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基本情報
登録情報 | データベース: PDB / ID: 6jqn | |||||||||||||||
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タイトル | Structure of PaaZ, a bifunctional enzyme in complex with NADP+ and OCoA | |||||||||||||||
![]() | Bifunctional protein PaaZ | |||||||||||||||
![]() | HYDROLASE / substrate channeling / bi-functional enzyme / dehydrogenase | |||||||||||||||
機能・相同性 | ![]() 3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase / oxepin-CoA hydrolase / hydrolase activity, acting on acid carbon-carbon bonds, in ketonic substances / ether hydrolase activity / oxidoreductase activity, acting on CH or CH2 groups, NAD or NADP as acceptor / phenylacetate catabolic process / oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor / enoyl-CoA hydratase activity / identical protein binding 類似検索 - 分子機能 | |||||||||||||||
生物種 | ![]() ![]() | |||||||||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.1 Å | |||||||||||||||
![]() | Gakher, L. / Vinothkumar, K.R. / Katagihallimath, N. / Sowdhamini, R. / Sathyanarayanan, N. / Cannone, G. | |||||||||||||||
資金援助 | ![]() ![]()
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![]() | ![]() タイトル: Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway. 著者: Nitish Sathyanarayanan / Giuseppe Cannone / Lokesh Gakhar / Nainesh Katagihallimath / Ramanathan Sowdhamini / Subramanian Ramaswamy / Kutti R Vinothkumar / ![]() ![]() ![]() 要旨: Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been ...Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues. | |||||||||||||||
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構造の表示
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構造ビューア | 分子: ![]() ![]() |
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-検証レポート
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-関連構造データ
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 73969.391 Da / 分子数: 6 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() #2: 化合物 | ChemComp-NAP / #3: 化合物 | ChemComp-CO8 / |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: PaaZ / タイプ: COMPLEX 詳細: PaaZ is a bifunctional enzyme that has hydrolase and dehydrogenase activity. Entity ID: #1 / 由来: RECOMBINANT | ||||||||||||
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分子量 | 値: 0.44 MDa / 実験値: NO | ||||||||||||
由来(天然) | 生物種: ![]() ![]() | ||||||||||||
由来(組換発現) | 生物種: ![]() ![]() | ||||||||||||
緩衝液 | pH: 7.4 詳細: Protein was purified and kept in 25mM Hepes buffer and 50 mM NaCl | ||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.015 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: The peak fraction from gel filtration was used for grid preparation. NADP+ and OCoA were added 10 fold excess and incubated for 15 minutes before freezing. | ||||||||||||
試料支持 | 詳細: Grids were glow discharged for 5 minutes and then graphene oxide was applied. Subsequently, grids were washed with water 3 times and dried. The graphene oxide grids were then used for freezing. グリッドの材料: GOLD / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: Quantifoil, UltrAuFoil, R1.2/1.3 | ||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 283.15 K 詳細: blotting force 10, blotting time 4 sec, waiting time 15 sec, drying time 0, blotting times 1. |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS / 詳細: Data was collected with EPU software |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 75000 X / 倍率(補正後): 132075 X / 最大 デフォーカス(公称値): 3200 nm / 最小 デフォーカス(公称値): 2200 nm / Cs: 2.7 mm / C2レンズ絞り径: 50 µm / アライメント法: COMA FREE |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER 最高温度: 80 K / 最低温度: 80 K |
撮影 | 平均露光時間: 60 sec. / 電子線照射量: 27 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 撮影したグリッド数: 1 / 実像数: 667 詳細: The 60 second exposure was saved into 75 frames with each frame ~0.36 e-. The frames were then grouped into 3 for alignment and summed images were used for data processing |
画像スキャン | サンプリングサイズ: 14 µm / 横: 4096 / 縦: 4096 |
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解析
ソフトウェア | 名称: PHENIX / バージョン: 1.13_2998: / 分類: 精密化 | ||||||||||||||||||||||||||||||||||||||||||||||||||
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EMソフトウェア |
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画像処理 | 詳細: counting mode was used | ||||||||||||||||||||||||||||||||||||||||||||||||||
CTF補正 | 詳細: CTF was corrected on each particle during refinement and reconstruction タイプ: NONE | ||||||||||||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 179346 | ||||||||||||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C3 (3回回転対称) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.1 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 101503 / アルゴリズム: BACK PROJECTION / クラス平均像の数: 1 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | B value: 59.9 / プロトコル: OTHER / 空間: REAL 詳細: Real space refinement with secondary structure enabled, minimization and adp |