Views
5 years ago

Methionine Biosynthesis in Lemna - Plant Physiology

Methionine Biosynthesis in Lemna - Plant Physiology

Methionine Biosynthesis in Lemna - Plant

Plant Physiol. (1982) 69, 1077-1083 0032-0889/69/82/1077/07/$00.50/0 Methionine Biosynthesis in Lemna STUDIES ON THE REGULATION OF CYSTATHIONINE y-SYNTHASE, O-PHOSPHOHOMOSERINE SULFHYDRYLASE, AND O-ACETYLSERINE SULFHYDRYLASE Received for publication June 23, 1981 and in revised form September 9, 1981 GREGORY A. THOMPSON, ANNE H. DATKO, S. HARVEY MUDD, AND JOHN GIOVANELLI National Institute of Mental Health, Laboratory of General and Comparative Biochemistry, Bethesda, Maryland 20205 ABSTRACT Regulation of enzymes of methionine bkosynthesis was investigated by measuring the specific activities of 0-p ot cys tathonine y-synthse, rim sulfhydrylase, and 0-acetylserine sulfhydrylase in Lemma paaeikesta Hegelm 6746 grown under various conditions. For cystathionine y-syntase, it was observed that (a) adding external methionlne (2 pM) decreased specific actvity to 15% of control, (b) blockingm e synthesis with 0.05 FiM L-n xy nylglycine or with 36 ,M lysine plus 4 pM threonine (Datko, Mudd 1961 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold Increase in specific activity, and (c) blockingm e synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cdtures was unaffected by addition ofm e, threonine, lysine p-s teonine, S-adenosyhne, or S-methynetoine sulfonium to the assay mixture. Parallel studies of 0-phosphohomoserine sulfhydrylase and O-acetyserine sulfhydrylase showed that O-phosphohomoserine sulfhydryase activity respond to growth conditions identically to cystathn synthase activity, whereas 0-acetylserine sulfhydrylase activity remained unaffected. Lemma extracts did not catalyze antho formation from O-acetylserne and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that 0-acetylserine sulfhydrylase has much higher activity and affinity for sulde than0-e sulfhydrylase. The results suggest that (a) methkoine, or one of its products, regulates the amount of active cystatbioine yBsynthase in Lenua, (b) 0-phosphohomoserine suihhydrylase and cystat y-synthase are probably activities of one enzyme that has low specificity for its sulfur-contahing substrate, and (c) 0-acetyiserine sulihydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of 0-acetylserine sulfhydrylase provides an explanation in molecular terms for tra aration, and not direct sulfhydration, being the dominant pathway for homocysteine biosynthesis. Methionine is synthesized in higher plants from (a) the fourcarbon moiety of aspartic acid via O-phoshohomoserine, (b) the sulfur of inorganic sulfate via cysteine, and (c) a methyl group from N5-methyltetrahydrofolate (triglutamyl derivative) (14) (Fig. 1). Datko and Mudd (7) have reported upon several compounds (or combinations of compounds) each of which inhibits a specific and different step in this pathway and which therefore impairs the endogenous synthesis of methionine by Lemna paucicostata. Such inhibitors are especially valuable because they can be used as tools to investigate whether or how plants adapt to conditions of limiting methionine. Specifically, one can determine whether the activities of certain key enzymes in the methionine pathway 14c . D77 are sensitive to changes in the physiological concentration of methionine or one of its products. In this regard, cystathionine y-synthase is of primary interest. The reaction it catalyzes (reaction 1) O-phosphohomoserine + cysteine -- cystathionine + Pi (1) is the central step joining branches of two separate pathways (Fig. 1). Both O-phosphohomoserine and cysteine are committed at this step to the formation of methionine rather than to their respective alternative fates of conversion to threonine or incorporation into protein and glutathione. Because regulation of biosynthetic pathways frequently occurs at committing steps, the cystathionine ysynthase reaction is a likely step at which to observe control by the end-product methionine. Another enzyme activity of interest is that which catalyzes direct sulfhiydration of O-phosphohomoserine (reaction 2): 0-phosphohomoserine + sulfide -. homocysteine + Pi (2) This reaction has been demonstrated in Lemna, Chlorella, and a number of other higher plants (8) and is a possible alternative to transsulfuration as a pathway for the formation of homocysteine (Fig. 1). However, studies of Lemna (15) and Chlorella (13) have failed to detect any homocysteine formation via this shunt and have shown that at least 90 to 95% of the homocysteine which is formed comes from cystathionine. These findings call into question the function of this sulfhydrylase. A third enzyme of interest is O-acetylserine sulfhydrylase which catalyzes reaction 3: O-acetylserine + sulfide -p cysteine + acetate (3) This enzyme is very active in many plants (14). Since the reaction it catalyzes is very similar to that catalyzed by 0-phosphohomoserine sulfliydrylase, it appeared appropriate to investigate the relationships between the O-acetylserine- and the O-phosphohomoserine-dependent activities. We have measured cystathionine y-synthase, O-phosphohomoserine sulfliydrylase, and O-acetylserine sulfliydrylase activities in extracts of Lemna grown in the presence of methionine or of inhibitors of methionine biosynthesis. We have also studied the effect of these compounds on enzyme activities in vitro. The results, reported here, provide new insight into several aspects of methionine biosynthesis in higher plants. A preliminary report upon this work has been made (27). MATERIALS AND METHODS Materials. Chemicals. The following chemicals were obtained from the sources indicated: DTT and L-cysteine- HCI from P-L Biochemicals; ninhydrin, Hepes, and S-adenosylmethionine from Sigma; 3-

Medicinal Plants Classification Biosynthesis and ... - Index of
Plant Physiology - Stony Brook University
Fungous diseases of plants, with chapters on physiology ... - Index of
Contents by subject areas - Plant and Cell Physiology
Chapter 8 Flowering and reproductive physiology in plant
Adaptation of Lemna paucicostata to Sublethal ... - Plant Physiology
Threonine Synthase of Lemna paucicostata ... - Plant Physiology
The S-Methylmethionine Cycle in Lemna ... - Plant Physiology
Uptake of Choline and Ethanolamine by Lemna ... - Plant Physiology
Biosynthesis of UDP-Xylose. Cloning and ... - Plant Physiology
Biosynthesis of t-Anethole in Anise ... - Plant Physiology
Control of the Protein Turnover Rates in Lemna ... - Plant Physiology
Induction of Jasmonate Biosynthesis in ... - Plant Physiology
Increased Arginine Biosynthesis during ... - Plant Physiology
Biosynthesis of Pectin1 - Plant Physiology
In Vivo Metabolism of 5'-Methylthioadenosine in ... - Plant Physiology
Regulation of Starch Biosynthesis in Response to a - Plant Physiology
Polyamines Inhibit Biosynthesis of Ethylene in ... - Plant Physiology
Gibberellin Biosynthesis in Developing Pumpkin ... - Plant Physiology
L-Ascorbic Acid Biosynthesis in Ochromonas ... - Plant Physiology
Xylan biosynthesis in grasses Corresponding ... - Plant Physiology
Folate Biosynthesis in Higher Plants. cDNA ... - Plant Physiology
Ethylene Biosynthesis-Inducing Xylanase' - Plant Physiology
Auxin Biosynthesis by the YUCCA Genes in Rice - Plant Physiology
Biosynthesis of Germacrene A Carboxylic Acid in ... - Plant Physiology
Phosphate Regulation of Lipid Biosynthesis in ... - Plant Physiology
Rice ERF OsEATB restricts GA biosynthesis - Plant Physiology
Storage cell wall: biosynthesis and degradation - Plant Physiology
The TIR2 gene and auxin biosynthesis ... - Plant Physiology
Biosynthesis of GA,, Methyl Ester in Lygocfium ... - Plant Physiology