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PDBsum entry 1a9x

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protein ligands metals Protein-protein interface(s) links
Amidotransferase PDB id
1a9x
Jmol
Contents
Protein chains
1058 a.a. *
379 a.a. *
Ligands
PO4 ×15
ADP ×8
ORN ×4
NET ×4
Metals
__K ×32
_MN ×12
_CL ×28
Waters ×4528
* Residue conservation analysis
PDB id:
1a9x
Name: Amidotransferase
Title: Carbamoyl phosphate synthetase: caught in the act of glutami hydrolysis
Structure: Carbamoyl phosphate synthetase (large chain). Chain: a, c, e, g. Engineered: yes. Carbamoyl phosphate synthetase (small chain). Chain: b, d, f, h. Engineered: yes. Mutation: yes
Source: Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562
Biol. unit: Dimer (from PQS)
Resolution:
1.80Å     R-factor:   0.191    
Authors: J.Thoden,H.Holden
Key ref:
J.B.Thoden et al. (1998). Carbamoyl phosphate synthetase: caught in the act of glutamine hydrolysis. Biochemistry, 37, 8825-8831. PubMed id: 9636022 DOI: 10.1021/bi9807761
Date:
14-Apr-98     Release date:   21-Oct-98    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P00968  (CARB_ECOLI) -  Carbamoyl-phosphate synthase large chain
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
1073 a.a.
1058 a.a.*
Protein chains
Pfam   ArchSchema ?
P0A6F1  (CARA_ECOLI) -  Carbamoyl-phosphate synthase small chain
Seq:
Struc:
382 a.a.
379 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 5 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: Chains A, B, C, D, E, F, G, H: E.C.6.3.5.5  - Carbamoyl-phosphate synthase (glutamine-hydrolyzing).
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

      Pathway:
Pyrimidine Biosynthesis
      Reaction: 2 ATP + L-glutamine + HCO3- + H2O = 2 ADP + phosphate + L-glutamate + carbamoyl phosphate
2 × ATP
+
L-glutamine
Bound ligand (Het Group name = ORN)
matches with 90.00% similarity
+ HCO(3)(-)
+ H(2)O
=
2 × ADP
Bound ligand (Het Group name = ADP)
corresponds exactly
+
phosphate
Bound ligand (Het Group name = PO4)
corresponds exactly
+ L-glutamate
+ carbamoyl phosphate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   3 terms 
  Biological process     metabolic process   12 terms 
  Biochemical function     catalytic activity     9 terms  

 

 
    reference    
 
 
DOI no: 10.1021/bi9807761 Biochemistry 37:8825-8831 (1998)
PubMed id: 9636022  
 
 
Carbamoyl phosphate synthetase: caught in the act of glutamine hydrolysis.
J.B.Thoden, S.G.Miran, J.C.Phillips, A.J.Howard, F.M.Raushel, H.M.Holden.
 
  ABSTRACT  
 
Carbamoyl phosphate synthetase from Escherichia coli catalyzes the production of carbamoyl phosphate from two molecules of Mg2+ATP, one molecule of bicarbonate, and one molecule of glutamine. The enzyme consists of two polypeptide chains referred to as the large and small subunits. While the large subunit provides the active sites responsible for the binding of nucleotides and other effector ligands, the small subunit contains those amino acid residues that catalyze the hydrolysis of glutamine to glutamate and ammonia. From both amino acid sequence analyses and structural studies it is now known that the small subunit belongs to the class I amidotransferase family of enzymes. Numerous biochemical studies have suggested that the reaction mechanism of the small subunit proceeds through the formation of the glutamyl thioester intermediate and that both Cys 269 and His 353 are critical for catalysis. Here we describe the X-ray crystallographic structure of carbamoyl phosphate synthetase from E. coli in which His 353 has been replaced with an asparagine residue. Crystals employed in the investigation were grown in the presence of glutamine, and the model has been refined to a crystallographic R-factor of 19.1% for all measured X-ray data from 30 to 1.8 A resolution. The active site of the small subunit clearly contains a covalently bound thioester intermediate at Cys 269, and indeed, this investigation provides the first direct structural observation of an enzyme intermediate in the amidotransferase family.
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
21134639 J.M.Lipchock, and J.P.Loria (2010).
Nanometer propagation of millisecond motions in V-type allostery.
  Structure, 18, 1596-1607.  
18458150 E.J.Hart, and S.G.Powers-Lee (2008).
Mutation analysis of carbamoyl phosphate synthetase: does the structurally conserved glutamine amidotransferase triad act as a functional dyad?
  Protein Sci, 17, 1120-1128.  
18069798 Y.Zhang, R.H.White, and S.E.Ealick (2008).
Crystal structure and function of 5-formaminoimidazole-4-carboxamide ribonucleotide synthetase from Methanocaldococcus jannaschii.
  Biochemistry, 47, 205-217.
PDB codes: 2r7k 2r7l 2r7m 2r7n 2r84 2r85 2r86 2r87
16251194 H.Arulanantham, N.J.Kershaw, K.S.Hewitson, C.E.Hughes, J.E.Thirkettle, and C.J.Schofield (2006).
ORF17 from the clavulanic acid biosynthesis gene cluster catalyzes the ATP-dependent formation of N-glycyl-clavaminic acid.
  J Biol Chem, 281, 279-287.  
16339145 M.Gengenbacher, T.B.Fitzpatrick, T.Raschle, K.Flicker, I.Sinning, S.Müller, P.Macheroux, I.Tews, and B.Kappes (2006).
Vitamin B6 biosynthesis by the malaria parasite Plasmodium falciparum: biochemical and structural insights.
  J Biol Chem, 281, 3633-3641.
PDB code: 2abw
15770625 E.M.Bulloch, and C.Abell (2005).
Detection of covalent intermediates formed in the reaction of 4-amino-4-deoxychorismate synthase.
  Chembiochem, 6, 832-834.  
15852022 M.J.Lenaeus, M.Vamvouka, P.J.Focia, and A.Gross (2005).
Structural basis of TEA blockade in a model potassium channel.
  Nat Struct Mol Biol, 12, 454-459.
PDB codes: 2bob 2boc
16030023 T.Raschle, N.Amrhein, and T.B.Fitzpatrick (2005).
On the two components of pyridoxal 5'-phosphate synthase from Bacillus subtilis.
  J Biol Chem, 280, 32291-32300.  
14585832 J.A.Bauer, E.M.Bennett, T.P.Begley, and S.E.Ealick (2004).
Three-dimensional structure of YaaE from Bacillus subtilis, a glutaminase implicated in pyridoxal-5'-phosphate biosynthesis.
  J Biol Chem, 279, 2704-2711.
PDB code: 1r9g
15296735 M.Goto, R.Omi, N.Nakagawa, I.Miyahara, and K.Hirotsu (2004).
Crystal structures of CTP synthetase reveal ATP, UTP, and glutamine binding sites.
  Structure, 12, 1413-1423.
PDB codes: 1vcm 1vcn 1vco
11839304 A.Douangamath, M.Walker, S.Beismann-Driemeyer, M.C.Vega-Fernandez, R.Sterner, and M.Wilmanns (2002).
Structural evidence for ammonia tunneling across the (beta alpha)(8) barrel of the imidazole glycerol phosphate synthase bienzyme complex.
  Structure, 10, 185-193.
PDB codes: 1gpw 1k9v
11756425 A.Saeed-Kothe, and S.G.Powers-Lee (2002).
Specificity determining residues in ammonia- and glutamine-dependent carbamoyl phosphate synthetases.
  J Biol Chem, 277, 7231-7238.  
11729189 B.W.Miles, J.B.Thoden, H.M.Holden, and F.M.Raushel (2002).
Inactivation of the amidotransferase activity of carbamoyl phosphate synthetase by the antibiotic acivicin.
  J Biol Chem, 277, 4368-4373.
PDB code: 1kee
11953431 H.Li, T.J.Ryan, K.J.Chave, and P.Van Roey (2002).
Three-dimensional structure of human gamma -glutamyl hydrolase. A class I glatamine amidotransferase adapted for a complex substate.
  J Biol Chem, 277, 24522-24529.
PDB code: 1l9x
12130656 J.B.Thoden, X.Huang, F.M.Raushel, and H.M.Holden (2002).
Carbamoyl-phosphate synthetase. Creation of an escape route for ammonia.
  J Biol Chem, 277, 39722-39727.
PDB code: 1m6v
11574542 A.Ahuja, C.Purcarea, H.I.Guy, and D.R.Evans (2001).
A novel carbamoyl-phosphate synthetase from Aquifex aeolicus.
  J Biol Chem, 276, 45694-45703.  
11435114 E.I.Scharff, J.Koepke, G.Fritzsch, C.Lücke, and H.Rüterjans (2001).
Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris.
  Structure, 9, 493-502.
PDB codes: 1e1a 2iao 2iap 2iaq 2iar 2ias 2iat 2iau
11371633 G.Spraggon, C.Kim, X.Nguyen-Huu, M.C.Yee, C.Yanofsky, and S.E.Mills (2001).
The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, L-tryptophan.
  Proc Natl Acad Sci U S A, 98, 6021-6026.
PDB codes: 1i7q 1i7s
11119647 M.M.Horvath, and N.V.Grishin (2001).
The C-terminal domain of HPII catalase is a member of the type I glutamine amidotransferase superfamily.
  Proteins, 42, 230-236.  
11395405 X.Huang, H.M.Holden, and F.M.Raushel (2001).
Channeling of substrates and intermediates in enzyme-catalyzed reactions.
  Annu Rev Biochem, 70, 149-180.  
10713991 K.A.Denessiouk, and M.S.Johnson (2000).
When fold is not important: a common structural framework for adenine and AMP binding in 12 unrelated protein families.
  Proteins, 38, 310-326.  
10852731 M.A.Rishavy, W.W.Cleland, and C.J.Lusty (2000).
15N isotope effects in glutamine hydrolysis catalyzed by carbamyl phosphate synthetase: evidence for a tetrahedral intermediate in the mechanism.
  Biochemistry, 39, 7309-7315.  
10966576 M.Y.Galperin, and N.V.Grishin (2000).
The synthetase domains of cobalamin biosynthesis amidotransferases cobB and cobQ belong to a new family of ATP-dependent amidoligases, related to dethiobiotin synthetase.
  Proteins, 41, 238-247.  
10727215 X.Huang, and F.M.Raushel (2000).
An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia.
  Biochemistry, 39, 3240-3247.  
10497179 A.Hewagama, H.I.Guy, J.F.Vickrey, and D.R.Evans (1999).
Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (cad).
  J Biol Chem, 274, 28240-28245.  
9931004 B.L.Braxton, L.S.Mullins, F.M.Raushel, and G.D.Reinhart (1999).
Allosteric dominance in carbamoyl phosphate synthetase.
  Biochemistry, 38, 1394-1401.  
10387030 F.M.Raushel, J.B.Thoden, and H.M.Holden (1999).
The amidotransferase family of enzymes: molecular machines for the production and delivery of ammonia.
  Biochemistry, 38, 7891-7899.  
10428826 J.B.Thoden, F.M.Raushel, G.Wesenberg, and H.M.Holden (1999).
The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase.
  J Biol Chem, 274, 22502-22507.
PDB code: 1ce8
10029528 J.B.Thoden, G.Wesenberg, F.M.Raushel, and H.M.Holden (1999).
Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding.
  Biochemistry, 38, 2347-2357.
PDB code: 1bxr
10587438 J.B.Thoden, X.Huang, F.M.Raushel, and H.M.Holden (1999).
The small subunit of carbamoyl phosphate synthetase: snapshots along the reaction pathway.
  Biochemistry, 38, 16158-16166.
PDB codes: 1c30 1c3o 1cs0
10591107 S.S.Brody, S.P.Gough, and C.G.Kannangara (1999).
Predicted structure and fold recognition for the glutamyl tRNA reductase family of proteins.
  Proteins, 37, 485-493.
PDB codes: 1b29 1b61
10449718 T.Knöchel, A.Ivens, G.Hester, A.Gonzalez, R.Bauerle, M.Wilmanns, K.Kirschner, and J.N.Jansonius (1999).
The crystal structure of anthranilate synthase from Sulfolobus solfataricus: functional implications.
  Proc Natl Acad Sci U S A, 96, 9479-9484.
PDB code: 1qdl
10587437 T.M.Larsen, S.K.Boehlein, S.M.Schuster, N.G.Richards, J.B.Thoden, H.M.Holden, and I.Rayment (1999).
Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product.
  Biochemistry, 38, 16146-16157.
PDB code: 1ct9
9858783 A.Hewagama, H.I.Guy, M.Chaparian, and D.R.Evans (1998).
The function of Glu338 in the catalytic triad of the carbamoyl phosphate synthetase amidotransferase domain.
  Biochim Biophys Acta, 1388, 489-499.  
9818189 F.M.Raushel, J.B.Thoden, G.D.Reinhart, and H.M.Holden (1998).
Carbamoyl phosphate synthetase: a crooked path from substrates to products.
  Curr Opin Chem Biol, 2, 624-632.  
9914247 H.M.Holden, J.B.Thoden, and F.M.Raushel (1998).
Carbamoyl phosphate synthetase: a tunnel runs through it.
  Curr Opin Struct Biol, 8, 679-685.  
9914248 J.L.Smith (1998).
Glutamine PRPP amidotransferase: snapshots of an enzyme in action.
  Curr Opin Struct Biol, 8, 686-694.  
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.