PDBsum entry 3pcd

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protein ligands metals Protein-protein interface(s) links
Dioxygenase PDB id
Protein chains
(+ 0 more) 200 a.a. *
(+ 0 more) 233 a.a. *
CO3 ×6
BME ×6
_FE ×6
Waters ×1422
* Residue conservation analysis
PDB id:
Name: Dioxygenase
Title: Protocatechuate 3,4-dioxygenase y447h mutant
Structure: Protocatechuate 3,4-dioxygenase. Chain: a, b, c, d, e, f. Engineered: yes. Mutation: yes. Protocatechuate 3,4-dioxygenase. Chain: m, n, o, p, q, r. Engineered: yes. Mutation: yes
Source: Pseudomonas putida. Organism_taxid: 303. Atcc: 23975. Gene: pcah. Expressed in: pseudomonas fluorescens. Expression_system_taxid: 294.
Biol. unit: 24mer (from PDB file)
2.10Å     R-factor:   0.180    
Authors: A.M.Orville,J.D.Lipscomb,D.H.Ohlendorf
Key ref:
R.W.Frazee et al. (1998). The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates. Biochemistry, 37, 2131-2144. PubMed id: 9485360 DOI: 10.1021/bi972047b
24-Nov-97     Release date:   27-May-98    
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Protein chains
Pfam   ArchSchema ?
P00436  (PCXA_PSEPU) -  Protocatechuate 3,4-dioxygenase alpha chain
201 a.a.
200 a.a.
Protein chains
Pfam   ArchSchema ?
P00437  (PCXB_PSEPU) -  Protocatechuate 3,4-dioxygenase beta chain
239 a.a.
233 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: Chains A, M, B, N, C, O, D, P, E, Q, F, R: E.C.  - Protocatechuate 3,4-dioxygenase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Benzoate Metabolism
      Reaction: 3,4-dihydroxybenzoate + O2 = 3-carboxy-cis,cis-muconate
+ O(2)
= 3-carboxy-cis,cis-muconate
      Cofactor: Iron
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     oxidation-reduction process   5 terms 
  Biochemical function     catalytic activity     8 terms  


DOI no: 10.1021/bi972047b Biochemistry 37:2131-2144 (1998)
PubMed id: 9485360  
The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates.
R.W.Frazee, A.M.Orville, K.B.Dolbeare, H.Yu, D.H.Ohlendorf, J.D.Lipscomb.
The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.

Literature references that cite this PDB file's key reference

  PubMed id Reference
21246129 N.Anitha, and M.Palaniandavar (2011).
Mononuclear iron(III) complexes of 3N ligands in organized assemblies: spectral and redox properties and attainment of regioselective extradiol dioxygenase activity.
  Dalton Trans, 40, 1888-1901.  
20835480 R.Mayilmurugan, M.Sankaralingam, E.Suresh, and M.Palaniandavar (2010).
Novel square pyramidal iron(III) complexes of linear tetradentate bis(phenolate) ligands as structural and reactive models for intradiol-cleaving 3,4-PCD enzymes: Quinone formation vs. intradiol cleavage.
  Dalton Trans, 39, 9611-9625.  
18003930 M.Y.Pau, J.D.Lipscomb, and E.I.Solomon (2007).
Substrate activation for O2 reactions by oxidized metal centers in biology.
  Proc Natl Acad Sci U S A, 104, 18355-18362.  
17256852 M.Y.Pau, M.I.Davis, A.M.Orville, J.D.Lipscomb, and E.I.Solomon (2007).
Spectroscopic and electronic structure study of the enzyme-substrate complex of intradiol dioxygenases: substrate activation by a high-spin ferric non-heme iron site.
  J Am Chem Soc, 129, 1944-1958.  
16317455 M.L.Neidig, and E.I.Solomon (2005).
Structure-function correlations in oxygen activating non-heme iron enzymes.
  Chem Commun (Camb), (), 5843-5863.  
15487948 C.K.Brown, M.W.Vetting, C.A.Earhart, and D.H.Ohlendorf (2004).
Biophysical analyses of designed and selected mutants of protocatechuate 3,4-dioxygenase1.
  Annu Rev Microbiol, 58, 555-585.
PDB codes: 2bum 2buq 2bur 2but 2buv
11594053 T.Sakurai, K.Fujimori, T.Ueda, H.Shindo, Y.Shibusawa, and M.Nakano (2001).
Sunlight induces N epsilon-(carboxymethyl)lysine formation from glycated polylysine-iron(III) complex.
  Photochem Photobiol, 74, 407-411.  
10736169 B.Schwartz, J.E.Dove, and J.P.Klinman (2000).
Kinetic analysis of oxygen utilization during cofactor biogenesis in a copper-containing amine oxidase from yeast.
  Biochemistry, 39, 3699-3707.  
10891075 M.W.Vetting, D.A.D'Argenio, L.N.Ornston, and D.H.Ohlendorf (2000).
Structure of Acinetobacter strain ADP1 protocatechuate 3, 4-dioxygenase at 2.2 A resolution: implications for the mechanism of an intradiol dioxygenase.
  Biochemistry, 39, 7943-7955.
PDB codes: 1eo2 1eo9 1eoa 1eob 1eoc
10801478 M.W.Vetting, and D.H.Ohlendorf (2000).
The 1.8 A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker.
  Structure, 8, 429-440.
PDB codes: 1dlm 1dlq 1dlt 1dmh
10467151 K.Sugimoto, T.Senda, H.Aoshima, E.Masai, M.Fukuda, and Y.Mitsui (1999).
Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions.
  Structure, 7, 953-965.
PDB codes: 1b4u 1bou
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