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

Abstract

(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...

The Hrp pathogenicity island of Erwinia amylovora and identification of three novel genes required for systemic infection‡

“…In support of this hypothesis, the homoarginine and ornithine moieties of phaseolotoxin in P. syringae pv. phaseolicola are produced by an amidinotransfer reaction from arginine to lysine (Hernandez‐Guzman and Alvarez‐Morales, 2001; Märkisch and Reuter, 1990). Whether E. amylovora produces an antimicrobial compound with phaseolotoxin‐like activity and whether the hsv genes are involved in its production have been explored.…”

In Pseudomonas syringae pv. phaseolicola, Expression of the argK Gene, Encoding the Phaseolotoxin-Resistant Ornithine Carbamoyltransferase, Is Regulated Indirectly by Temperature and Directly by a Precursor Resembling Carbamoylphosphate

“…Amidinotransferase is an enzyme that transfers the amidino group of L-arginine to L-lysine to produce homoarginine and ornithine (13); both of these amino acids are present in the tripeptide moiety of phaseolotoxin. Therefore, in the absence of amidinotransferase, the NЈ-sulfodiaminophosphinyl group may accumulate because of the lack of homoarginine and possibly limiting amounts of ornithine.…”

“…Arginine is a key amino acid in phaseolotoxin synthesis, since it is substrate of an amidinotransferase that catalyzes the synthesis of homoarginine, a constituent of the organic moiety of phaseolotoxin (13,26). This amidinotransferase is encoded by amtA, whose expression is coordinated with phaseolotoxin synthesis and argK expression.…”

Advances in Molecular Biology of Phytotoxin Production and Its Involvement in Plant Pathogenesis

“…The biosynthesis of sulphodiamino phosphinyl moiety has not been identified. However, Markisch and Reuter (1990) demonstrated that homoarginine and ornithine residues are synthesised by a transamidination reaction from arginine and lysine.…”