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PDBsum entry 2hm7

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Hydrolase PDB id
2hm7
Jmol
Contents
Protein chain
308 a.a. *
Ligands
SO4
Waters ×162
* Residue conservation analysis
PDB id:
2hm7
Name: Hydrolase
Title: Crystal structure analysis of the g84s est2 mutant
Structure: Carboxylesterase. Chain: a. Engineered: yes. Mutation: yes
Source: Alicyclobacillus acidocaldarius. Organism_taxid: 405212. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Resolution:
2.00Å     R-factor:   0.196     R-free:   0.222
Authors: V.Menchise,V.Alterio,G.De Simone
Key ref:
L.Mandrich et al. (2007). Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius. Proteins, 71, 1721-1731. PubMed id: 18076040 DOI: 10.1002/prot.21877
Date:
11-Jul-06     Release date:   26-Jun-07    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
Q7SIG1  (Q7SIG1_ALIAC) -  Hydrolase
Seq:
Struc:
310 a.a.
308 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   1 term 
  Biochemical function     hydrolase activity     1 term  

 

 
DOI no: 10.1002/prot.21877 Proteins 71:1721-1731 (2007)
PubMed id: 18076040  
 
 
Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius.
L.Mandrich, V.Menchise, V.Alterio, G.De Simone, C.Pedone, M.Rossi, G.Manco.
 
  ABSTRACT  
 
Recent mutagenic and molecular modelling studies suggested a role for glycine 84 in the putative oxyanion loop of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. A 114 times decrease of the esterase catalytic activity of the G84S mutant was observed, without changes in the thermal stability. The recently solved three-dimensional (3D) structure of EST2 in complex with a HEPES molecule permitted to demonstrate that G84 (together with G83 and A156) is involved in the stabilization of the oxyanion through a hydrogen bond from its main chain NH group. The structural data in this case did not allowed us to rationalize the effect of the mutation, since this hydrogen bond was predicted to be unaltered in the mutant. Since the mutation could shed light on the role of the oxyanion loop in the HSL family, experiments to elucidate at the mechanistic level the reasons of the observed drop in k (cat) were devised. In this work, the kinetic and structural features of the G84S mutant were investigated in more detail. The optimal temperature and pH for the activity of the mutated enzyme were found significantly changed (T = 65 degrees C and pH = 5.75). The catalytic constants K (M) and V(max) were found considerably altered in the mutant, with ninefold increased K (M) and 14-fold decreased V(max), at pH 5.75. At pH 7.1, the decrease in k (cat) was much more dramatic. The measurement of kinetic constants for some steps of the reaction mechanism and the resolution of the mutant 3D structure provided evidences that the observed effects were partly due to the steric hindrance of the S84-OH group towards the ester substrate and partly to its interference with the nucleophilic attack of a water molecule on the second tetrahedral intermediate. Proteins 2007. (c) 2007 Wiley-Liss, Inc.
 
  Selected figure(s)  
 
Figure 3.
Figure 3. EST2 reaction mechanism.
Figure 8.
Figure 8. Ball and stick representation of the second tetrahedral intermediate models of (A) EST2-octanoate and (B) G84S-octanoate.
 
  The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2007, 71, 1721-1731) copyright 2007.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20383468 S.Yu, B.Zheng, X.Zhao, and Y.Feng (2010).
Gene cloning and characterization of a novel thermophilic esterase from Fervidobacterium nodosum Rt17-B1.
  Acta Biochim Biophys Sin (Shanghai), 42, 288-295.  
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.