Purification and Characterization of Glutamate 1-Semialdehyde Aminotransferase from Barley Expressed in Escherichia coli.

The immediate precursor in the synthesis of tetrapyrroles is Delta-aminolevulinate (ALA). ALA is synthesized from glutamate in higher plants, algae, and certain bacteria. Glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8) (GSA-AT), the third enzyme involved in this metabolic pathway, catalyzes the transamination of GSA to form ALA. The gene encoding this aminotransferase has previously been isolated from barley (Hordeum vulgare) and inserted into an Escherichia coli expression vector. We describe herein the purification of this recombinant barley GSA-AT expressed in Escherichia coli. Coexpression of GroEL and GroES is required for isolation of active aminotransferase from the soluble protein fraction of Escherichia coli. Purified GSA-AT exhibits absorption maxima characteristic of vitamin B(6)-containing enzymes. GSA-AT is primarily in the pyridoxamine form when isolated and can be interconverted between this and the pyridoxal form by addition of 4,5-dioxovalerate and 4,5-diaminovalerate. The conversion of GSA to ALA under steady-state conditions exhibited typical Michaelis-Menten kinetics. Values for K(m) (d,l-GSA) and k(cat) were determined to be 25 micromolar and 0.11 per second, respectively, by nonlinear regression analysis. Stimulation of ALA synthesis by increasing concentrations of d,l-GSA at various fixed concentrations of 4,5-diaminovalerate supports the hypothesis that 4,5-diaminovalerate is the intermediate in the synthesis of ALA.