PDBsum entry 2bri

Transferase

PDB id: 2bri

Contents

Protein chains

225 a.a.

Ligands

ANP ×2

Metals

_MG ×2

Waters ×14

References listed in PDB file

Key reference

Title

The crystal structure of pyrococcus furiosus ump kinase provides insight into catalysis and regulation in microbial pyrimidine nucleotide biosynthesis.

Authors

C.Marco-Marín, F.Gil-Ortiz, V.Rubio.

Ref.

J Mol Biol, 2005, 352, 438-454. [DOI no: 10.1016/j.jmb.2005.07.045]

PubMed id

16095620

Abstract

UMP kinase (UMPK), the enzyme responsible for microbial UMP phosphorylation, plays a key role in pyrimidine nucleotide biosynthesis, regulating this process via feed-back control and via gene repression of carbamoyl phosphate synthetase (the first enzyme of the pyrimidine biosynthesis pathway). We present crystal structures of Pyrococcus furiosus UMPK, free or complexed with AMPPNP or AMPPNP and UMP, at 2.4 A, 3 A and 2.55 A resolution, respectively, providing a true snapshot of the catalytically competent bisubstrate complex. The structure proves that UMPK does not resemble other nucleoside monophosphate kinases, including the UMP/CMP kinase found in animals, and thus UMPK may be a potential antimicrobial target. This enzyme has a homohexameric architecture centred around a hollow nucleus, and is organized as a trimer of dimers. The UMPK polypeptide exhibits the amino acid kinase family (AAKF) fold that has been reported in carbamate kinase and acetylglutamate kinase. Comparison with acetylglutamate kinase reveals that the substrates bind within each subunit at equivalent, adequately adapted sites. The UMPK structure contains two bound Mg ions, of which one helps stabilize the transition state, thus having the same catalytic role as one lysine residue found in acetylglutamate kinase, which is missing from P.furiosus UMPK. Relative to carbamate kinase and acetylglutamate kinase, UMPK presents a radically different dimer architecture, lacking the characteristic 16-stranded beta-sheet backbone that was considered a signature of AAKF enzymes. Its hexameric architecture, also a novel trait, results from equatorial contacts between the A and B subunits of adjacent dimers combined with polar contacts between A or B subunits, and may be required for the UMPK regulatory functions, such as gene regulation, proposed here to be mediated by hexamer-hexamer interactions with the DNA-binding protein PepA.

Figure 1.
Figure 1. Pyrimidine nucleotide biosynthesis and the roles of UMPK demonstrated in Escherichia coli. Prokaryotic UMPK is highlighted within a grey box. The eukaryotic CMP/UMPK is included also. Multiple arrows denote multiple steps. The broken and dotted arrows denote feed-back inhibition and activation, respectively. The double-lined arrow indicates repression by UMPK of the carAB genes, which encode carbamoyl phosphate synthetase. The involvement of the DNA-binding proteins PepA and IHF in this process is indicated. Figure 1. Pyrimidine nucleotide biosynthesis and the roles of UMPK demonstrated in Escherichia coli. Prokaryotic UMPK is highlighted within a grey box. The eukaryotic CMP/UMPK is included also. Multiple arrows denote multiple steps. The broken and dotted arrows denote feed-back inhibition and activation, respectively. The double-lined arrow indicates repression by UMPK of the carAB genes, which encode carbamoyl phosphate synthetase. The involvement of the DNA-binding proteins PepA and IHF in this process is indicated.

Figure 4.
Figure 4. Substrate binding in UMPK. (a) Stereo view of the C^α trace of the substrate-binding sites, with bound Mg[2]AMPPNP and UMP coloured. Amino acid side-chains are also in colour, in thinner trace. (b) Stereoscopic detailed representation of the phosphoryl group transfer site in the complex with Mg[2]AMPPNP and UMP. Mg ions and water molecules are drawn as purple and cyan spheres, respectively. Nearby protein residues are shown in thinner trace. Hydrogen bonds and coordination bonds with Mg are shown as red lines, indicating the interatomic distances (in Å). The interatomic distance between the attacking O atom of UMP and the γ-P atom is represented with a blue line. (c) and (d) Plots of the interactions between the protein and (c) UMP or (d) Mg[2]AMPPNP in the ternary complex. The letter W denotes a water molecule. Distances are in Å. Figure 4. Substrate binding in UMPK. (a) Stereo view of the C^α trace of the substrate-binding sites, with bound Mg[2]AMPPNP and UMP coloured. Amino acid side-chains are also in colour, in thinner trace. (b) Stereoscopic detailed representation of the phosphoryl group transfer site in the complex with Mg[2]AMPPNP and UMP. Mg ions and water molecules are drawn as purple and cyan spheres, respectively. Nearby protein residues are shown in thinner trace. Hydrogen bonds and coordination bonds with Mg are shown as red lines, indicating the interatomic distances (in Å). The interatomic distance between the attacking O atom of UMP and the γ-P atom is represented with a blue line. (c) and (d) Plots of the interactions between the protein and (c) UMP or (d) Mg[2]AMPPNP in the ternary complex. The letter W denotes a water molecule. Distances are in Å. Parts (c) and (d) were drawn with LIGPLOT.[3]^50

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 352, 438-454) copyright 2005.