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PDBsum entry 1iin

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protein ligands Protein-protein interface(s) links
Transferase PDB id
1iin
Jmol
Contents
Protein chains
289 a.a. *
Ligands
UPG ×4
Waters ×762
* Residue conservation analysis
PDB id:
1iin
Name: Transferase
Title: Thymidylyltransferase complexed with udp-glucose
Structure: Glucose-1-phosphate thymidylyltransferase. Chain: a, b, c, d. Synonym: thymidylyltransferase. Engineered: yes
Source: Salmonella enterica. Organism_taxid: 28901. Strain: lt2. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Tetramer (from PQS)
Resolution:
2.10Å     R-factor:   0.184     R-free:   0.224
Authors: W.A.Barton,J.Lesniak,J.B.Biggins,P.D.Jeffrey,J.Jiang,K.R.Raj J.S.Thorson,D.B.Nikolov
Key ref:
W.A.Barton et al. (2001). Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization. Nat Struct Biol, 8, 545-551. PubMed id: 11373625 DOI: 10.1038/88618
Date:
23-Apr-01     Release date:   09-May-01    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
No UniProt id for this chain
Struc: 289 a.a.
Key:    Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.2.7.7.24  - Glucose-1-phosphate thymidylyltransferase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

      Pathway:
6-Deoxyhexose Biosynthesis
      Reaction: dTTP + alpha-D-glucose 1-phosphate = diphosphate + dTDP-alpha-D-glucose
dTTP
+ alpha-D-glucose 1-phosphate
= diphosphate
+
dTDP-alpha-D-glucose
Bound ligand (Het Group name = UPG)
matches with 94.59% similarity
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     biosynthetic process   5 terms 
  Biochemical function     transferase activity     5 terms  

 

 
    reference    
 
 
DOI no: 10.1038/88618 Nat Struct Biol 8:545-551 (2001)
PubMed id: 11373625  
 
 
Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization.
W.A.Barton, J.Lesniak, J.B.Biggins, P.D.Jeffrey, J.Jiang, K.R.Rajashankar, J.S.Thorson, D.B.Nikolov.
 
  ABSTRACT  
 
Metabolite glycosylation is affected by three classes of enzymes: nucleotidylyltransferases, which activate sugars as nucleotide diphospho-derivatives, intermediate sugar-modifying enzymes and glycosyltransferases, which transfer the final derivatized activated sugars to aglycon substrates. One of the first crystal structures of an enzyme responsible for the first step in this cascade, alpha-D-glucopyranosyl phosphate thymidylyltransferase (Ep) from Salmonella, in complex with product (UDP-Glc) and substrate (dTTP) is reported at 2.0 A and 2.1 A resolution, respectively. These structures, in conjunction with the kinetic characterization of Ep, clarify the catalytic mechanism of this important enzyme class. Structure-based engineering of Ep produced modified enzymes capable of utilizing 'unnatural' sugar phosphates not accepted by wild type Ep. The demonstrated ability to alter nucleotidylyltransferase specificity by design is an integral component of in vitro glycosylation systems developed for the production of diverse glycorandomized libraries.
 
  Selected figure(s)  
 
Figure 1.
Figure 1. Potential of E[p] for glycosylation. a, Examples of pharmacologically important glycosylated metabolites. The putative nucleotidylyltransferase-catalyzed formation of NDP-sugars is indicated in the box; the carbohydrate ligands of each metabolite are red. Note that the natural substrate for E[p] is glucose-1-phosphate and that the difference between erythromycin from Saccharopolyspora erythrea and megalomicin from Micromonospora megalomicea is the addition of a third sugar megosamine (indicated by the arrow). b, Sugar phosphates accepted by wild type E[p]. The natural substrate ( -D-glucopyranosyl phosphate) is shown in the box, and the corresponding deviations from this structure in each unnatural substrate are highlighted in red. These substrates can be converted by E[p] in the presence of both dTTP and UTP.
Figure 3.
Figure 3. The E[p] active site. Hydrogen bonds are depicted by dashed lines. All panels are in stereo. a, Interactions between E[p] and the nucleosides of the dTTP substrate (upper) and the UDP-Glc product (lower). b, Interactions between E[p] and the glucose moiety in the sugar binding pocket. c, The position of the catalytic metal in the active site. d, dTTP bound in the 'accessory' site at the monomer interface. The different chains of the tetramer are green (chain A) or magenta (chain B). The -phosphate of dTTP hydrogen bonds with both His 117 from chain A and Gly 221 from chain B.
 
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2001, 8, 545-551) copyright 2001.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
18807197 D.Simkhada, T.J.Oh, E.M.Kim, J.C.Yoo, and J.K.Sohng (2009).
Cloning and characterization of CalS7 from Micromonospora echinospora sp. calichensis as a glucose-1-phosphate nucleotidyltransferase.
  Biotechnol Lett, 31, 147-153.  
  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.  
18627619 C.J.Zea, G.Camci-Unal, and N.L.Pohl (2008).
Thermodynamics of binding of divalent magnesium and manganese to uridine phosphates: implications for diabetes-related hypomagnesaemia and carbohydrate biocatalysis.
  Chem Cent J, 2, 15.  
18798210 C.Zhang, R.Moretti, J.Jiang, and J.S.Thorson (2008).
The in vitro characterization of polyene glycosyltransferases AmphDI and NysDI.
  Chembiochem, 9, 2506-2514.  
18678278 G.J.Williams, R.W.Gantt, and J.S.Thorson (2008).
The impact of enzyme engineering upon natural product glycodiversification.
  Curr Opin Chem Biol, 12, 556-564.  
18219417 M.P.Huestis, G.A.Aish, J.P.Hui, E.C.Soo, and D.L.Jakeman (2008).
Lipophilic sugar nucleotide synthesis by structure-based design of nucleotidylyltransferase substrates.
  Org Biomol Chem, 6, 477-484.  
18387352 R.Moretti, and J.S.Thorson (2008).
A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay.
  Anal Biochem, 377, 251-258.  
17434970 D.Aragão, A.M.Fialho, A.R.Marques, E.P.Mitchell, I.Sá-Correia, and C.Frazão (2007).
The complex of Sphingomonas elodea ATCC 31461 glucose-1-phosphate uridylyltransferase with glucose-1-phosphate reveals a novel quaternary structure, unique among nucleoside diphosphate-sugar pyrophosphorylase members.
  J Bacteriol, 189, 4520-4528.
PDB code: 2ux8
17322528 J.B.Thoden, and H.M.Holden (2007).
The molecular architecture of glucose-1-phosphate uridylyltransferase.
  Protein Sci, 16, 432-440.
PDB code: 2e3d
17567737 J.B.Thoden, and H.M.Holden (2007).
Active site geometry of glucose-1-phosphate uridylyltransferase.
  Protein Sci, 16, 1379-1388.
PDB code: 2pa4
18033579 J.S.Rokem, A.E.Lantz, and J.Nielsen (2007).
Systems biology of antibiotic production by microorganisms.
  Nat Prod Rep, 24, 1262-1287.  
17434871 R.Moretti, and J.S.Thorson (2007).
Enhancing the latent nucleotide triphosphate flexibility of the glucose-1-phosphate thymidylyltransferase RmlA.
  J Biol Chem, 282, 16942-16947.  
  16946483 D.Aragão, A.R.Marques, C.Frazão, F.J.Enguita, M.A.Carrondo, A.M.Fialho, I.Sá-Correia, and E.P.Mitchell (2006).
Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 62, 930-934.  
16538696 S.A.Borisova, C.Zhang, H.Takahashi, H.Zhang, A.W.Wong, J.S.Thorson, and H.W.Liu (2006).
Substrate specificity of the macrolide-glycosylating enzyme pair DesVII/DesVIII: opportunities, limitations, and mechanistic hypotheses.
  Angew Chem Int Ed Engl, 45, 2748-2753.  
16085866 E.Silva, A.R.Marques, A.M.Fialho, A.T.Granja, and I.Sá-Correia (2005).
Proteins encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG genes, involved in gellan gum biosynthesis, exhibit both dTDP- and UDP-glucose pyrophosphorylase activities.
  Appl Environ Microbiol, 71, 4703-4712.  
16206230 J.Bae, K.H.Kim, D.Kim, Y.Choi, J.S.Kim, S.Koh, S.I.Hong, and D.S.Lee (2005).
A practical enzymatic synthesis of UDP sugars and NDP glucoses.
  Chembiochem, 6, 1963-1966.  
15975511 J.Yang, X.Fu, J.Liao, L.Liu, and J.S.Thorson (2005).
Structure-based engineering of E. coli galactokinase as a first step toward in vivo glycorandomization.
  Chem Biol, 12, 657-664.  
15678424 M.Yang, M.Brazier, R.Edwards, and B.G.Davis (2005).
High-throughput mass-spectrometry monitoring for multisubstrate enzymes: determining the kinetic parameters and catalytic activities of glycosyltransferases.
  Chembiochem, 6, 346-357.  
15634670 N.M.Koropatkin, W.W.Cleland, and H.M.Holden (2005).
Kinetic and structural analysis of alpha-D-Glucose-1-phosphate cytidylyltransferase from Salmonella typhi.
  J Biol Chem, 280, 10774-10780.
PDB code: 1wvc
15598657 Z.Zhang, M.Tsujimura, J.Akutsu, M.Sasaki, H.Tajima, and Y.Kawarabayasi (2005).
Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7.
  J Biol Chem, 280, 9698-9705.  
15062779 J.Chen, and J.Stubbe (2004).
Bleomycins: new methods will allow reinvestigation of old issues.
  Curr Opin Chem Biol, 8, 175-181.  
14695508 J.S.Thorson, W.A.Barton, D.Hoffmeister, C.Albermann, and D.B.Nikolov (2004).
Structure-based enzyme engineering and its impact on in vitro glycorandomization.
  Chembiochem, 5, 16-25.  
15239058 J.Yang, L.Liu, and J.S.Thorson (2004).
Structure-based enhancement of the first anomeric glucokinase.
  Chembiochem, 5, 992-996.  
15292268 N.M.Koropatkin, and H.M.Holden (2004).
Molecular structure of alpha-D-glucose-1-phosphate cytidylyltransferase from Salmonella typhi.
  J Biol Chem, 279, 44023-44029.
PDB code: 1tzf
12837772 A.Matte, J.Sivaraman, I.Ekiel, K.Gehring, Z.Jia, and M.Cygler (2003).
Contribution of structural genomics to understanding the biology of Escherichia coli.
  J Bacteriol, 185, 3994-4002.  
12512086 C.G.Hyun, T.Bililign, J.Liao, and J.S.Thorson (2003).
The biosynthesis of indolocarbazoles in a heterologous E. coli host.
  Chembiochem, 4, 114-117.  
14612558 D.Hoffmeister, J.Yang, L.Liu, and J.S.Thorson (2003).
Creation of the first anomeric D/L-sugar kinase by means of directed evolution.
  Proc Natl Acad Sci U S A, 100, 13184-13189.  
12740816 J.Jiang, C.Albermann, and J.S.Thorson (2003).
Application of the nucleotidylyltransferase Ep toward the chemoenzymatic synthesis of dTDP-desosamine analogues.
  Chembiochem, 4, 443-446.  
12209778 B.D.Martin, E.R.Welsh, J.C.Mastrangelo, and R.Aggarwal (2002).
General O-glycosylation of 2-furfuryl alcohol using beta-glucuronidase.
  Biotechnol Bioeng, 80, 222-227.  
11706035 B.Y.Kwak, Y.M.Zhang, M.Yun, R.J.Heath, C.O.Rock, S.Jackowski, and H.W.Park (2002).
Structure and mechanism of CTP:phosphocholine cytidylyltransferase (LicC) from Streptococcus pneumoniae.
  J Biol Chem, 277, 4343-4350.
PDB codes: 1jyk 1jyl
12171937 J.Sivaraman, V.Sauvé, A.Matte, and M.Cygler (2002).
Crystal structure of Escherichia coli glucose-1-phosphate thymidylyltransferase (RffH) complexed with dTTP and Mg2+.
  J Biol Chem, 277, 44214-44219.
PDB code: 1mc3
11986319 M.Ouzzine, S.Gulberti, N.Levoin, P.Netter, J.Magdalou, and S.Fournel-Gigleux (2002).
The donor substrate specificity of the human beta 1,3-glucuronosyltransferase I toward UDP-glucuronic acid is determined by two crucial histidine and arginine residues.
  J Biol Chem, 277, 25439-25445.  
12374866 W.A.Barton, J.B.Biggins, J.Jiang, J.S.Thorson, and D.B.Nikolov (2002).
Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering.
  Proc Natl Acad Sci U S A, 99, 13397-13402.
PDB codes: 1mp3 1mp4 1mp5
12045109 X.M.He, and H.W.Liu (2002).
Formation of unusual sugars: mechanistic studies and biosynthetic applications.
  Annu Rev Biochem, 71, 701-754.  
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.