TY - JOUR
T1 - Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii
AU - Kadziola, Anders
AU - Jepsen, Clemens H.
AU - Johansson, Eva
AU - McGuire, Jim
AU - Larsen, Sine
AU - Hove-Jensen, Bjarne
N1 - Funding Information: We thank Martin Willemoës for invaluable discussions and Tonny D. Hansen for excellent technical assistance. We acknowledge the access to synchrotron radiation at beamline BW7A, EMBL outstation Hamburg and beamline I711, MAX-laboratory, Lund. Visits to the synchrotrons were supported by the European Community's Access to Research Infrastructure (ARI) program and the Danish Natural Science Research Council through a grant to Dansync. The work was funded by the Danish National Research Foundation and the Danish Natural Science Research Council.
PY - 2005/12/9
Y1 - 2005/12/9
N2 - The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M. jannaschii PRPP synthase has been purified, characterised, crystallised, and the crystal structure determined. The enzyme is activated by phosphate ions and only ATP or dATP serve as diphosphoryl donors. The K m values are determined as 2.6 mM and 2.8 mM for ATP and ribose 5-phosphate, respectively, and the V max value as 2.20 mmol (min x mg of protein) -1. ADP is a potent inhibitor of activity while GDP has no effect. A single ADP binding site, the active site, is present per subunit. The crystal structure of the enzyme reveals a more compact subunit than that of the enzyme from the mesophile Bacillus subtilis, caused by truncations at the N and C terminus as well as shorter loops in the M. jannaschii enzyme. The M. jannaschii enzyme displays a tetrameric quaternary structure in contrast to the hexameric quaternary structure of B. subtilis PRPP synthase. Soaking of the crystals with 5′-AMP and PRPP revealed the position of the former compound as well as that of ribose 5-phosphate. The properties of M. jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases.
AB - The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M. jannaschii PRPP synthase has been purified, characterised, crystallised, and the crystal structure determined. The enzyme is activated by phosphate ions and only ATP or dATP serve as diphosphoryl donors. The K m values are determined as 2.6 mM and 2.8 mM for ATP and ribose 5-phosphate, respectively, and the V max value as 2.20 mmol (min x mg of protein) -1. ADP is a potent inhibitor of activity while GDP has no effect. A single ADP binding site, the active site, is present per subunit. The crystal structure of the enzyme reveals a more compact subunit than that of the enzyme from the mesophile Bacillus subtilis, caused by truncations at the N and C terminus as well as shorter loops in the M. jannaschii enzyme. The M. jannaschii enzyme displays a tetrameric quaternary structure in contrast to the hexameric quaternary structure of B. subtilis PRPP synthase. Soaking of the crystals with 5′-AMP and PRPP revealed the position of the former compound as well as that of ribose 5-phosphate. The properties of M. jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases.
KW - Kinetics
KW - Nucleotide metabolism
KW - PRPP
KW - Phosphate ion-activation
KW - Thermophile
UR - http://www.scopus.com/inward/record.url?scp=28444484987&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2005.10.001
DO - 10.1016/j.jmb.2005.10.001
M3 - Journal article
SN - 0022-2836
VL - 354
SP - 815
EP - 828
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -