Homocysteine Biosynthesis in Green Plants

• Published in: The Journal of biological chemistry 1974-02, Vol.249 (4), p.1139-1155
• Main Authors: Datko, Anne H., Giovanelli, John, Mudd, S. Harvey
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)42953-0

Optimal conditions for biosynthesis of cystathionine with crude extracts of green plants were determined. The specific activities observed under these conditions are considerably higher than those initially detected (Giovanelli, J., and Mudd, S. H. (1966) Biochem. Biophys. Res. Commun. 25, 366), and compare favorably with those of bacterial extracts. At near saturating concentrations of homoserine esters, crude extracts of tissues representing most of the major phylogenetic divisions of green plants generally catalyze the highest rates of cystathionine synthesis in the presence of O-malonylhomoserine, intermediate rates in the presence of O-oxalyl-, O-succinyl-, and O-phosphorylhomoserine, and very low rates with O-acetylhomoserine. Green plants are unique among the organisms studied by us or other workers in their capacity to use O-phosphorylhomoserine in cystathionine synthesis. Enzymic assay systems permitting the detection of compounds capable of serving as α-aminobutyryl donors in the synthesis of cystathionine were developed, taking advantage of the fact that crude preparations of plant cystathionine γ-synthase are active with a wide range of compounds. An analogous system designed to detect O-acetylhomoserine with greater sensitivity was developed through the use of an enzyme preparation from Bacillus subtilis. An α-amino-butyryl donor was purified by ion exchange chromatography and paper electrophoresis from tissues of green plants ranging from the green alga Chlorella to the higher flowering plants. Only a single α-aminobutyryl donor was detected among these green plants. This compound was characterized as O-phosphorylhomoserine on the basis of its base stability, its behavior during ion exchange chromatography and paper electrophoresis, and a rate of hydrolysis by highly purified alkaline phosphatase that was indistinguishable from that of authentic O-phosphorylhomoserine. It is proposed that O-phosphorylhomoserine is the dominant physiological precursor of cystathionine in green plants. Some of the implications of this finding are discussed. During the course of this work, studies on the relative activity of cystathionine γ-synthase with various substrates were extended to a number of lower organisms. The patterns of substrate activity with crude extracts of a blue-green alga and Escherichia coli are very similar, and resemble the pattern for cystathionine γ-synthase of Salmonella typhimurium (Kaplan, M. M., and Flavin, M. (1966) J. Biol. Chem.