrate acting as an indirect modulator of Triton-X100 inhibition of the fusion protein. Our study demonstrates that the introduction of ligand-dependent regulatory control by domain fusion is surprisingly facile, suggesting that the catalytic mechanism of some enzymes may be evolutionary optimized in a way that it can easily be perturbed by small conformational changes. |
登録者 |
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-構造の表示
構造ビューア | 分子: MolmilJmol/JSmol |
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-ダウンロードとリンク
-モデル
モデル #2027 | タイプ: dummy / ソフトウェア: (2.5) / ダミー原子の半径: 2.30 A / カイ2乗値: 3.243601 Omokage検索でこの集合体の類似形状データを探す (詳細) |
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モデル #2028 | タイプ: dummy / ダミー原子の半径: 3.25 A / カイ2乗値: 3.243601 Omokage検索でこの集合体の類似形状データを探す (詳細) |
-試料
試料 | 名称: Citrate-binding PAS domain from the sensor histidine kinase, CitA, fused to lipase EstA 試料濃度: 4.98 mg/ml |
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バッファ | 名称: 10 mM glycine buffer, 10 mM NaCl / pH: 10 |
要素 #1079 | 名称: CitAP-BsLA / タイプ: protein 記述: citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA 分子量: 38.495 / 分子数: 2 配列: MGSSHHHHHH SSGLVPRGSH MDITEERLHY QVGQRALIQA MQISAMPELV EAVQKRDLAR IKALIDPMRS FSDATYITVG DASGQRLYHV NPDEIGKSME GGDSDEALIN AKSYVSVRKG SLGSSLRGKS PIQDATGKVI GIVSVGYTIE QLENYEKLLE DSLTEITALS ...配列: MGSSHHHHHH SSGLVPRGSH MDITEERLHY QVGQRALIQA MQISAMPELV EAVQKRDLAR IKALIDPMRS FSDATYITVG DASGQRLYHV NPDEIGKSME GGDSDEALIN AKSYVSVRKG SLGSSLRGKS PIQDATGKVI GIVSVGYTIE QLENYEKLLE DSLTEITALS IEGREAEHNP VVMVHGIGGA SFNFAGIKSY LVSQGWSRDK LYAVDFWDKT GTNYNNGPVL SRFVQKVLDE TGAKKVDIVA HSMGGANTLY YIKNLDGGNK VANVVTLGGA NRLTTGKALP GTDPNQKILY TSIYSSADMI VMNYLSRLDG ARNVQIHGVG HIGLLYSSQV NSLIKEGLNG GGQNTN |
-実験情報
ビーム | 設備名称: ESRF BM29 / 地域: Grenoble / 国: France / 線源: X-ray synchrotron / 波長: 0.09919 Å / スペクトロメータ・検出器間距離: 2.849 mm | |||||||||||||||||||||||||||||
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検出器 | 名称: Pilatus 1M | |||||||||||||||||||||||||||||
スキャン | タイトル: Citrate-binding PAS domain from the sensor histidine kinase, CitA, fused to lipase EstA 測定日: 2014年2月6日 / 保管温度: 20 °C / セル温度: 20 °C / 照射時間: 1 sec. / フレーム数: 10 / 単位: 1/nm /
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距離分布関数 P(R) | ソフトウェア P(R): GNOM 5.0 / ポイント数: 463 /
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結果 | カーブのタイプ: single_conc コメント: The protein is a fusion of: i) CitAP - the PAS domain from Sensor histidine kinase, CitA, from Klebsiella pneumoniae; UniProt: P52687 (Amino acid range 45-177); ii) BsLA - the Lipase ...コメント: The protein is a fusion of: i) CitAP - the PAS domain from Sensor histidine kinase, CitA, from Klebsiella pneumoniae; UniProt: P52687 (Amino acid range 45-177); ii) BsLA - the Lipase EstA from Bacillus subtilis 168; UniProt: I6V559 (Amino acid range 32-212) and; iii) A linker connecting CitAP with EstA - derived from the blue light photoreceptor, YtvA, from Bacillus subtilis 168; UniProt: O34627 (Amino acid range 132-147).
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