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- PDB-5zc2: Acinetobacter baumannii p-hydroxyphenylacetate 3-hydroxylase (HPA... -

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Basic information

Entry
Database: PDB / ID: 5zc2
TitleAcinetobacter baumannii p-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component (C1)
Componentsp-hydroxyphenylacetate 3-hydroxylase, reductase component
KeywordsFLAVOPROTEIN / Flavin reductase / MarR
Function / homology
Function and homology information


flavin reductase (NADH) / flavin reductase (NADH) activity / riboflavin reductase (NADPH) activity / : / FMN binding
Similarity search - Function
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
Similarity search - Domain/homology
FLAVIN MONONUCLEOTIDE / p-hydroxyphenylacetate 3-hydroxylase, reductase component
Similarity search - Component
Biological speciesAcinetobacter baumannii (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.898 Å
AuthorsYuenyao, A. / Petchyam, N. / Chaiyen, P. / Pakotiprapha, D.
Funding support Thailand, 2items
OrganizationGrant numberCountry
Other governmentRTA5980001 Thailand
Other governmentIRG598008 Thailand
CitationJournal: 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 ...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.
History
DepositionFeb 14, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 8, 2018Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: p-hydroxyphenylacetate 3-hydroxylase, reductase component
A: p-hydroxyphenylacetate 3-hydroxylase, reductase component
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,8234
Polymers70,9102
Non-polymers9132
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS, Scattering curve generated from the crystal structure using CRYSOL from ATSAS package agrees well with the experimental SAXS data.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12990 Å2
ΔGint-109 kcal/mol
Surface area24010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)76.851, 76.851, 185.784
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 14 through 20 or resid 22...
21(chain B and (resid 14 through 20 or resid 22...

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ILEILEARGARG(chain A and (resid 14 through 20 or resid 22...AB14 - 2014 - 20
12ALAALATHRTHR(chain A and (resid 14 through 20 or resid 22...AB22 - 3722 - 37
13SERSERSERSER(chain A and (resid 14 through 20 or resid 22...AB3939
14ILEILEILEILE(chain A and (resid 14 through 20 or resid 22...AB14 - 31514 - 315
15ILEILEILEILE(chain A and (resid 14 through 20 or resid 22...AB14 - 31514 - 315
16ILEILEILEILE(chain A and (resid 14 through 20 or resid 22...AB14 - 31514 - 315
17ILEILEILEILE(chain A and (resid 14 through 20 or resid 22...AB14 - 31514 - 315
21ILEILEARGARG(chain B and (resid 14 through 20 or resid 22...BA14 - 2014 - 20
22ALAALATHRTHR(chain B and (resid 14 through 20 or resid 22...BA22 - 3722 - 37
23SERSERSERSER(chain B and (resid 14 through 20 or resid 22...BA3939
24ILEILEILEILE(chain B and (resid 14 through 20 or resid 22...BA14 - 31514 - 315
25ILEILEILEILE(chain B and (resid 14 through 20 or resid 22...BA14 - 31514 - 315
26ILEILEILEILE(chain B and (resid 14 through 20 or resid 22...BA14 - 31514 - 315
27ILEILEILEILE(chain B and (resid 14 through 20 or resid 22...BA14 - 31514 - 315

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Components

#1: Protein p-hydroxyphenylacetate 3-hydroxylase, reductase component / 4-HPA 3-monooxygenase small component / Flavin:NAD(+) oxidoreductase / p-hydroxyphenylacetate 3- ...4-HPA 3-monooxygenase small component / Flavin:NAD(+) oxidoreductase / p-hydroxyphenylacetate 3-hydroxylase C1 component


Mass: 35455.227 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baumannii (bacteria) / Gene: C1-hpah / Plasmid: pET11a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q6Q271, flavin reductase (NADH)
#2: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE / Flavin mononucleotide


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.23 Å3/Da / Density % sol: 45 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 0.1M HEPES pH 7.5, 1.0M sodium citrate trihydrate, 2mM HPA.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: ENRAF-NONIUS FR591 / Wavelength: 1.5418 Å
DetectorType: Nonius Kappa CCD / Detector: CCD / Date: Mar 29, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.898→50 Å / Num. obs: 14373 / % possible obs: 97.1 % / Redundancy: 4.3 % / Biso Wilson estimate: 22.63 Å2 / Rmerge(I) obs: 0.144 / Χ2: 2.545 / Net I/σ(I): 10.1
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsΧ2Diffraction-ID% possible all
2.9-32.30.44812752.833189.4
3-3.122.50.37713492.725193.8
3.12-3.272.80.33514053.078196.1
3.27-3.4430.27613873.224196.2
3.44-3.653.50.21914603.252198
3.65-3.944.40.15814283.049198.6
3.94-4.334.80.12514652.85199.3
4.33-4.965.20.0914732.436199.8
4.96-6.246.10.10915112.033199.9
6.24-507.50.06816201.962199.7

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation4.88 Å45.34 Å
Translation4.88 Å45.34 Å

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Processing

Software
NameVersionClassification
PHENIX1.13_2998: ???refinement
HKL-2000data reduction
HKL-2000data scaling
PHASER2.7.17phasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.898→45.337 Å / SU ML: 0.38 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 26.43
RfactorNum. reflection% reflection
Rfree0.2889 734 5.13 %
Rwork0.2534 --
obs0.2552 14317 97.03 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 62.59 Å2 / Biso mean: 20.0061 Å2 / Biso min: 8.7 Å2
Refinement stepCycle: final / Resolution: 2.898→45.337 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4726 0 62 0 4788
Biso mean--12.87 --
Num. residues----596
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2652X-RAY DIFFRACTION2.564TORSIONAL
12B2652X-RAY DIFFRACTION2.564TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 5

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.8982-3.12190.36671410.35012479262091
3.1219-3.4360.32681500.3212633278396
3.436-3.9330.28711390.25092740287998
3.933-4.95410.25661580.20542774293299
4.9541-45.34280.24521460.207729573103100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.40070.42710.69761.38820.06491.397-0.14930.3908-0.1121-0.01810.1252-0.41370.10890.62660.33520.14240.07660.00530.15710.02130.12954.7254119.6072202.8001
20.4655-1.33390.06763.7116-0.15861.4333-0.08270.1797-0.16060.19720.08810.71180.037-0.4782-0.41330.21130.0642-0.11630.3499-0.00280.217620.4074127.8232192.0713
30.85810.11210.11742.4915-0.23131.52530.15220.2633-0.6014-0.6527-0.4279-0.5460.18730.3067-2.08890.1341-0.0341-0.0510.1054-0.1638-0.05138.139111.9724178.593
42.26041.14180.47963.48490.86981.44460.0095-0.9416-0.51411.0534-0.25870.38330.3827-0.2374-2.5305-0.1290.2008-0.2585-0.05630.5192-0.854436.1557117.2562212.0095
50.3799-0.04660.09120.88720.22941.1398-0.0329-0.2405-0.3780.25360.08360.37970.1222-0.428-0.71150.0999-0.05920.12420.11780.12550.292434.9812108.436210.4809
62.4562-1.82941.15533.3730.97522.21050.15420.54960.4522-0.6822-0.302-0.5575-0.66090.3123-0.75460.3004-0.0265-0.1220.2548-0.18150.253642.6638119.9859175.5887
70.2833-0.32260.02381.40380.42620.7062-0.0169-0.0120.2317-0.00560.00490.2537-0.0109-0.1431-00.0742-0.0016-0.00720.1716-0.02030.177226.6775133.6947193.6761
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 14 through 165 )A14 - 165
2X-RAY DIFFRACTION2chain 'A' and (resid 166 through 191 )A166 - 191
3X-RAY DIFFRACTION3chain 'A' and (resid 192 through 315 )A192 - 315
4X-RAY DIFFRACTION4chain 'B' and (resid 14 through 47 )B14 - 47
5X-RAY DIFFRACTION5chain 'B' and (resid 48 through 165 )B48 - 165
6X-RAY DIFFRACTION6chain 'B' and (resid 166 through 191 )B166 - 191
7X-RAY DIFFRACTION7chain 'B' and (resid 192 through 315 )B192 - 315

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