UVM Theses and Dissertations
Format:
Print
Author:
Champagne, Karen S.
Dept./Program:
Microbiology and Molecular Genetics
Year:
2005
Degree:
PhD
Abstract:
The N1-5'-phosphoribosyl ATP transferase (ATP-PRTase) encoded by the hisG locus catalyzes the condensation of ATP with 5'-phosphoribosyl pyrophosphate, the first reaction in the biosynthesis of histidine. Unlike the homohexameric forms of the enzyme found in Escherichia coli and Salmonella typhimurium, the ATP-PRTase from Lactococcus lactis and a number of other bacterial species consists of two different polypeptides, both of which are required for catalytic activity (Sissler et al. 1999). The first of these is a truncated version of HisG that is approximately 100 amino acids shorter than the canonical versions. HisZ constitutes the second of the two polypeptides, and is a 328-residue version of a class II aminoacyl-tRNA synthetase catalytic domain that possesses no aminoacylation function. We have determined the molecular weight and subunit composition of the L. lactis HisZ-HisG heteromeric ATP-PRTase using size exclusion chromatography, and quantitative protein sequencing.
The results indicate that the HisZG ATP-PRTase from L. lactis is a 250 kDa multimeric enzyme complex consisting of four HisG and four HisZ subunits. We also present the first structure of a PRPP-bound ATP-PRT at 2.9 Å, and provide a structural model for allosteric activation based on comparisons with other inhibited and activated ATP-PRTs from both the hetero-octameric and hexameric families. The activated state of the octameric enzyme is characterized by an interstitial phosphate ion in the HisZ-HisG interface, and new contacts between the HisZ motif 2 loop and the HisG[subscript s] dimer interface. These contacts restructure the interface to recruit conserved residues to the active site, where they activate pyrophosphate to promote catalysis. Additionally, mutational analysis identifies the histidine binding sites within a region highly conserved between HisZ and the functional HisRS. We examined the steady-state kinetic parameters for the L. lactis ATP-PRTase to characterize the hetero-octameric enzymatic reaction. The K[subscript m] for ATP appears to be much higher in this enzyme, and is close to the intracellular concentrations of ATP.
This indicates that the biosynthesis of histidine may be determined by the availability of ATP in organisms possessing the hetero-octameric enzyme. Alanine mutations based on the crystal structure were used identify residues that are essential for PRPP binding. El58 forms a salt bridge with R10, which creates a binding pocket for the adenine ring of the ATP substrate. K8 is not involved in the direct binding of substrate molecules. However, it is positioned suitably for activation of the PPi leaving group during catalysis, and likely plays a key role in stabilization of the transition state during the reaction. These results provide evidence for how a histidyl-tRNA synthetaselike domain can be recruited to a novel function in amino acid biosynthesis. This direct corroboration of the catalytic and regulatory mechanisms predicted from the L. lactis ATP-PRTase crystal structure are essential first steps in understanding the intricate allosteric mechanism that has evolved to control the first committed step of the histidine biosynthetic pathway.
The results indicate that the HisZG ATP-PRTase from L. lactis is a 250 kDa multimeric enzyme complex consisting of four HisG and four HisZ subunits. We also present the first structure of a PRPP-bound ATP-PRT at 2.9 Å, and provide a structural model for allosteric activation based on comparisons with other inhibited and activated ATP-PRTs from both the hetero-octameric and hexameric families. The activated state of the octameric enzyme is characterized by an interstitial phosphate ion in the HisZ-HisG interface, and new contacts between the HisZ motif 2 loop and the HisG[subscript s] dimer interface. These contacts restructure the interface to recruit conserved residues to the active site, where they activate pyrophosphate to promote catalysis. Additionally, mutational analysis identifies the histidine binding sites within a region highly conserved between HisZ and the functional HisRS. We examined the steady-state kinetic parameters for the L. lactis ATP-PRTase to characterize the hetero-octameric enzymatic reaction. The K[subscript m] for ATP appears to be much higher in this enzyme, and is close to the intracellular concentrations of ATP.
This indicates that the biosynthesis of histidine may be determined by the availability of ATP in organisms possessing the hetero-octameric enzyme. Alanine mutations based on the crystal structure were used identify residues that are essential for PRPP binding. El58 forms a salt bridge with R10, which creates a binding pocket for the adenine ring of the ATP substrate. K8 is not involved in the direct binding of substrate molecules. However, it is positioned suitably for activation of the PPi leaving group during catalysis, and likely plays a key role in stabilization of the transition state during the reaction. These results provide evidence for how a histidyl-tRNA synthetaselike domain can be recruited to a novel function in amino acid biosynthesis. This direct corroboration of the catalytic and regulatory mechanisms predicted from the L. lactis ATP-PRTase crystal structure are essential first steps in understanding the intricate allosteric mechanism that has evolved to control the first committed step of the histidine biosynthetic pathway.