Nitrogen supply and abiotic stress influence canavanine synthesis and the productivity of in vitro regenerated Sutherlandia frutescens microshoots.

Environmental stresses can significantly alter the synthesis of both primary and secondary metabolites, resulting in medicinal plants with unpredictable biological activity. Here, in vitro shoot cultures of the medicinal plant Sutherlandia frutescens were used to study the impact of three abiotic stresses (nitrogen availability, drought and salinity), primarily on l-canavanine synthesis. This compound, a non-protein amino acid, is amongst those metabolites linked to the health benefits of Sutherlandia extracts. Nitrogen supplied to microplants positively correlated with canavanine levels, exhibited by a fourfold reduction when nitrates provided were halved. Although the biomass generated was lowered under these conditions, a higher capacity for rooting (52%) in comparison to the controls (37%) became evident. Only a small increase of the canavanine content in microplants growing on 100mM NaCl medium was detected, indicating that salinity stress was not a major limitation on cavanine production, but that it played more of a role in vitro on plantlet morphogenesis. Similarly, PEG as a supplement had little to no effect on canavanine synthesis. We conclude that a deeper understanding of the nutritional requirements for the agricultural crop management of S. frutescens, which serves the herbal products industry, is needed.

Conversion of canavanine to alpha-keto-gamma-guanidinooxybutyrate and to vinylglyoxylate and 2-hydroxyguanidine.

It was observed previously that hydroxyguanidine is formed in the reaction of canavanine(2-amino-4-guanidinooxybutanoate) with amino acid oxidases. The present work shows that hydroxyguanidine is formed by a nonenzymatic beta,gamma-elimination reaction following enzymatic oxidation at the alpha-C and that the abstraction of the beta-H is general-base catalyzed. The elimination reaction requires the presence in the alpha-position of an anion-stabilizing group--the protonated imino group (iminium ion group) or the carbonyl group. The iminium ion group is more activating than the carbonyl group. Elimination is further facilitated by protonation of the guanidinooxy group. The other product formed in the elimination reaction was identified as vinylglyoxylate (2-oxo-3-butenoate), a very highly electrophilic substance. The product resulting from hydrolysis following oxidation was identified as alpha-keto-gamma-guanidinooxybutyrate (ketocanavanine). The ratio of hydroxyguanidine to ketocanavanine depended upon the concentration and degree of basicity of the basic catalyst and on pH. In the presence of semicarbazide, the elimination reaction was prevented because the imino group in the semicarbazone derivative of ketocanavanine is not significantly protonated. Incubation of canavanine with 5'-deoxypyridoxal also yielded hydroxyguanidine. Since the elimination reactions take place under mild conditions, they may occur in vivo following oxidation at the alpha-C of L-canavanine (ingested or formed endogenously) or of other amino acids with a good leaving group in the gamma-position (e.g., S-adenosylmethionine, methionine sulfoximine, homocyst(e)ine, or cysteine-homocysteine mixed disulfide) by an L-amino acid oxidase, a transaminase, or a dehydrogenase. Therefore, vinylglyoxylate may be a normal metabolite in mammals which at elevated concentrations may contribute to the in vivo toxicity of canavanine and of some of the other above-mentioned amino acids.

Use of gas chromatography for determining catabolic products of arginine by bacteria.

A rapid and sensitive procedure for determining catabolic products of arginine metabolism by bacteria was developed. The method consists of inoculating a solution of L-arginine with a heavy cell suspension of the test organism. After a 2-hr incubation period, dissimilation products (citrulline, ornithine, agmatine, putrescine) are converted to volatile derivatives and analyzed by gas-liquid chromatography. Compared with conventional microbiological tests, the new procedure is rapid and can be used for sensitive quantitative measurements of specific metabolites from arginine.