ATP-phosphoribosyltransferase (ATPPRT) catalyses the first step in histidine biosynthesis, the condensation of ATP and 5-phospho--D-ribosyl-1-pyrophosphate to generate N 1 -(5-phospho--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. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was solved at 2.34-Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-to 4-fold increase in kcat.Reaction profiles for both enzymes are indistinguishable by 31 P-NMR, indicating that the same reaction is catalysed. Temperature dependence of kcat shows deviation from Arrhenius behaviour 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 K and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterisation of a psychrophilic ATPPRT. 4Adenosine 5ʹ-triphosphate phosphoribosyltransferase (ATPPRT) (EC 2.4.2.17) catalyses the reversible Mg 2+ -dependent reaction between adenosine 5ʹ-triphosphate (ATP) and(PR-ATP) and inorganic pyrophosphate (PPi) (Scheme 1), the first step in histidine biosynthesis. 1 The chemical equilibrium of the reaction strongly favours reactants, 2 and the enzyme is allosterically inhibited by histidine. 1 In addition to being a model for understanding allostery, 2-4 ATPPRT is of biotechnological interest as a tool for histidine production, provided that histidine feedback inhibition can be overcome. [5][6][7] Two forms of ATPPRT, encoded by the hisG gene, are found in nature. Fungi, plants, and most bacteria possess a long, homo-hexameric protein, HisGL, each subunit consisting of two domains that make up the catalytic core and a C-terminal regulatory domain that mediates feedback inhibition by histidine. 8 Some bacteria and archaea have a short version of the protein, HisGS, which lacks the C-terminal regulatory domain and is insensitive to histidine. In these organisms, a catalytically inactive regulatory protein, HisZ, the product of the hisZ gene, is present. 9 HisZ, which shares a common ancestry with histidyl-tRNA synthetase (HisRS), binds HisGS to form ...

Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg2+-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisGS is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS’s catalytic rate, but decreases the hetero-octamer’s, indicating that chemistry and product release are rate-limiting for HisGS and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.

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