SASDDT7: p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant...

Basic information

Entry

Database: SASBDB / ID: SASDDT7

Sample

p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of E248A/E251A C1 in the presence of 1 mM p-hydroxyphenylacetic acid (HPA)

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

Function / homology

Function:

flavin reductase (NADH) / flavin reductase (NADH) activity / catabolic process / FMN binding

Domain/homology:

: / 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

Component:

p-hydroxyphenylacetate 3-hydroxylase, reductase component

• Biological species: Acinetobacter baumannii (bacteria)
• Citation: Journal: Arch Biochem Biophys / Year: 2018; Title: 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.; Authors: Anan Yuenyao / Nopphon Petchyam / Nuntaporn Kamonsutthipaijit / Pimchai Chaiyen / Danaya Pakotiprapha / ; Abstract: 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.

Contact author

• Anan Yuenyao (Mahidol University)

Models

Sample

• Sample: Name: p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of E248A/E251A C1 in the presence of 1 mM p-hydroxyphenylacetic acid (HPA); Specimen concentration: 8 mg/ml
• Buffer: Name: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, and 5% glycerol; pH: 7

Entity #1094

Name: E248A/E251A C1 / Type: protein
Description: p-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A/E251A C1
Formula weight: 35.296 / Num. of mol.: 2 / Source: Acinetobacter baumannii / References: UniProt: Q6Q271
Sequence:

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

Experimental information

• Beam: Instrument name: Synchrotron Light Research Institute (SLRI) BL1.3W; City: Nakhon Ratchasima / 国: Thailand / Type of source: X-ray synchrotron / Wavelength: 0.13776 Å / Dist. spec. to detc.: 1.332 mm
• Detector: Name: Rayonix SX165 / Type: CCD / Pixsize x: 15 mm

Scan

Title: p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of E248A/E251A C1 in the presence of 1 mM p-hydroxyphenylacetic acid (HPA)
Measurement date: May 26, 2018 / Storage temperature: 4 °C / Cell temperature: 16 °C / Exposure time: 600 sec. / Number of frames: 1 / Unit: 1/A /

	Min	Max
Q	0.0298	0.2752

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 898 /

	Min	Max
Q	0.0322751	0.275188
P(R) point	1	898
R	0	87.99

Result

Type of curve: single_conc /

	Experimental	Porod
MW	65 kDa	-
Volume	-	88 nm3

	P(R)	Guinier	Guinier error
Forward scattering, I0	243.3	234.51	0.48
Radius of gyration, Rg	2.87 nm	2.749 nm	0.007

	Min	Max
D	-	8.8
Guinier point	10	64