(Friedrich-Schiller-Universitiit Jena, Sektion Biologie --M ikrobielle Biochemie)Biosynthesis of homoarginine and ornithine as precursors of the phytoeffector phaseolotoxin by the amidinotransfer from arginine to lysine catalyzed by an amidinotransferase in Pseudomonas syringae pv. phaseolicola U. MARKISCH and G. REUTER f Receiuetl 16 March lY8YIAccepfed 1 DecemherlY89)In the phytopathogenic Pseudomonos syrinycre pv. phaseolicola an amidinotransferase was detected and characterized which catalyzes the amidinotransfer from arginine to lysine producing the phaseolotoxin precursors homoarginine and ornithine. The enzyme was purified 419-fold by affinity chromatography on homoarginine-Sepharose. It had an apparent mol. wt. of 200000 and exhibited MICHAELIS-MENTEN kinetics with K,,, 7.7 mM for arginine and K,,, 4.2 mM for lysine. Ornithine competitively inhibited the enzyme,with a Ki of 0.8 mM. During cultivation of the pseudomonad the highest enzyme activity corresponds with the highest production rate of phaseolotoxin. At low toxin production, mutants exhibit a low enzyme activity and vice versa. Only those strains of different pathovarieties which are able to produce phaseolotoxin contain the enzyme. At 30 "C cultivation temperature, the enzyme activity was 50 7; of that at 18 "C, and toxin production decreased with increasing temperature. When microbes were grown on ribose, their enzyme activity was 60',', lower than grown on glucose or glycerol. Toxin production was decreased to about 10 "/d when growing on ribose. Amidinotransferase is considered to be one of the key enzymes of phaseolotoxin biosynthesis.Pseudonzonas syringae pv. phaseolicola ( P . phaseolicola) causes the halo blight disease in bean, Pltaseouls vulgaris (RUDOLPH 1976, DURBIN 1985. It produces phaseolotoxin, a tripeptide consisting of homoarginine, alanine and ornithine which is substituted in its b-aminogroup, om-P(0) (NHJ-NH-S0,H. Furthermore it produces this substituted ornithine (PNSOrn) 1977, PATIL et al. 1970, TAM and PATIL 1972. Searching for the molecular basis of the resistance of P. phaseolicola against the selfproduced toxin we found a second OCT, which is insensitive to the toxin (JAHN el at. 1985(JAHN el at. , 1987(JAHN el at. , 1989a(JAHN el at. , 1989b. It is suggested that PNSOrn is the decisive ingredient of phaseolotoxin and that alanyl-homoarginine functions as a toxin vehicle in its transport to the plant cell (MITCHELL 1976).Until now nothing was known about the in vitro biosynthesis of phaseolotoxin and its precursor homoarginine. Arginine is wellknown as a common amidine donor for guanidines. So we did expect that homoarginine and ornithine, two of the phaseolotoxin precursors, were prepared by a transamidination reation from lysine and arginine. The aim of this study was to find out and to characterize an amidinotfansferase in cell free extracts of P. phaseolicola, to rnofify methods for the purification of this enzyme opening possibilities for its additional characterization, and to test the conncection between enz...
