TSRI's main web site PROMISE mirror at TSRI Metalloprotein DB site Created: 27 February 1998
Last modified: 11 November 1998

Glutamine PRPP amidotransferase
Iron-sulphur cluster Formal oxidation/spin states
Fe4S4 image
[Fe4S4](SgammaCys)4
[Fe4S4]2+ (S=0); [Fe4S4]+ (mainly S=3/2; minor S=1/2, S=5/2)

Glutamine amidotransferases (GATases) transfer the glutamine amide nitrogen to variety of substrates [1-3]. Most GATases can use ammonia (NH3) as an alternative nitrogen source. Sites for glutamine binding and for NH3­dependent synthesis are localised in different domains (sometimes in different subunits), termed `glutamine' and `transferase' domains. Phosphoribosyltransferases (PRTases) are involved in the biosynthesis and metabolism of nucleotides. They catalyse reactions at the 1­position of ribose with phosphoribosylpyrophosphate (PRPP) and an enzyme specific amine as substrates [4].

Glutamine PRPP amidotransferase (GPATase; EC 2.4.2.14) is a member of both enzyme families. It catalyses the first step in de novo biosynthesis of purine nucleotides and is the key regulatory enzyme in the pathway [5].

GPATase exists as a tetramer of identical subunits, so that each subunit includes both the glutamine and transferase domains. In Bacillus subtilis GPATase, each subunit contains a [Fe4S4] cluster, which presumably has a regulatory but not a catalytic function [6]. The native [Fe4S4]2+ state can be easily reduced to the [Fe4S4]+ state; both forms are enzymatically active [7]. Oxygen­dependent inactivation of the enzyme is accompanied by the decomposition of the iron-sulphur cluster and denaturation of the protein. The homologous enzyme from Escherichia coli (39% sequence identity with B. subtilis GPATase) does not contain the iron-sulphur cluster and is not oxygen­sensitive in vitro [2].

The 3­D structures of GPATases from B. subtilis [8] and E. coli [9] have been determined. The B. subtilis tetramer is a `doughnut' of approximate dimensions 105×95×55 Å (see Figure 1AO0 a). The subunits contain two alpha+ß domains: the N­terminal `glutamine' domain (residues 1-230) and the C­terminal `transferase' domain (residues 231-465) (Figure 1AO0 d and e, respectively). The four [Fe4S4] clusters are situated at the corners of the tetramer between the N­ and C­terminal domains of each subunit. The cluster is coordinated by four Cys sulphur ligands (residues 236, 382, 437 and 440) (Figure 1AO0 b). Remarkably, the peptides surrounding the iron-sulfur cluster in B. subtilis GPATase and their homologues in E. coli enzyme adopt the identical conformation. It is suggested that a common ancestor of of the metal­containing and metal­free GPATases contained an iron-sulfur cluster [9].

In both E. coli and B. subtilis GPATases, the active site of the glutamine domain is inaccessible to bulk solvent. Cys­1 is the catalytic residue involved in glutamine utilisation. Cys­1 functions as a nucleophile that attacks the carboxamide of glutamine yielding a gamma­glutamyl thioether intermediate [10].

The transferase domain contains two types of nucleotide­binding sites, catalytic (C) and allosteric (A). The C site consists of a strand­loop­helix structure containing a sequence motif (`PRPP­loop') that is common to several PRTases and has been proposed as a binding site for the ribose 5­phosphate of PRPP, of PRTase products and of inhibitors [5, 9]. A 30­residue flexible loop (called the `flag' in the case of B. subtilis enzyme) interacts with the glutamine domain of the neighbouring subunit and forms a major part of the A site, which is adapted for binding feedback inhibitors [9].

Glutamine PRPP amidotransferase in enzyme databases

ENZYME LIGAND BRENDA Official name Alternative names
2.4.2.14 2.4.2.14 2.4.2.14 Amidophosphoribosyltransferase Glutamine phosphoribosylpyrophosphate amidotransferase; phosphoribosyldiphosphate 5­amidotransferase

Glutamine PRPP amidotransferase in motif databases

PRINTS ID PRINTS AC PROSITE/BLOCKS ID PROSITE AC BLOCKS AC
-
-
 PUR_PYR_PR_TRANSFER PS00103 BL00103
-
-
 GATASE_TYPE_II PS00443 BL00443

Glutamine PRPP amidotransferase in alignment databases

Protein Superfamily Pfam LPFC 3­D alignment
00286; amidophosphoribosyltransferase PF00310; GATase_2
-

Glutamine PRPP amidotransferase in 3­D databases

GPATase from B. subtilis contains a single cubane­like [Fe4S4] cluster (see Figure 1AO0); GPATase from E. coli (*) is metal­free.

PDB scop BSMRELI
Base		 Header MMS Abstract ¹
1ao0
-
 1ao0 1ao0 Glutamine PRPP amidotransferase (complex with GMP, ADP and Mg2+); Bacillus subtilis (recombinant form expressed in Escherichia coli)
-
1ecf* 1ecf* 1ecf* 1ecf* Glutamine PRPP amidotransferase [complex with piperazine­N,N'­bis(2­ethanesulphonic acid)]; Escherichia coli
-
1ecg* 1ecg* 1ecg* 1ecg* Glutamine PRPP amidotransferase [complex with piperazine­N,N'­bis(2­ethanesulphonic acid) and 5­oxo­L­norleucine]; Escherichia coli
-
1gph 1gph 1gph 1gph Glutamine PRPP amidotransferase (complex with AMP); Bacillus subtilis (recombinant form expressed in Escherichia coli) MS5GL8

¹ Macromolecular Structures abstract. Full text is available to BioMedNet Members

References

  1. Buchanan, J.M. (1973) The amidotransferases. Adv. Enzymol. Relat. Areas Mol. Biol. 39, 91-183.
  2. Zalkin, H. (1983) Structure, function, and regulation of amidophosphoribosyltransferase from prokaryotes. Adv. Enzyme Regul. 21, 225-237.
  3. Zalkin, H. (1993) The amidotransferases. Adv. Enzymol. Relat. Areas Mol. Biol. 66, 203-309.
  4. Musick, W.D.L. (1981) Structural features of the phosphoribosyltransferases and their relationship to the human deficiency disorders of purine and pyrimidine metabolism. CRC Crit. Rev. Biochem. 11, 1-34.
  5. Smith, J.L. (1995a) Enzymes of nucleotide synthesis. Curr. Opin. Struct. Biol. 5, 752-757.
  6. Switzer, R.L. (1989) Non­redox roles for iron-sulfur clusters in enzymes. BioFactors 2, 77-86.
  7. Vollmer, S.J., Switzer, R.L. and Debrunner, P.G. (1983) Oxidation-reduction properties of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem. 258, 14284-14293.
  8. Smith, J.L., Zaluzec, E.J., Wery, J.­P., Niu, L., Switzer, R.L., Zalkin, H. and Satow, Y. (1994) Structure of the allosteric regulatory enzyme of purine biosynthesis. Science 264, 1427-1433.
  9. Muchmore, C.R.A., Krahn, J.M., Kim, J.H., Zalkin, H. and Smith, J.L. (1998) Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Protein Science 7, 39-51.
  10. Smith, J.L. (1995b) Structures of glutamine amidotransferases from the purine biosynthetic pathway. Biochem. Soc. Trans. 23, 894-898.
Bibliography on structural studies of glutamine PRPP amidotransferase