SASDDN7: p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant...

基本情報

登録情報

データベース: SASBDB / ID: SASDDN7

試料

p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of E248A/E251A C1 in the absence of p-hydroxyphenylacetic acid (HPA)

• p-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A/E251A C1 (protein), E248A/E251A C1, Acinetobacter baumannii

機能・相同性

分子機能:

flavin reductase (NADH) / flavin reductase (NADH) activity / : / FMN binding

ドメイン・相同性:

Flavin reductase like domain / Flavin reductase like domain / Flavin reductase like domain / FMN-binding split barrel / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily

構成要素:

p-hydroxyphenylacetate 3-hydroxylase, reductase component

• 生物種: Acinetobacter baumannii (バクテリア)
• 引用: ジャーナル: Arch Biochem Biophys / 年: 2018; タイトル: Crystal structure of the flavin reductase of Acinetobacter baumannii p-hydroxyphenylacetate 3-hydroxylase (HPAH) and identification of amino acid residues underlying its regulation by aromatic ligands.; 著者: Anan Yuenyao / Nopphon Petchyam / Nuntaporn Kamonsutthipaijit / Pimchai Chaiyen / Danaya Pakotiprapha / ; 要旨: The first step in the degradation of p-hydroxyphenylacetic acid (HPA) is catalyzed by the two-component enzyme p-hydroxyphenylacetate 3-hydroxylase (HPAH). The two components of Acinetobacter baumannii HPAH are known as C and C, respectively. C is a flavin reductase that uses NADH to generate reduced flavin mononucleotide (FMNH), which is used by C in the hydroxylation of HPA. Interestingly, although HPA is not directly involved in the reaction catalyzed by C, the presence of HPA dramatically increases the FMN reduction rate. Amino acid sequence analysis revealed that C contains two domains: an N-terminal flavin reductase domain, and a C-terminal MarR domain. Although MarR proteins typically function as transcription regulators, the MarR domain of C was found to play an auto-inhibitory role. Here, we report a crystal structure of C and small-angle X-ray scattering (SAXS) studies that revealed that C undergoes a substantial conformational change in the presence of HPA, concomitant with the increase in the rate of flavin reduction. Amino acid residues that are important for HPA binding and regulation of C activity were identified by site-directed mutagenesis. Amino acid sequence similarity analysis revealed several as yet uncharacterized flavin reductases with N- or C-terminal fusions.

登録者

• Anan Yuenyao (Mahidol University)

モデル

試料

• 試料: 名称: p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of E248A/E251A C1 in the absence of p-hydroxyphenylacetic acid (HPA); 試料濃度: 2 mg/ml
• バッファ: 名称: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, and 5% glycerol; pH: 7

要素 #1094

名称: E248A/E251A C1 / タイプ: protein
記述: p-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A/E251A C1
分子量: 35.296 / 分子数: 2 / 由来: Acinetobacter baumannii / 参照: UniProt: Q6Q271
配列:

MNQLNTAIVE KEVIDPMAFR RALGNFATGV TIMTAQTSSG ERVGVTANSF NSVSLDPALV LWSIDKKSSS YRIFEEATHF GVNILSAAQI ELSNRFARRS EDKFANIEFD LGVGNIPLFK NCSAAFECER YNIVEGGDHW IIIGRVVKFH DHGRSPLLYH QGAYSAVLPH PSLNMKSETA EGVFPGRLYD NMYYLLTQAV RAYQNDYQPK QLASGFRTSE ARLLLVLESK TASSKCDLQR EVAMPIRAIE AATKILSEKG LLIDNGQHYE LTEQGNACAH MLYKIAESHQ EEVFAKYTVD ERKLFKNMLK DLIGI

実験情報

• ビーム: 設備名称: Synchrotron Light Research Institute (SLRI) BL1.3W; 地域: Nakhon Ratchasima / 国: Thailand / 線源: X-ray synchrotronシンクロトロン / 波長: 0.13776 Å / スペクトロメータ・検出器間距離: 1.332 mm
• 検出器: 名称: Rayonix SX165 / タイプ: CCD / Pixsize x: 15 mm

スキャン

タイトル: p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of E248A/E251A C1 in the absence of p-hydroxyphenylacetic acid (HPA)
測定日: 2018年5月26日 / 保管温度: 4 °C / セル温度: 16 °C / 照射時間: 600 sec. / フレーム数: 1 / 単位: 1/A /

	Min	Max
Q	0.0298	0.2752

距離分布関数 P(R)

ソフトウェア P(R): GNOM 5.0 / ポイント数: 872 /

	Min	Max
Q	0.0393264	0.275188
P(R) point	1	872
R	0	67.16

結果

カーブのタイプ: single_conc /

	実験値	Porod
分子量	50 kDa	-
体積	-	88 nm3

	P(R)	Guinier	Guinier error
前方散乱 I0	57.94	53.66	0.32
慣性半径, Rg	2.46 nm	2.31 nm	0.016

	Min	Max
D	-	6.72
Guinier point	36	97