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PDBsum entry 1k0i

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Hydrolase PDB id
1k0i
Jmol
Contents
Protein chain
394 a.a. *
Ligands
SO4 ×8
SO3
FAD
PHB ×2
Waters ×256
* Residue conservation analysis
PDB id:
1k0i
Name: Hydrolase
Title: Pseudomonas aeruginosa phbh r220q in complex with 100mm phb
Structure: P-hydroxybenzoate hydroxylase. Chain: a. Engineered: yes. Mutation: yes
Source: Pseudomonas aeruginosa. Organism_taxid: 287. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PDB file)
Resolution:
1.80Å     R-factor:   0.207     R-free:   0.240
Authors: J.Wang,M.Ortiz-Maldonado,B.Entsch,D.Ballou,D.L.Gatti
Key ref:
J.Wang et al. (2002). Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase. Proc Natl Acad Sci U S A, 99, 608-613. PubMed id: 11805318 DOI: 10.1073/pnas.022640199
Date:
19-Sep-01     Release date:   27-Feb-02    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P20586  (PHHY_PSEAE) -  p-hydroxybenzoate hydroxylase
Seq:
Struc:
394 a.a.
394 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     metabolic process   5 terms 
  Biochemical function     oxidoreductase activity     4 terms  

 

 
DOI no: 10.1073/pnas.022640199 Proc Natl Acad Sci U S A 99:608-613 (2002)
PubMed id: 11805318  
 
 
Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.
J.Wang, M.Ortiz-Maldonado, B.Entsch, V.Massey, D.Ballou, D.L.Gatti.
 
  ABSTRACT  
 
para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide. These conflicting requirements are met by the coordination of multiple movements involving the protein, the two cofactors, and the substrate. Here, using the R220Q mutant form of para-hydroxybenzoate hydroxylase, we show that in the absence of substrate, the large beta alpha beta domain (residues 1-180) and the smaller sheet domain (residues 180-270) separate slightly, and the flavin swings out to a more exposed position to open an aqueous channel from the solvent to the protein interior. Substrate entry occurs by first binding at a surface site and then sliding into the protein interior. In our study of this mutant, the structure of the complex with pyridine nucleotide was obtained. This cofactor binds in an extended conformation at the enzyme surface in a groove that crosses the binding site of FAD. We postulate that for stereospecific reduction, the flavin swings to an out position and NADPH assumes a folded conformation that brings its nicotinamide moiety into close contact with the isoalloxazine moiety of the flavin. This work clearly shows how complex dynamics can play a central role in catalysis by enzymes.
 
  Selected figure(s)  
 
Figure 1.
Fig. 1. The active site of R220Q PHBH. The C trace and side chains of the mutant enzyme are shown with salmon and ivory bonds, respectively; nitrogen is blue, and oxygen is red. The backbones of residues 44-47 and 297-300 are shown with sticks colored according to atom type. The C trace and the Arg-220 side chain of the wild-type enzyme are shown with transparent bonds. FAD is shown with green bonds, its re face pointing to the right. Hydrogen bonds are shown as dotted light blue lines. Coordinates for this figure are from PDB entry 1IUW (wild type) and 1K0L (R220Q PHBH, this study). Figures were generated with MOLSCRIPT (32), BOBSCRIPT (33), and RASTER3D (34).
Figure 3.
Fig. 3. Cofactor dynamics in PHBH. (A) Stereoview of the NADPH binding site. The C trace and side chains of the R220Q enzyme are shown with salmon and ivory bonds, respectively. FAD is shown with green bonds. NADPH is shown as ball-and-sticks with gold bonds, surrounded by a sigmaA-weighted omit electron density map contoured at 0.9 . Sulfate ions are shown as ball-and-sticks with yellow bonds. Nitrogen, blue; oxygen, red; sulfur, yellow; phosphorus, pale blue. Note how the FAD ring is slightly bent and occupies the "in" position. Coordinates are from PDB entry 1K0J (this study). (B) Modeling of the reduction step of the catalytic cycle. The FAD ring is "out," and the nicotinamide moiety of NADPH swings "in" such that its R side faces the re side of the flavin. Only a few side chains are drawn for reference. Coordinates for this panel were derived by energy minimization after the flavin and nicotinamide rings had been manually repositioned.
 
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21402075 G.Volkers, G.J.Palm, M.S.Weiss, G.D.Wright, and W.Hinrichs (2011).
Structural basis for a new tetracycline resistance mechanism relying on the TetX monooxygenase.
  FEBS Lett, 585, 1061-1066.
PDB codes: 2xdo 2xyo 2y6q 2y6r
21183988 Y.W.Tan, and H.Yang (2011).
Seeing the forest for the trees: fluorescence studies of single enzymes in the context of ensemble experiments.
  Phys Chem Chem Phys, 13, 1709-1721.  
20029835 J.C.Lambry, E.Beaumont, B.Tarus, M.Blanchard-Desce, and A.Slama-Schwok (2010).
Selective probing of a NADPH site controlled light-induced enzymatic catalysis.
  J Mol Recognit, 23, 379-388.  
20055497 U.E.Ukaegbu, A.Kantz, M.Beaton, G.T.Gassner, and A.C.Rosenzweig (2010).
Structure and ligand binding properties of the epoxidase component of styrene monooxygenase .
  Biochemistry, 49, 1678-1688.
PDB code: 3ihm
19664062 C.R.Pudney, S.Hay, and N.S.Scrutton (2009).
Bipartite recognition and conformational sampling mechanisms for hydride transfer from nicotinamide coenzyme to FMN in pentaerythritol tetranitrate reductase.
  FEBS J, 276, 4780-4789.  
19317437 K.M.McCulloch, T.Mukherjee, T.P.Begley, and S.E.Ealick (2009).
Structure of the PLP degradative enzyme 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase from Mesorhizobium loti MAFF303099 and its mechanistic implications.
  Biochemistry, 48, 4139-4149.
PDB codes: 3gmb 3gmc
19243237 T.Senda, M.Senda, S.Kimura, and T.Ishida (2009).
Redox control of protein conformation in flavoproteins.
  Antioxid Redox Signal, 11, 1741-1766.  
18176933 E.V.Kudryashova, A.J.Visser, and W.J.van Berkel (2008).
Monomer formation and function of p-hydroxybenzoate hydroxylase in reverse micelles and in dimethylsulfoxide/water mixtures.
  Chembiochem, 9, 413-419.  
18071645 Y.Huang, K.X.Zhao, X.H.Shen, C.Y.Jiang, and S.J.Liu (2008).
Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum.
  Appl Microbiol Biotechnol, 78, 75-83.  
17873060 K.S.Ryan, A.R.Howard-Jones, M.J.Hamill, S.J.Elliott, C.T.Walsh, and C.L.Drennan (2007).
Crystallographic trapping in the rebeccamycin biosynthetic enzyme RebC.
  Proc Natl Acad Sci U S A, 104, 15311-15316.
PDB codes: 2r0c 2r0g 2r0p
16275925 C.Siebold, N.Berrow, T.S.Walter, K.Harlos, R.J.Owens, D.I.Stuart, J.R.Terman, A.L.Kolodkin, R.J.Pasterkamp, and E.Y.Jones (2005).
High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule.
  Proc Natl Acad Sci U S A, 102, 16836-16841.
PDB codes: 2bry 2c4c
16275926 M.Nadella, M.A.Bianchet, S.B.Gabelli, J.Barrila, and L.M.Amzel (2005).
Structure and activity of the axon guidance protein MICAL.
  Proc Natl Acad Sci U S A, 102, 16830-16835.
PDB code: 2bra
16267294 X.Gao, C.L.Tan, C.C.Yeo, and C.L.Poh (2005).
Molecular and biochemical characterization of the xlnD-encoded 3-hydroxybenzoate 6-hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9867.
  J Bacteriol, 187, 7696-7702.  
15010540 E.Hitt, and M.L.Ludwig (2004).
Biography of Martha L. Ludwig.
  Proc Natl Acad Sci U S A, 101, 3727-3728.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.