spacer
spacer

PDBsum entry 3b8u

Go to PDB code: 
protein ligands Protein-protein interface(s) links
Isomerase PDB id
3b8u
Jmol
Contents
Protein chains
359 a.a. *
Ligands
SO4 ×12
PLP ×4
Waters ×354
* Residue conservation analysis
PDB id:
3b8u
Name: Isomerase
Title: Crystal structure of escherichia coli alaine racemase mutant
Structure: Alanine racemase. Chain: a, b, c, d. Engineered: yes. Mutation: yes
Source: Escherichia coli. Organism_taxid: 83333. Strain: jm109. Gene: alr. Expressed in: escherichia coli.
Resolution:
3.00Å     R-factor:   0.215     R-free:   0.224
Authors: D.Wu,T.Hu,L.Zhang,H.Jiang,X.Shen
Key ref:
D.Wu et al. (2008). Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis. Protein Sci, 17, 1066-1076. PubMed id: 18434499 DOI: 10.1110/ps.083495908
Date:
02-Nov-07     Release date:   08-Jul-08    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P0A6B4  (ALR1_ECOLI) -  Alanine racemase, biosynthetic
Seq:
Struc:
359 a.a.
359 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.5.1.1.1  - Alanine racemase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: L-alanine = D-alanine
L-alanine
= D-alanine
      Cofactor: Pyridoxal 5'-phosphate
Pyridoxal 5'-phosphate
Bound ligand (Het Group name = PLP) matches with 93.75% similarity
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     cell wall organization   5 terms 
  Biochemical function     catalytic activity     4 terms  

 

 
    reference    
 
 
DOI no: 10.1110/ps.083495908 Protein Sci 17:1066-1076 (2008)
PubMed id: 18434499  
 
 
Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E. coli: Enzymatic characterization with crystal structure analysis.
D.Wu, T.Hu, L.Zhang, J.Chen, J.Du, J.Ding, H.Jiang, X.Shen.
 
  ABSTRACT  
 
Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr.
 
  Selected figure(s)  
 
Figure 3.
Substrate entryway of EcAlr. Stereo pictures of the conserved residues at the substrate entryway in both side view (A) and top view (B). Residues forming the entryway from the N-terminal domain, C-terminal domain, and C-terminal domain of the second monomer are colored in orange, yellow, and pink the same as in Figure 1B Figure 1.-, respectively. The catalytic residues Lys34, Tyr255[prime prime or minute], and PLP are colored in cyan. The rest of the part of the EcAlr dimer is shown as a ribbon diagram and colored in white. (C) Superposition of the residues chosen for mutation (plus nearby secondary structures) in EcAlr (cyan) with the counterparts in PaDadX (green) and MtAlr (magenta). The residues are labeled in the corresponding colors. (D) Superposition of the loop containing mutations from wild-type EcAlr (cyan), P219A (brown), E221A (sky blue), E221K (yellow), and E221P (purple). The residues after mutation are labeled in the corresponding colors.
Figure 4.
Electrostatic surfaces around the substrate entryways of EcAlr wild type (A), E221K (B), E221P (C), PaDadX (D), GsAlr (E), MtAlr (F), PLP --L-Ala-bound (G), and PLP --D-Ala-bound GsAlr (H). Red colored to blue suggests the charges from negative to positive. The charged residues around the entrance and PLP or PLP --Ala in the active sites are shown in sticks and labeled. The sulfate ions in EcAlr structures are also shown in sticks.
 
  The above figures are reprinted from an Open Access publication published by the Protein Society: Protein Sci (2008, 17, 1066-1076) copyright 2008.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
22194336 E.R.Scaletti, S.R.Luckner, and K.L.Krause (2012).
Structural features and kinetic characterization of alanine racemase from Staphylococcus aureus (Mu50).
  Acta Crystallogr D Biol Crystallogr, 68, 82-92.
PDB code: 4a3q
20971724 J.Ju, S.Xu, Y.Furukawa, Y.Zhang, H.Misono, T.Minamino, K.Namba, B.Zhao, and K.Ohnishi (2011).
Correlation between catalytic activity and monomer-dimer equilibrium of bacterial alanine racemases.
  J Biochem, 149, 83-89.  
20445930 J.Lowther, B.A.Yard, K.A.Johnson, L.G.Carter, V.T.Bhat, M.C.Raman, D.J.Clarke, B.Ramakers, S.A.McMahon, J.H.Naismith, and D.J.Campopiano (2010).
Inhibition of the PLP-dependent enzyme serine palmitoyltransferase by cycloserine: evidence for a novel decarboxylative mechanism of inactivation.
  Mol Biosyst, 6, 1682-1693.
PDB code: 2xbn
19695097 R.M.Couñago, M.Davlieva, U.Strych, R.E.Hill, and K.L.Krause (2009).
Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames).
  BMC Struct Biol, 9, 53.
PDB code: 3ha1
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.