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PDBsum entry 1dmq

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protein links
Isomerase PDB id
1dmq
Jmol
Contents
Protein chain
123 a.a. *
Waters ×47
* Residue conservation analysis
PDB id:
1dmq
Name: Isomerase
Title: Crystal structure of mutant enzyme y32f of ketosteroid isomerase from pseudomonas putida biotype b
Structure: Steroid delta-isomerase. Chain: a. Engineered: yes. Mutation: yes
Source: Pseudomonas putida. Organism_taxid: 303. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PDB file)
Resolution:
2.15Å     R-factor:   0.182     R-free:   0.234
Authors: D.H.Kim,D.S.Jang,G.H.Nam,B.H.Oh,K.Y.Choi
Key ref:
D.H.Kim et al. (2000). Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B. Biochemistry, 39, 4581-4589. PubMed id: 10769113 DOI: 10.1021/bi992119u
Date:
14-Dec-99     Release date:   23-May-00    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P07445  (SDIS_PSEPU) -  Steroid Delta-isomerase
Seq:
Struc:
131 a.a.
123 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.5.3.3.1  - Steroid Delta-isomerase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: A 3-oxo-Delta5-steroid = a 3-oxo-Delta4-steroid
3-oxo-Delta(5)-steroid
= 3-oxo-Delta(4)-steroid
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     intracellular   1 term 
  Biological process     transport   3 terms 
  Biochemical function     isomerase activity     2 terms  

 

 
    Added reference    
 
 
DOI no: 10.1021/bi992119u Biochemistry 39:4581-4589 (2000)
PubMed id: 10769113  
 
 
Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B.
D.H.Kim, D.S.Jang, G.H.Nam, G.Choi, J.S.Kim, N.C.Ha, M.S.Kim, B.H.Oh, K.Y.Choi.
 
  ABSTRACT  
 
Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
22388816 K.Hotta, X.Chen, R.S.Paton, A.Minami, H.Li, K.Swaminathan, I.I.Mathews, K.Watanabe, H.Oikawa, K.N.Houk, and C.Y.Kim (2012).
Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis.
  Nature, 483, 355-358.
PDB code: 3rga
20937603 S.Ayuso-Tejedor, O.Abián, and J.Sancho (2011).
Underexposed polar residues and protein stabilization.
  Protein Eng Des Sel, 24, 171-177.  
20080683 D.A.Kraut, P.A.Sigala, T.D.Fenn, and D.Herschlag (2010).
Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole.
  Proc Natl Acad Sci U S A, 107, 1960-1965.
PDB code: 3ipt
19799395 D.K.Chakravorty, A.V.Soudackov, and S.Hammes-Schiffer (2009).
Hybrid quantum/classical molecular dynamics simulations of the proton transfer reactions catalyzed by ketosteroid isomerase: analysis of hydrogen bonding, conformational motions, and electrostatics.
  Biochemistry, 48, 10608-10619.  
18424811 H.J.Lee, Y.J.Yoon, d.o. .S.Jang, C.Kim, H.J.Cha, B.H.Hong, K.Y.Choi, and H.C.Lee (2008).
15N NMR relaxation studies of Y14F mutant of ketosteroid isomerase: the influence of mutation on backbone mobility.
  J Biochem, 144, 159-166.  
15819891 Y.S.Yun, G.H.Nam, Y.G.Kim, B.H.Oh, and K.Y.Choi (2005).
Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B.
  FEBS J, 272, 1999-2011.
PDB code: 1w6y
11274465 D.H.Kim, G.H.Nam, D.S.Jang, S.Yun, G.Choi, H.C.Lee, and K.Y.Choi (2001).
Roles of dimerization in folding and stability of ketosteroid isomerase from Pseudomonas putida biotype B.
  Protein Sci, 10, 741-752.  
11274474 Y.Gholizadeh, M.Prevost, F.Van Bambeke, B.Casadewall, P.M.Tulkens, and P.Courvalin (2001).
Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium.
  Protein Sci, 10, 836-844.  
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 code is shown on the right.