PDBsum entry 1xff

Transferase

PDB id: 1xff

Contents

Protein chains

238 a.a. *

Ligands

ACT

GLU ×2

Metals

_NA ×2

Waters ×283

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Substrate binding is required for assembly of the active conformation of the catalytic site in ntn amidotransferases: evidence from the 1.8 a crystal structure of the glutaminase domain of glucosamine 6-Phosphate synthase.

Authors

M.N.Isupov, G.Obmolova, S.Butterworth, M.A.Badet-Denisot, B.Badet, I.Polikarpov, J.A.Littlechild, A.Teplyakov.

Ref.

Structure, 1996, 4, 801-810. [DOI no: 10.1016/S0969-2126(96)00087-1]

PubMed id

8805567

Abstract

BACKGROUND: Amidotransferases use the amide nitrogen of glutamine in a number of important biosynthetic reactions. They are composed of a glutaminase domain, which catalyzes the hydrolysis of glutamine to glutamate and ammonia, and a synthetase domain, catalyzing amination of the substrate. To gain insight into the mechanism of nitrogen transfer, we examined the structure of the glutaminase domain of glucosamine 6-phosphate synthase (GLMS). RESULTS: The crystal structures of the enzyme complexed with glutamate and with a competitive inhibitor, Glu-hydroxamate, have been determined to 1.8 A resolution. The protein fold has structural homology to other members of the superfamily of N-terminal nucleophile (Ntn) hydrolases, being a sandwich of antiparallel beta sheets surrounded by two layers of alpha helices. CONCLUSIONS: The structural homology between the glutaminase domain of GLMS and that of PRPP amidotransferase (the only other Ntn amidotransferase whose structure is known) indicates that they may have diverged from a common ancestor. Cys1 is the catalytic nucleophile in GLMS, and the nucleophilic character of its thiol group appears to be increased through general base activation by its own alpha-amino group. Cys1 can adopt two conformations, one active and one inactive; glutamine binding locks the residue in a predetermined conformation. We propose that when a nitrogen acceptor is present Cys1 is kept in the active conformation, explaining the phenomenon of substrate-induced activation of the enzyme, and that Arg26 is central in this coupling.

Figure 5.
Figure 5. Active site of the glutaminase domain of GLMS with the bound product, glutamate, shown in outline. Hydrogen bonds are indicated by dashed lines. The loop 73-78 is in the closed conformation. Cys1 and Asn98 are in the inactive conformation.

Figure 6.
Figure 6. Proposed catalytic mechanism of glutamine hydrolysis by amidotransferases. Residue numbers are those of GLMS. The nucleophilicity of Cys1 is enhanced by its own free a-amino group. This interaction is mediated by a bridging water molecule which serves as a virtual base. Deacylation involves another water molecule which is activated through the same mechanism. Residues Asn98 and Gly99 form the oxyanion hole for the tetrahedral intermediates.

The above figures are reprinted by permission from Cell Press: Structure (1996, 4, 801-810) copyright 1996.

Secondary reference #1

Title

Crystallization and preliminary X-Ray analysis of the two domains of glucosamine-6-Phosphate synthase from escherichia coli.

Authors

G.Obmolova, M.A.Badet-Denisot, B.Badet, A.Teplyakov.

Ref.

J Mol Biol, 1994, 242, 703-705.

PubMed id

7932726

Secondary reference #2

Title

Channeling of ammonia in glucosamine-6-Phosphate synthase.

Authors

A.Teplyakov, G.Obmolova, B.Badet, M.A.Badet-Denisot.

Ref.

J Mol Biol, 2001, 313, 1093-1102. [DOI no: 10.1006/jmbi.2001.5094]

PubMed id

11700065

Figure 1.
Figure 1. The reaction catalysed by GlmS. Ammonia produced by glutamine hydrolysis is utilised for sugar phosphate amination.

Figure 3.
Figure 3. GlmS dimer as viewed along the molecular 2-fold axis. Glutaminase domains are shown in green, isomerase domains are red and yellow. Active sites are indicated by ball-and-stick models of the substrates.

The above figures are reproduced from the cited reference with permission from Elsevier