SASDFH7: Pseudomonas putida CBB5 NdmAB complex (Methylxanthine N1-demethyl...

基本情報

• 登録情報: データベース: SASBDB / ID: SASDFH7

試料

Pseudomonas putida CBB5 NdmAB complex

• Methylxanthine N1-demethylase NdmA (protein), Pseudomonas putida
• Methylxanthine N3-demethylase NdmB (protein), Pseudomonas putida

機能・相同性

分子機能:

methylxanthine N3-demethylase / methylxanthine N1-demethylase / alkaloid catabolic process / demethylase activity / 2 iron, 2 sulfur cluster binding / oxidoreductase activity / metal ion binding / cytoplasm

ドメイン・相同性:

Vanillate O-demethylase oxygenase-like, C-terminal catalytic domain / Vanillate O-demethylase oxygenase C-terminal domain / : / Rieske [2Fe-2S] domain / Rieske [2Fe-2S] iron-sulphur domain / Rieske [2Fe-2S] iron-sulfur domain profile. / Rieske [2Fe-2S] iron-sulphur domain superfamily

構成要素:

Methylxanthine N1-demethylase NdmA / Methylxanthine N3-demethylase NdmB

• 生物種: Pseudomonas putida (バクテリア)
• 引用: ジャーナル: J Mol Biol / 年: 2019; タイトル: Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex.; 著者: Jun Hoe Kim / Bong Heon Kim / Shelby Brooks / Seung Yeon Kang / Ryan M Summers / Hyun Kyu Song / ; 要旨: Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N- and N-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications.

登録者

• Jun Hoe Kim (Korea University, South Korea)

モデル

• モデル #3134: ; タイプ: dummy / ダミー原子の半径: 3.75 A / 対称性: P3 / カイ2乗値: 1.384 / P-value: 0.002663; 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: Pseudomonas putida CBB5 NdmAB complex / 試料濃度: 21.6 mg/ml / Entity id: 1709 / 1710
• バッファ: 名称: 20 mM HEPES 150 mM NaCl 2 mM TCEP / pH: 7.5 / コメント: gel filtration buffer

要素 #1709

タイプ: protein / 記述: Methylxanthine N1-demethylase NdmA / 分子量: 42.327 / 分子数: 3 / 由来: Pseudomonas putida / 参照: UniProt: H9N289
配列:

MGSSHHHHHH ENLYFQGSME QAIINDEREY LRHFWHPVCT VTELEKAHPS SLGPLAVKLL NEQLVVAKLG DEYVAMRDRC AHRSAKLSLG TVSGNRLQCP YHGWQYDTHG ACQLVPACPN SPIPNKAKVD RFDCEERYGL IWIRLDSSFD CTEIPYFSAA NDPRLRIVIQ EPYWWDATAE RRWENFTDFS HFAFIHPGTL FDPNNAEPPI VPMDRFNGQF RFVYDTPEDM AVPNQAPIGS FSYTCSMPFA INLEVSKYSS SSLHVLFNVS CPVDSHTTKN FLIFAREQSD DSDYLHIAFN DLVFAEDKPV IESQWPKDAP ADEVSVVADK VSIQYRKWLR ELKEAHKEGS QAFRSALLDP VIESDRSYI

要素 #1710

タイプ: protein / 記述: Methylxanthine N3-demethylase NdmB / 分子量: 43.012 / 分子数: 3 / 由来: Pseudomonas putida / 参照: UniProt: H9N290
配列:

MGSSHHHHHH ENLYFQGSMK EQLKPLLEDK TYLRHFWHPV CTLNEFERAN ASGHGPMGVT LLGEKLVLAR LNSKIIAAAD RCAHRSAQLS IGRVCSNAGK DYLECPYHGW RYDEAGACQL IPACPDKSIS PRAKISSFDC EVKYDIVWVR LDNSFDCTQI PYLSDFDNPD MQVIVADSYI WETVAERRWE NFTDFSHFAF VHPGTLYDPF FASHPTVYVN RVDGELQFKL APPREMKGIP PEAPMGDFTY RCTMPYSVNL EIKLWKDDSR FVLWTTASPV DNKSCRNFMI IVREKDNQPD HMHLAFQKRV LDEDQPVIES QWPLEIQTSE VSVATDKISV QFRKWHKELS LSAVEGREAF RDSVLTNVIE EEQ

実験情報

• ビーム: 設備名称: Photon Factory (PF), High Energy Accelerator Research Organization (KEK) BL-10C; 地域: Tsukuba / 国: Japan / 線源: X-ray synchrotron / 波長: 0.15 Å / スペクトロメータ・検出器間距離: 3 mm
• 検出器: 名称: Pilatus3 2M / Pixsize x: 0.172 mm

スキャン

タイトル: Pseudomonas putida CBB5 NdmAB complex / 測定日: 2018年5月21日 / 保管温度: 4 °C / セル温度: 18 °C / 照射時間: 10 sec. / フレーム数: 48 / 単位: 1/A /

	Min	Max
Q	0.0033	0.2809

距離分布関数 P(R)

ソフトウェア P(R): GNOM 5.0 / ポイント数: 715 /

	Min	Max
Q	0.0068966	0.176578
P(R) point	1	715
R	0	108.5

結果

Experimental MW: 227 kDa / D max: 10.86 / カーブのタイプ: sec

	P(R)	Guinier
前方散乱 I0	2.727	2.72
慣性半径, Rg	4.14 nm	4.191 nm