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PDBsum entry 1spa

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Transferase(aminotransferase) PDB id
1spa
Jmol
Contents
Protein chain
396 a.a. *
Ligands
NPL
Waters ×100
* Residue conservation analysis
PDB id:
1spa
Name: Transferase(aminotransferase)
Title: Role of asp222 in the catalytic mechanism of escherichia col aspartate aminotransferase: the amino acid residue which en function of the enzyme-bound coenzyme pyridoxal 5'-phosphat
Structure: Aspartate aminotransferase. Chain: a. Engineered: yes
Source: Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562
Biol. unit: Dimer (from PQS)
Resolution:
2.00Å     R-factor:   0.209    
Authors: Y.Hinoue,T.Yano,D.E.Metzler,I.Miyahara,K.Hirotsu,H.Kagamiyam
Key ref:
T.Yano et al. (1992). Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate. Biochemistry, 31, 5878-5887. PubMed id: 1610831 DOI: 10.1021/bi00140a025
Date:
26-Jan-93     Release date:   31-Oct-93    
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 1 residue position (black cross)

 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
= oxaloacetate
+ L-glutamate
      Cofactor: Pyridoxal 5'-phosphate
Pyridoxal 5'-phosphate
Bound ligand (Het Group name = NPL) matches with 83.33% similarity
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/bi00140a025 Biochemistry 31:5878-5887 (1992)
PubMed id: 1610831  
 
 
Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate.
T.Yano, S.Kuramitsu, S.Tanase, Y.Morino, H.Kagamiyama.
 
  ABSTRACT  
 
Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
  21332942 H.J.Wu, Y.Yang, S.Wang, J.Q.Qiao, Y.F.Xia, Y.Wang, W.D.Wang, S.F.Gao, J.Liu, P.Q.Xue, and X.W.Gao (2011).
Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3.
  FEBS J, 278, 1345-1357.  
21081698 M.Koutmos, O.Kabil, J.L.Smith, and R.Banerjee (2010).
Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine {beta}-synthase.
  Proc Natl Acad Sci U S A, 107, 20958-20963.
PDB codes: 3pc2 3pc3 3pc4
17989071 Y.Yoshikane, N.Yokochi, M.Yamasaki, K.Mizutani, K.Ohnishi, B.Mikami, H.Hayashi, and T.Yagi (2008).
Crystal structure of pyridoxamine-pyruvate aminotransferase from Mesorhizobium loti MAFF303099.
  J Biol Chem, 283, 1120-1127.
PDB codes: 2z9u 2z9v 2z9w 2z9x
16141215 M.Goto, I.Miyahara, K.Hirotsu, M.Conway, N.Yennawar, M.M.Islam, and S.M.Hutson (2005).
Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin.
  J Biol Chem, 280, 37246-37256.
PDB codes: 2a1h 2cog 2coi 2coj
12672110 M.Allert, and L.Baltzer (2003).
Noncovalent binding of a reaction intermediate by a designed helix-loop-helix motif-implications for catalyst design.
  Chembiochem, 4, 306-318.  
14674749 P.LeMagueres, H.Im, A.Dvorak, U.Strych, M.Benedik, and K.L.Krause (2003).
Crystal structure at 1.45 A resolution of alanine racemase from a pathogenic bacterium, Pseudomonas aeruginosa, contains both internal and external aldimine forms.
  Biochemistry, 42, 14752-14761.
PDB code: 1rcq
11294630 K.Haruyama, T.Nakai, I.Miyahara, K.Hirotsu, H.Mizuguchi, H.Hayashi, and H.Kagamiyama (2001).
Structures of Escherichia coli histidinol-phosphate aminotransferase and its complexes with histidinol-phosphate and N-(5'-phosphopyridoxyl)-L-glutamate: double substrate recognition of the enzyme.
  Biochemistry, 40, 4633-4644.
PDB codes: 1gew 1gex 1gey
10684605 T.Fujii, M.Maeda, H.Mihara, T.Kurihara, N.Esaki, and Y.Hata (2000).
Structure of a NifS homologue: X-ray structure analysis of CsdB, an Escherichia coli counterpart of mammalian selenocysteine lyase.
  Biochemistry, 39, 1263-1273.
PDB code: 1c0n
10079072 A.A.Morollo, G.A.Petsko, and D.Ringe (1999).
Structure of a Michaelis complex analogue: propionate binds in the substrate carboxylate site of alanine racemase.
  Biochemistry, 38, 3293-3301.
PDB code: 2sfp
10584065 A.Poupon, F.Jebai, G.Labesse, F.Gros, J.Thibault, J.P.Mornon, and M.Krieger (1999).
Structure modelling and site-directed mutagenesis of the rat aromatic L-amino acid pyridoxal 5'-phosphate-dependent decarboxylase: a functional study.
  Proteins, 37, 191-203.  
9930994 P.W.van Ophem, D.Peisach, S.D.Erickson, K.Soda, D.Ringe, and J.M.Manning (1999).
Effects of the E177K mutation in D-amino acid transaminase. Studies on an essential coenzyme anchoring group that contributes to stereochemical fidelity.
  Biochemistry, 38, 1323-1331.
PDB code: 5daa
  10595543 W.Blankenfeldt, C.Nowicki, M.Montemartini-Kalisz, H.M.Kalisz, and H.J.Hecht (1999).
Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode.
  Protein Sci, 8, 2406-2417.
PDB code: 1bw0
9790670 H.Hayashi, H.Mizuguchi, and H.Kagamiyama (1998).
The imine-pyridine torsion of the pyridoxal 5'-phosphate Schiff base of aspartate aminotransferase lowers its pKa in the unliganded enzyme and is crucial for the successive increase in the pKa during catalysis.
  Biochemistry, 37, 15076-15085.  
9521672 J.Gong, G.A.Hunter, and G.C.Ferreira (1998).
Aspartate-279 in aminolevulinate synthase affects enzyme catalysis through enhancing the function of the pyridoxal 5'-phosphate cofactor.
  Biochemistry, 37, 3509-3517.  
  9761478 S.Pascarella, S.Angelaccio, R.Contestabile, S.Delle Fratte, M.Di Salvo, and F.Bossa (1998).
The structure of serine hydroxymethyltransferase as modeled by homology and validated by site-directed mutagenesis.
  Protein Sci, 7, 1976-1982.  
9063881 J.P.Shaw, G.A.Petsko, and D.Ringe (1997).
Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1.9-A resolution.
  Biochemistry, 36, 1329-1342.
PDB code: 1sft
8916896 J.Gong, C.J.Kay, M.J.Barber, and G.C.Ferreira (1996).
Mutations at a glycine loop in aminolevulinate synthase affect pyridoxal phosphate cofactor binding and catalysis.
  Biochemistry, 35, 14109-14117.  
7744828 A.L.Osterman, L.N.Kinch, N.V.Grishin, and M.A.Phillips (1995).
Acidic residues important for substrate binding and cofactor reactivity in eukaryotic ornithine decarboxylase identified by alanine scanning mutagenesis.
  J Biol Chem, 270, 11797-11802.  
  8563634 M.D.Toney, S.Pascarella, and D.De Biase (1995).
Active site model for gamma-aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities.
  Protein Sci, 4, 2366-2374.  
8513804 P.K.Mehta, T.I.Hale, and P.Christen (1993).
Aminotransferases: demonstration of homology and division into evolutionary subgroups.
  Eur J Biochem, 214, 549-561.  
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.