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PDBsum entry 1toi

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Transferase PDB id
1toi
Jmol
Contents
Protein chain
396 a.a. *
Ligands
HCI
Waters ×230
* Residue conservation analysis
PDB id:
1toi
Name: Transferase
Title: Hydrocinnamic acid-bound structure of hexamutant + a293d mut coli aspartate aminotransferase
Structure: Aspartate aminotransferase. Chain: a. Synonym: transaminase a, aspat. Engineered: yes. Mutation: yes
Source: Escherichia coli. Organism_taxid: 562. Gene: aspc, b0928. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PDB file)
Resolution:
1.90Å     R-factor:   0.175     R-free:   0.197
Authors: M.A.Chow,K.E.Mcelroy,K.D.Corbett,J.M.Berger,J.F.Kirsch
Key ref:
M.A.Chow et al. (2004). Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase. Biochemistry, 43, 12780-12787. PubMed id: 15461450 DOI: 10.1021/bi0487544
Date:
14-Jun-04     Release date:   05-Oct-04    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P00509  (AAT_ECOLI) -  Aspartate aminotransferase
Seq:
Struc:
396 a.a.
396 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 8 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.2.6.1.1  - Aspartate transaminase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: L-aspartate + 2-oxoglutarate = oxaloacetate + L-glutamate
L-aspartate
+
2-oxoglutarate
Bound ligand (Het Group name = HCI)
matches with 50.00% similarity
= oxaloacetate
+ L-glutamate
      Cofactor: Pyridoxal 5'-phosphate
Pyridoxal 5'-phosphate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   2 terms 
  Biological process     biosynthetic process   4 terms 
  Biochemical function     catalytic activity     8 terms  

 

 
    reference    
 
 
DOI no: 10.1021/bi0487544 Biochemistry 43:12780-12787 (2004)
PubMed id: 15461450  
 
 
Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase.
M.A.Chow, K.E.McElroy, K.D.Corbett, J.M.Berger, J.F.Kirsch.
 
  ABSTRACT  
 
Several mutant Escherichia coli aspartate aminotransferases (eAATases) have been characterized in the attempt to evolve or rationally redesign the substrate specificity of eAATase into that of E. coli tyrosine aminotransferase (eTATase). These include HEX (designed), HEX + A293D (design followed by directed evolution), and SRHEPT (directed evolution). The A293D mutation realized from directed evolution of HEX is here imported into the SRHEPT platform by site-directed mutagenesis, resulting in an enzyme (SRHEPT + A293D) with nearly the same ratio of k(cat)/K(m)(Phe) to k(cat)/K(m)(Asp) as that of wild-type eTATase. The A293D substitution is an important specificity determinant; it selectively disfavors interactions with dicarboxylic substrates and inhibitors compared to aromatic ones. Context dependence analysis is generalized to provide quantitative comparisons of a common substitution in two or more different protein scaffolds. High-resolution crystal structures of ligand complexes of HEX + A293D, SRHEPT, and SRHEPT + A293D were determined. We find that in both SRHEPT + A293D and HEX + A293D, the additional mutation holds the Arg 292 side chain away from the active site to allow increased specificity for phenylalanine over aspartate. The resulting movement of Arg 292 allows greater flexibility of the small domain in HEX + A293D. While HEX is always in the closed conformation, HEX + A293D is observed in both the closed and a novel open conformation, allowing for more rapid product release.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
20826162 I.Campeotto, A.H.Bolt, T.A.Harman, C.Dennis, C.H.Trinh, S.E.Phillips, A.Nelson, A.R.Pearson, and A.Berry (2010).
Structural insights into substrate specificity in variants of N-acetylneuraminic Acid lyase produced by directed evolution.
  J Mol Biol, 404, 56-69.
PDB codes: 2wnn 2wnq 2wnz 2wo5 2wpb 2xfw
17680656 B.K.Cho, H.Y.Park, J.H.Seo, J.Kim, T.J.Kang, B.S.Lee, and B.G.Kim (2008).
Redesigning the substrate specificity of omega-aminotransferase for the kinetic resolution of aliphatic chiral amines.
  Biotechnol Bioeng, 99, 275-284.  
17964807 K.E.Muratore, J.R.Srouji, M.A.Chow, and J.F.Kirsch (2008).
Recombinant expression of twelve evolutionarily diverse subfamily Ialpha aminotransferases.
  Protein Expr Purif, 57, 34-44.  
17469798 T.D.Turbeville, J.Zhang, G.A.Hunter, and G.C.Ferreira (2007).
Histidine 282 in 5-aminolevulinate synthase affects substrate binding and catalysis.
  Biochemistry, 46, 5972-5981.  
16923533 J.Kaur, and R.Sharma (2006).
Directed evolution: an approach to engineer enzymes.
  Crit Rev Biotechnol, 26, 165-199.  
16611635 L.Chávez-Gutiérrez, E.Matta-Camacho, J.Osuna, E.Horjales, P.Joseph-Bravo, B.Maigret, and J.L.Charli (2006).
Homology modeling and site-directed mutagenesis of pyroglutamyl peptidase II. Insights into omega-versus aminopeptidase specificity in the M1 family.
  J Biol Chem, 281, 18581-18590.  
16672228 M.Pieren, A.E.Prota, C.Ruch, D.Kostrewa, A.Wagner, K.Biedermann, F.K.Winkler, and K.Ballmer-Hofer (2006).
Crystal structure of the Orf virus NZ2 variant of vascular endothelial growth factor-E. Implications for receptor specificity.
  J Biol Chem, 281, 19578-19587.
PDB code: 2gnn
16503426 M.Royo, and S.Colette Daubner (2006).
Kinetics of regulatory serine variants of tyrosine hydroxylase with cyclic AMP-dependent protein kinase and extracellular signal-regulated protein kinase 2.
  Biochim Biophys Acta, 1764, 786-792.  
15857778 L.G.Otten, and W.J.Quax (2005).
Directed evolution: selecting today's biocatalysts.
  Biomol Eng, 22, 1-9.  
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.