Biosynthesis of Proline in Pseudomonas

y-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of L-glutamic acid 5-semialdehyde from y-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized L-l-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)+. The apparent Km values were: NAD+, 0.36mM; NADP+, 0.31mM; L-1-pyrroline-5carboxylic acid, 4mM with NADP+ and 8mm with NAD+; Pi, 28mm. 3-(Phosphonoacetylamido)-L-alanine, a structural analogue of y-glutamyl phosphate, inhibited this enzyme competitively (K, = 7mM). I-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH4)2SO4 fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced L-1pyrroline-5-carboxylate with NAD(P)H as a cofactor to L-proline. NADH (Km = 0.05mM) was a better substrate than NADPH (Ku, = 0.02mM). The apparent Km values for L-1pyrroline-5-carboxylate were 0.12mM with NADPH and 0.09mM with NADH. The 3acetylpyridine analogue of NAD+ at 2mM caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)+, heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of y-glutamyl kinase and yglutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.