the cofactor of ScGAD is verified to be either pyridoxal 5'-phosphate (PLP) or pyridoxal. The optimal concentration of either cofactor is 50 mg/l. GAD requires a coenzyme for activity while inactive apo-GAD forms a GAD-PLP complex (holo-GAD) to obtain its activity
GAD can be regulated in vivo by Ca2+/calmodulin atpH 5.8. The addition of bovine calmodulin and Ca2+ to the reaction mix causes an activation of GAD activity of 32%
a unique feature of plant GAD is the presence of a calmodulin (CaM)-binding domain at its C-terminus. In plants, transient elevation of cytosolic Ca2+ in response to different types of stress is responsible for GAD activation via CaM
activity of GST-GAD54, GAD65 and 6*His-GAD65 is nearly zero without exogenous pyridoxal 5'-phosphate. Activity of GST-GAD67 is 20% without exogenous pyridoxal 5'-phosphate. Activity of GAD67 is 32% without exogenous pyridoxal 5'-phosphate. Activity of 6*His-GAD67 is 30% without exogenous pyridoxal 5'-phosphate
although GAD65 and GAD67 interact differently with pyridoxal 5'-phosphate, their cofactor-binding sites contain the same set of nine putative cofactor-binding residues and has the same basic structural fold. Thus the cofactor-binding differences cannot by attributed to fundamental structural differences between the GADs but must result from subtle modifications of the basic cofactor-binding fold
slower binding of pyridoxal 5'-phosphate to GAD67 than to GAD65, and less ease to dissociate pyridoxal 5'-phosphate from holoGAD67 than holoGAD54. TGAD67 is more highly saturated by the cofactor than GAD65. The two binding sites of GAD65 exhibit similar affinities for pyridoxal 5'-phosphate. One binding site of GAD67 exhibits a significantly higher affinity for pyridoxal 5'-phosphate than the other binding site
the pyridine ring of pyridoxal 5'-phosphate is sandwiched between residues Ala248 and Gln166. Residues Lys279 and His278 are required for the binding of cofactor pyridoxal 5'-phosphate to form a Schiff base as in many other pyridoxal 5'-phosphate-dependent enzymes
PLP, plays a key role in the acquisition of a folding necessary for the canonical catalytic activity. Preparation of recombinant apoenzyme, and reconstitution with pyridoxal 5'-phosphate, overview
the GAD from Lactobacillus sakei strain A156 contains a highly conserved catalytic domain that belongs to the pyridoxal 5'-phosphate-dependent decarboxylase superfamily. The domain includes a lysine residue essential for pyridoxal 5'-phosphate binding, designated as PLP lysine