N5-Carboxyaminoimidazole ribonucleotide synthetase (N 5-CAIR synthetase), a key enzyme in microbial de novo purine biosynthesis, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to N 5-CAIR. To date, this enzyme has been observed only in microorganisms, and thus, it represents an ideal target for antimicrobial drug development. Here, we report structural and functional studies on the Aspergillus clavatus N5-CAIR synthetase and identification of inhibitors for the enzyme. In collaboration with Dr. Hazel Holden of the University of Wisconsin, the three-dimensional structure of Aspergillus clavatus N5-CAIR synthetase was solved in the presence of either Mg2ATP or MgADP and AIR. These structures, determined to 2.1 and 2.0 Å resolution, respectively, revealed that AIR binds in a pocket analogous to that observed for other ATP-grasp enzymes involved in purine metabolism. On the basis of these models, a site-directed mutagenesis study was subsequently conducted that focused on five amino acid residues located in the active site region of the enzyme. These investigations demonstrated that Asp153 and Lys353 play critical roles in catalysis without affecting substrate binding. All other mutations affected substrate binding and, in some instances, catalysis as well. Taken together, the structural and kinetic data presented here suggest a catalytic mechanism whereby Mg2ATP and bicarbonate first react to form the unstable intermediate carboxyphosphate. This intermediate subsequently decarboxylates to CO2 and inorganic phosphate, and the amino group of AIR, through general base assistance by Asp153, attacks CO2 to form N5-CAIR.
To identify the inhibitors for this enzyme, we have conducted high-throughput screening (HTS) against Escherichia coli N5-CAIR synthetase using a highly reproducible phosphate assay. HTS of 48,000 compounds identified 14 compounds that inhibited the enzyme. The hits identified could be classified into three classes based on chemical structure. Class I contains compounds with an indenedione core. Class II contains an indolinedione group, and class III contains compounds that are structurally unrelated to other inhibitors in the group. We determined the Michaelis-Menten kinetics for five compounds representing each of the classes. Examination of compounds belonging to class I indicates that these compounds do not follow normal Michaelis-Menten kinetics. Instead, these compounds inhibit N5-CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the class II families of compounds are non-competitive with both AIR and ATP. One compound in class III is competitive with AIR but uncompetitive with ATP, whereas the other is non-competitive with both substrates. Finally, these compounds display no inhibition of human AIR carboxylase indicating that these agents are selective inhibitors of N5-CAIR synthetase.
Given the importance of the class II, non-competitive inhibitors, we developed a diazirine-based photocrosslinking agent to identify the binding site of these inhibitors. These studies revealed that the isatin core of class II inhibitors is capable of undergoing photochemical conversion to isatoic anhydride. Once formed, the anhydride is capable of reacting with the protein. Treatment of N5-CAIR synthetase with the photoreactive agent lead to the dimerization of two monomers of the synthetase. Proteomic analysis of the crosslinked protein identified serine 227 as a possible site of modification. These studies also revealed two peptides that were missing in the dimerized protein sample. These two peptides were located near serine 227. While compelling, the location of the missing peptides and serine 227 is 20 Å away from the dimerization interface observed in the crystal structure. Thus, our photocrosslinking studies suggest that N5-CAIR synthetase may exist in multiple dimer conformations.
|Advisor:||Firestine, Steven M.|
|Commitee:||Chow, Christine, Kowluru, Anjaneyulu, Woster, Patrick|
|School:||Wayne State University|
|School Location:||United States -- Michigan|
|Source:||DAI-B 71/09, Dissertation Abstracts International|
|Subjects:||Biochemistry, Pharmacy sciences|
|Keywords:||Antibacterial target, Medicinal chemistry, Ribonucleotide synthetase|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be