Kinetics studies of archaeal PurO and PurH2-related cyclohydrolases

Author: Matt Johnson
Major: Biochemistry
Approved: Fall 2017
Status: Completed

The ability to complete the de novo biosynthesis of purines is important for the production of numerous essential purine products including the purine bases of DNA and RNA, energy carriers ATP and GTP, and coenzymes NAD and FAD. While all three domains of life contain the ability to perform purine biosynthesis, the archaeal pathway is the most variable. The final two steps of purine biosynthesis, the conversion of 5-aminoimadazole-4-carboxamide ribonucleotide (AICAR) to 5-formamidoimidazole-4-carboxamide ribonucleotide (FAICAR) and FAICAR to inosine 5’-monophosphate (IMP), illustrate this contrast. Eukarya and bacteria complete the final two steps of purine biosynthesis using the bifunctional PurH protein. Archaea, in contrast, complete these steps using the separate enzymes PurP or PurH1 (AICAR formyltransferases) and PurO or PurH2 (IMP cyclohydrolases). This study optimized an expression system to enzymatically attain purified FAICAR. E. coli cells were transformed with a plasmid for a PurP enzyme (from Methanocaldococcus jannaschii locus MJ0136), and active PurP expression was induced. PurP proceeded through purifications before incubation with assay reagents in order to produce the product FAICAR. FAICAR was isolated using HPLC. Future studies will use isolated FAICAR obtained from this experiment to kinetically study PurO and PurH2-related enzymes and examine additional IMP cyclohydrolase candidates.