SASDFJ7: Pseudomonas putida CBB5 mCherry-NdmA/ECFP-NdmB complex - static

基本情報

• 登録情報: データベース: SASBDB / ID: SASDFJ7

試料

Pseudomonas putida CBB5 mCherry-NdmA/ECFP-NdmB complex - static

• 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 #3140: ; タイプ: dummy / ダミー原子の半径: 6.75 A / カイ2乗値: 0.419; 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 mCherry-NdmA/ECFP-NdmB complex - static; 試料濃度: 4.40-11.00 / Entity id: 1700 / 1718
• バッファ: 名称: 20 mM HEPES 150 mM NaCl 2 mM TCEP 10% v/v glycerol / pH: 7.5

要素 #1700

タイプ: protein / 記述: Methylxanthine N1-demethylase NdmA / 分子量: 69.001 / 分子数: 3 / 由来: Pseudomonas putida / 参照: UniProt: H9N289
配列:

MGHHHHHHMV SKGEEDNMAI IKEFMRFKVH MEGSVNGHEF EIEGEGEGRP YEGTQTAKLK VTKGGPLPFA WDILSPQFMY GSKAYVKHPA DIPDYLKLSF PEGFKWERVM NFEDGGVVTV TQDSSLQDGE FIYKVKLRGT NFPSDGPVMQ KKTMGWEASS ERMYPEDGAL KGEIKQRLKL KDGGHYDAEV KTTYKAKKPV QLPGAYNVNI KLDITSHNED YTIVEQYERA EGRHSTGGMD ELYKSGENLY FQGSMEQAII NDEREYLRHF WHPVCTVTEL EKAHPSSLGP LAVKLLNEQL VVAKLGDEYV AMRDRCAHRS AKLSLGTVSG NRLQCPYHGW QYDTHGACQL VPACPNSPIP NKAKVDRFDC EERYGLIWIR LDSSFDCTEI PYFSAANDPR LRIVIQEPYW WDATAERRWE NFTDFSHFAF IHPGTLFDPN NAEPPIVPMD RFNGQFRFVY DTPEDMAVPN QAPIGSFSYT CSMPFAINLE VSKYSSSSLH VLFNVSCPVD SHTTKNFLIF AREQSDDSDY LHIAFNDLVF AEDKPVIESQ WPKDAPADEV SVVADKVSIQ YRKWLRELKE AHKEGSQAFR SALLDPVIES DRSYI

要素 #1718

タイプ: protein / 記述: Methylxanthine N3-demethylase NdmB / 分子量: 69.188 / 分子数: 3 / 由来: Pseudomonas putida / 参照: UniProt: H9N290
配列:

MGHHHHHHMV SKGEELFTGV VPILVELDGD VNGHKFSVSG EGEGDATYGK LTLKFICTTG KLPVPWPTLV TTLTWGVQCF SRYPDHMKQH DFFKSAMPEG YVQERTIFFK DDGNYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNYISHNVY ITADKQKNGI KANFKIRHNI EDGSVQLADH YQQNTPIGDG PVLLPDNHYL STQSALSKDP NEKRDHMVLL EFVTAAGITL GMDELYKSGL GSMKEQLKPL LEDKTYLRHF WHPVCTLNEF ERANASGHGP MGVTLLGEKL VLARLNSKII AAADRCAHRS AQLSIGRVCS NAGKDYLECP YHGWRYDEAG ACQLIPACPD KSISPRAKIS SFDCEVKYDI VWVRLDNSFD CTQIPYLSDF DNPDMQVIVA DSYIWETVAE RRWENFTDFS HFAFVHPGTL YDPFFASHPT VYVNRVDGEL QFKLAPPREM KGIPPEAPMG DFTYRCTMPY SVNLEIKLWK DDSRFVLWTT ASPVDNKSCR NFMIIVREKD NQPDHMHLAF QKRVLDEDQP VIESQWPLEI QTSEVSVATD KISVQFRKWH KELSLSAVEG REAFRDSVLT NVIEEEQ

実験情報

• ビーム: 設備名称: Pohang Accelerator Laboratory 4C / 地域: Pohang / 国: South Korea / 線源: X-ray synchrotron / 波長: 0.0733 Å
• 検出器: 名称: Rayonix SX165 / タイプ: CCD / Pixsize x: 15 mm

スキャン

タイトル: Pseudomonas putida CBB5 mCherry-NdmA/ECFP-NdmB complex - static
測定日: 2018年7月27日 / 保管温度: 18 °C / セル温度: 4 °C / 照射時間: 5 sec. / フレーム数: 6 / 単位: 1/A /

	Min	Max
Q	0.0064	0.3399

距離分布関数 P(R)

ソフトウェア P(R): GNOM 5.0 / ポイント数: 1231 /

	Min	Max
Q	0.0137116	0.143956
P(R) point	1	1231
R	0	191.4

結果

Experimental MW: 444 kDa / カーブのタイプ: extrapolated

	P(R)	Guinier
前方散乱 I0	222.8	219.33
慣性半径, Rg	5.71 nm	5.552 nm

	Min	Max
D	-	19.14
Guinier point	50	141