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PDBsum entry 1e7q

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Epimerase/reductase PDB id
1e7q
Jmol
Contents
Protein chain
314 a.a. *
Ligands
NAP
UVW
SO4 ×4
TRS
Waters ×314
* Residue conservation analysis
PDB id:
1e7q
Name: Epimerase/reductase
Title: Gdp 4-keto-6-deoxy-d-mannose epimerase reductase s107a
Structure: Gdp-fucose synthetase. Chain: a. Synonym: gdp-4-keto 6-deoxy-mannose 3,5-epimerase 4-reductase, gmer. Engineered: yes. Mutation: yes. Other_details: unknown molecule labeled as acetylphosphate
Source: Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562
Biol. unit: Homo-Dimer (from PDB file)
Resolution:
1.6Å     R-factor:   0.138     R-free:   0.182
Authors: C.Rosano,G.Izzo,M.Bolognesi
Key ref:
C.Rosano et al. (2000). Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants. J Mol Biol, 303, 77-91. PubMed id: 11021971 DOI: 10.1006/jmbi.2000.4106
Date:
07-Sep-00     Release date:   13-Feb-07    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P32055  (FCL_ECOLI) -  GDP-L-fucose synthase
Seq:
Struc:
321 a.a.
314 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.1.1.1.271  - GDP-L-fucose synthase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

      Pathway:
GDP-L-Fucose and GDP-mannose Biosynthesis
      Reaction: GDP-beta-L-fucose + NADP+ = GDP-4-dehydro-6-deoxy-alpha-D-mannose + NADPH
GDP-beta-L-fucose
+
NADP(+)
Bound ligand (Het Group name = NAP)
corresponds exactly
= GDP-4-dehydro-6-deoxy-alpha-D-mannose
+ NADPH
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   1 term 
  Biological process     metabolic process   5 terms 
  Biochemical function     catalytic activity     6 terms  

 

 
    reference    
 
 
DOI no: 10.1006/jmbi.2000.4106 J Mol Biol 303:77-91 (2000)
PubMed id: 11021971  
 
 
Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants.
C.Rosano, A.Bisso, G.Izzo, M.Tonetti, L.Sturla, A.De Flora, M.Bolognesi.
 
  ABSTRACT  
 
GDP-4-keto-6-deoxy-d-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-l-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-l-fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy-d-mannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-specific mutant forms of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and refined at 1. 45-1.60 A resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The refined protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy-d-mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.
 
  Selected figure(s)  
 
Figure 1.
Figure 1. The GDP- Image -fucose biosynthetic pathway and the products obtained after NaBH[4] reduction of the intermediate compounds. GMD, GDP- Image -mannose 4,6 dehydratase; GMER, GDP-4-keto-6-deoxy- Image -mannose epimerase/reductase; R-, GDP.
Figure 6.
Figure 6. Stereo view of the proposed binding mode for a nucleotide-sugar molecule (shown as a space-filling model) relative to active-site residues discussed in the text. The C-2 and C-3 centers can be recognized as those closest to His179 side-chain; the C-4 center falls next to the nicotinamide carboxamido group.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 303, 77-91) copyright 2000.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
18398479 A.R.Kinjo, and H.Nakamura (2008).
Nature of protein family signatures: insights from singular value analysis of position-specific scoring matrices.
  PLoS ONE, 3, e1963.  
18422615 B.Liu, Y.A.Knirel, L.Feng, A.V.Perepelov, S.N.Senchenkova, Q.Wang, P.R.Reeves, and L.Wang (2008).
Structure and genetics of Shigella O antigens.
  FEMS Microbiol Rev, 32, 627-653.  
  19058170 C.J.Thibodeaux, C.E.Melançon, and H.W.Liu (2008).
Natural-product sugar biosynthesis and enzymatic glycodiversification.
  Angew Chem Int Ed Engl, 47, 9814-9859.  
17634983 K.Imada, T.Tamura, R.Takenaka, I.Kobayashi, K.Namba, and K.Inagaki (2008).
Structure and quantum chemical analysis of NAD+-dependent isocitrate dehydrogenase: hydride transfer and co-factor specificity.
  Proteins, 70, 63-71.
PDB code: 2d4v
19011750 K.L.Kavanagh, H.Jörnvall, B.Persson, and U.Oppermann (2008).
Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.
  Cell Mol Life Sci, 65, 3895-3906.  
17625271 C.Louime, M.Abazinge, E.Johnson, L.Latinwo, C.Ikediobi, and A.M.Clark (2007).
Molecular cloning and biochemical characterization of a family-9 endoglucanase with an unusual structure from the gliding bacteria Cytophaga hutchinsonii.
  Appl Biochem Biotechnol, 141, 127-138.  
17950751 J.D.King, N.J.Harmer, A.Preston, C.M.Palmer, M.Rejzek, R.A.Field, T.L.Blundell, and D.J.Maskell (2007).
Predicting protein function from structure--the roles of short-chain dehydrogenase/reductase enzymes in Bordetella O-antigen biosynthesis.
  J Mol Biol, 374, 749-763.
PDB codes: 2pzj 2pzk 2pzl 2pzm 2q1s 2q1t 2q1u 2q1w
16650000 S.Rhomberg, C.Fuchsluger, D.Rendić, K.Paschinger, V.Jantsch, P.Kosma, and I.B.Wilson (2006).
Reconstitution in vitro of the GDP-fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster.
  FEBS J, 273, 2244-2256.  
12954627 B.A.Wolucka, and M.Van Montagu (2003).
GDP-mannose 3',5'-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants.
  J Biol Chem, 278, 47483-47490.  
14551189 L.S.Forsberg, K.D.Noel, J.Box, and R.W.Carlson (2003).
Genetic locus and structural characterization of the biochemical defect in the O-antigenic polysaccharide of the symbiotically deficient Rhizobium etli mutant, CE166. Replacement of N-acetylquinovosamine with its hexosyl-4-ulose precursor.
  J Biol Chem, 278, 51347-51359.  
12192068 C.A.Bottoms, P.E.Smith, and J.J.Tanner (2002).
A structurally conserved water molecule in Rossmann dinucleotide-binding domains.
  Protein Sci, 11, 2125-2137.  
12004063 C.Creuzenet, R.V.Urbanic, and J.S.Lam (2002).
Structure-function studies of two novel UDP-GlcNAc C6 dehydratases/C4 reductases. Variation from the SYK dogma.
  J Biol Chem, 277, 26769-26778.  
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.