The Investigation on the Effect Asn123Gly mutation in the Enzyme Pyrroline-5-Carboxylate Reductase.

In this experiment, we are studying the enzyme pyrroline-5-carboxylate reductase from Sinorhizobium melloti. Bioinformatics proposed that the enzyme catalyzes the reduction of pyrroline-5-carboxylate to L-proline using NAD(P)H, which is the final step in proline biosynthesis. The purpose of this experiment is to study the effect of the point mutation of asparagine 123 to glycine on the enzymatic activity of the enzyme. The mutation was generated using site-directed mutagenesis to change the GGT codon to CCA in the proC gene. The mutation was confirmed via DNA sequencing and the gene was recombinantly expressed in Escherichia coli Rosetta 2 (DE3) cells for protein overproduction. The protein was purified using Ni-NTA affinity chromatography to >90% purity (determined by SDS-PAGE). The effect of the mutation on the function of the enzyme was determined using a continuous kinetic assay following the oxidation of the L-proline analog L-thiazolidine-4-carboxylate using NAD(P) and monitoring the formation of NAD(P)H at 340 nm on a UV-visible spectrophotometer. Preliminary investigations show that the mutation results in a decreased kinetic activity. Current work is underway to identify the underlying structural reason why the Asn123Gly variant has decreased activity.