SASDD84: p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant...

Basic information

Entry

Database: SASBDB / ID: SASDD84

Sample

p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of S172A C1 in the absence of p-hydroxyphenylacetic acid (HPA)

• p-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component S172A mutant (protein), S172A C1 reductase, Acinetobacter baumannii

• 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): 4 mg/ml of S172A C1 in the absence of p-hydroxyphenylacetic acid (HPA); Specimen concentration: 4 mg/ml
• Buffer: Name: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, and 5% glycerol / pH: 7

Entity #983

Name: S172A C1 reductase / Type: protein
Description: p-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component S172A mutant
Formula weight: 35.396 / Num. of mol.: 2 / Source: Acinetobacter baumannii
Sequence:

MNQLNTAIVE KEVIDPMAFR RALGNFATGV TIMTAQTSSG ERVGVTANSF NSVSLDPALV LWSIDKKSSS YRIFEEATHF GVNILSAAQI ELSNRFARRS EDKFANIEFD LGVGNIPLFK NCSAAFECER YNIVEGGDHW IIIGRVVKFH DHGRSPLLYH QGAYSAVLPH PALNMKSETA EGVFPGRLYD NMYYLLTQAV RAYQNDYQPK QLASGFRTSE ARLLLVLESK TASSKCDLQR EVAMPIREIE EATKILSEKG 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.: 1.3323 mm
• Detector: Name: Rayonix SX165 / Type: CCD / Pixsize x: 15 mm

Scan

Title: p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of S172A C1 in the absence of p-hydroxyphenylacetic acid (HPA)
Measurement date: Mar 7, 2017 / Storage temperature: 4 °C / Cell temperature: 16 °C / Exposure time: 600 sec. / Number of frames: 1 / Unit: 1/A /

	Min	Max
Q	0.03	0.2781

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 906 /

	Min	Max
Q	0.0318774	0.278067
P(R) point	1	906
R	0	71.39

Result

Type of curve: single_conc /

	Experimental	Porod
MW	71.5 kDa	69 kDa
Volume	-	111 nm3

	P(R)	Guinier	Guinier error
Forward scattering, I0	206.7	195.21	0.41
Radius of gyration, Rg	2.52 nm	2.382 nm	0.006

	Min	Max
D	-	7.14
Guinier point	11	90