(228ff) Biochemical Characterization of the N-Acetylglutamate Kinase (ArgB) from Lactobacillus Plantarum

Arginine is one of the twenty standard amino acids and considered a conditionally essential amino acid. Most mammals can produce arginine in vivo, but it is generally not sufficient to meet nutritional needs. Arginine is important because of the beneficial health effects it offers to mammals, specifically in the immunological, neurological, and cardiovascular systems. In addition, arginine plays an important role in embryogenesis, implantation of blastocysts, placental cell growth, and nutrient transfer to the fetus. Arginine has also been used to help rescue compromised pregnancies.

Probiotics, specifically Lactobacillus, offer the opportunity to deliver arginine-producing bacteria to mammalian gastrointestinal tracts. Arginine biosynthesis in prokaryotes is encoded by the arg operon and involves eight enzymatic steps starting at glutamate. The second step is catalyzed by a N-acetylglutamtate kinase, encoded by the argB gene, that phosphorylates an acetylated glutamate molecule. Based on literature, arginine exhibits feedback inhibition toward the ArgB enzyme, which is one of the key methods in how the cell regulates the production of arginine in many organisms. In order to increase the production of arginine, this feedback inhibition must be fully understood and characterized.

The argB gene was isolated from Lactobacillus plantarum, cloned into the pET32a(+) plasmid with a C-terminal His₆ tag, and transformed into E. coli BL21(DE3) for expression. The resultant recombinant proteins were recovered from cell lysates by affinity chromatography using a Ni-NTA column and subjected to full biochemical characterization. In future work, results will be used to relieve arginine feedback inhibition of ArgB and enable the construction of arginine-producing L. plantarum.