title: Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase.
abstract: Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyzes the first step in histidine biosynthesis, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to generate N1-(5-phospho-β-d-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain and cannot bind histidine. HisGSinstead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGScatalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGSwas thought to be catalytically inactive without HisZ. Here, recombinant HisGSand HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was determined at 2.34 Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both the ATPPRT holoenzyme and HisGSare catalytically active. Surprisingly, HisZ confers only a modest 2-4-fold increase in kcat. Reaction profiles for both enzymes cannot be distinguished by31P nuclear magnetic resonance, indicating that the same reaction is catalyzed. The temperature dependence of kcatshows deviation from Arrhenius behavior at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterization of a psychrophilic ATPPRT.
authors.affiliation: School of Engineering and Applied Science, Rotterdam University of Applied Science , G. J. de Jonghweg 4-6, 3015 GG Rotterdam, The Netherlands.
authors.forename: Rozanne
authors.initials: R
authors.lastname: Stroek
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
authors.forename: Paul D
authors.initials: PD
authors.lastname: Talbot
authors.forename: Mateusz K
authors.initials: MK
authors.lastname: Glok
authors.forename: Klaudia E
authors.initials: KE
authors.lastname: Bernaś
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Thomson
authors.forename: Eoin R
authors.initials: ER
authors.lastname: Gould
authors.forename: Magnus S
authors.initials: MS
authors.lastname: Alphey
authors.forename: Huanting
authors.initials: H
authors.lastname: Liu
authors.forename: Gordon J
authors.initials: GJ
authors.lastname: Florence
authors.forename: James H
authors.identifier: 0000-0001-6744-5061
authors.initials: JH
authors.lastname: Naismith
authors.forename: Rafael G
authors.identifier: 0000-0002-1308-8190
authors.initials: RG
authors.lastname: da Silva
mesh_terms: D001276:ATP Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000064:Acclimatization; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000494:Allosteric Regulation; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003080:Cold Temperature; D018360:Crystallography, X-Ray; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000096:biosynthesis / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010754:Phosphoribosyl Pyrophosphate / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D044096:Psychrobacter / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D011756:Diphosphates; C106638:HisG protein, bacteria; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; C003130:phosphoribosyladenosine triphosphate; C107241:diphosphoric acid; D006639:Histidine; D010754:Phosphoribosyl Pyrophosphate; D000255:Adenosine Triphosphate; D001276:ATP Phosphoribosyltransferase; D000604:Amino Acyl-tRNA Synthetases