Characterization of Cysteine Synthase in Echinochloa crus-galli L. and Its Inhibition by Substrate Analogues

Abstract

Abstract Cysteine synthase [CS; EC 4.2.99.8] catalyzes the terminal step of cysteine biosynthesis whereby cysteine is formed from O -acetylserine (OAS) and sulfide. CS was extracted from the leaves of Echinochloa crus-galli L., fractionated with ammonium sulfate, and then characterized. The optimum assay temperature of CS was 50°C, suggesting thermal stability. The activity was relatively low at pH 6.0, but increased sharply when the pH increased from 6.4 to 7.4. Maximal activity seemed between pH 7.4 and 9.0. The apparent K m value was 2.53 mM for OAS and 0.88 mM for sulfide, which indicates that CS has a low affinity for OAS. Two inhibitors of pyridoxal-5′-phosphate (PLP)-dependent enzymes, amino-oxyacetate (AOA) and cycloserine, inhibited this enzyme by 76 and 42%, respectively, at 10 mM. An analogue of OAS, O -phosphoserine, inhibited CS slightly, but other analogues (e.g., O -phosphothreonine) were not significantly inhibitory, even at 30 mM. The I 50 value of AOA was about 3 mM. When CS and AOA were preincubated together at 30°C, AOA inhibited CS in a time-dependent manner, by 51% with 0.1 mM AOA after a 4-h incubation. CS was stable during this incubation period. AOA and cycloserine inhibited the shoot elongation of E. crus-galli by 72 and 31%, respectively, at 5 kg a.i./ha postemergence. Growth inhibition by other compounds was less than 10%. These data suggest a correlation between CS inhibition and growth reduction; however, the supplementation of exogenous cysteine failed to reverse this inhibition in a test tube bioassay. This indicates that the growth inhibition could not be entirely attributed to the starvation of cysteine which might be caused by the inhibitor.