PDBsum entry 2j5t

Transferase

PDB id: 2j5t

Contents

Protein chains

(+ 1 more) 355 a.a.

337 a.a.

Ligands

GLU ×12

SO4 ×8

Metals

_MG ×8

_CL ×20

Waters ×103

References listed in PDB file

Key reference

Title

A novel two-Domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of escherichia coli glutamate 5-Kinase.

Authors

C.Marco-Marín, F.Gil-Ortiz, I.Pérez-Arellano, J.Cervera, I.Fita, V.Rubio.

Ref.

J Mol Biol, 2007, 367, 1431-1446. [DOI no: 10.1016/j.jmb.2007.01.073]

PubMed id

17321544

Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a perfect match.

Abstract

Glutamate 5-kinase (G5K) makes the highly unstable product glutamyl 5-phosphate (G5P) in the initial, controlling step of proline/ornithine synthesis, being feedback-inhibited by proline or ornithine, and causing, when defective, clinical hyperammonaemia. We determined two crystal structures of G5K from Escherichia coli, at 2.9 A and 2.5 A resolution, complexed with glutamate and sulphate, or with G5P, sulphate and the proline analogue 5-oxoproline. E. coli G5K presents a novel tetrameric (dimer of dimers) architecture. Each subunit contains a 257 residue AAK domain, typical of acylphosphate-forming enzymes, with characteristic alpha(3)beta(8)alpha(4) sandwich topology. This domain is responsible for catalysis and proline inhibition, and has a crater on the beta sheet C-edge that hosts the active centre and bound 5-oxoproline. Each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic beta(5)beta(4) sandwich fold and three alpha helices. The AAK and PUA domains of one subunit associate non-canonically in the dimer with the same domains of the other subunit, leaving a negatively charged hole between them that hosts two Mg ions in one crystal, in line with the G5K requirement for free Mg. The tetramer, formed by two dimers interacting exclusively through their AAK domains, is flat and elongated, and has in each face, pericentrically, two exposed active centres in alternate subunits. This would permit the close apposition of two active centres of bacterial glutamate-5-phosphate reductase (the next enzyme in the proline/ornithine-synthesising route), supporting the postulated channelling of G5P. The structures clarify substrate binding and catalysis, justify the high glutamate specificity, explain the effects of known point mutations, and support the binding of proline near glutamate. Proline binding may trigger the movement of a loop that encircles glutamate, and which participates in a hydrogen bond network connecting active centres, which is possibly involved in the cooperativity for glutamate.

Figure 1.
Figure 1. Pathway of proline synthesis in microorganisms and plants, and of ornithine synthesis in mammals. Enzymes are enclosed in grey boxes. Feed-back inhibition of microbial and plant G5Ks by proline and of animal G5Ks by ornithine is indicated with broken arrows. The dotted arrow indicates the spontaneous cyclization of G5P to 5-oxoproline that is an abortive side-reaction.

Figure 9.
Figure 9. Possible interaction between the glutamate 5-kinase (G5K) and the glutamyl 5-phosphate reductase (G5PR). Surface representation of two perpendicular views of the G5K tetramer, with the substrates in space-filling representation. A dimer of the G5PR from T. maritima is shown (ribbon representation) with one subunit (orange) presenting the open conformation as observed in the crystal structure of this enzyme in the absence of substrates (PDB 1O20), and with the other subunit (yellow) presenting a closed conformation modelled from class 3 aldehyde dehydrogenase complexed to NAD (PDB 1AD3). The catalytic and NADPH-binding domains of G5PR are identified.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 367, 1431-1446) copyright 2007.