Pyridoxal 5’-phosphate supplementation modulates the heterologous expression and activity of a PLP dependent model protein in E.coli

Abstract

AbstractPLP is a biologically active form of Vitamin B6 and is required for carbohydrates, amino acids and fatty acid metabolism. Many of the PLP dependent proteins are important drug targets and effector molecules, and thus, their heterologous overexpression is of industrial importance and has commercial value. We have predicted the docking site of PLP on O-acetyl serine sulfhydrylase protein (OASS) of H.contortus and determined that lysine-47 is very important for the binding of PLP in the enzyme pocket. We have used this protein as a model protein for testing the effect of PLP on the expression of PLP dependent proteins by E.coli. We have tested the effect of supplementation of PLP in the media on the expression of PLP dependent model protein in E.coli. Soluble recombinant protein could be purified from each of the culture vials grown with variable amount of PLP [0 mM (Group I), 0.01mM (Group II), 0.025mM (Group III), 0.05mM (Group IV) and 0.1mM (Group V)]. There was approximately 4.2%, 7.2%, 10.5% and 18% increase in purified protein yield in Group II, III, IV and V, respectively, in comparison to group I. We studied the relative incorporation of PLP into the purified protein by scanning the changes in internal fluorescence of purified proteins. There was a significant quenching of tryptophan fluorescence emission in groups II, III, IV and V compared to group I (Purified protein without PLP addition). There was a linear increase in the activity of protein purified from cultures of group I to group V. This was due to greater availability of PLP, thus, allowing higher incorporation of the cofactor in the apoenzyme to form holoenzyme complexes. PLP is not known to be directly imported into E.coli. We could find a PLP concentration-dependent increase in expression and catalytic activity of the enzyme signifying the probable transport of PLP across the membrane. The mechanism of transport of PLP in the light of the current experiment is still unknown and should be a subject of future studies.