O-acetylserine (thiol) lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in Arabidopsis thaliana.
The synthesis of cysteine is positioned at a decisive stage of assimilatory sulphate reduction, marking the fixation of inorganic sulphide into a carbon skeleton. O-acetylserine (thiol) lyase (OAS-TL) catalyses the reaction of inorganic sulphide with O-acetylserine (OAS). Despite its prominent position in the pathway OAS-TL is generally regarded as a non-limiting enzyme without regulatory function, due to low substrate affinities and semi-constitutive expression patterns. To resolve this apparent contradiction, the kinetic properties of three OAS-TLs from Arabidopsis thaliana, localized in the cytosol (A), plastids (B), and mitochondria (C), were analysed. The recombinant expressed OAS-TLs were purified to apparent homogeneity without any fusion tag to maintain their native forms. The proteins displayed high specific activities of 550-900 micromol min(-1) mg(-1). Using an improved and highly sensitive assay method for cysteine determination, the apparent K(m)(sulphide) was 3-6 microM for OAS-TL A, B, and C and thus 10-100 times lower than previously reported for plant OAS-TLs. K(m)(OAS) was between 310 microM and 690 microM for OAS-TL isoform A, B, and C, whereas the apparent dissociation binding constant for OAS was much lower (K(d)<1 microM OAS). A HPLC method was developed for OAS quantification that revealed fast increases of the cellular OAS concentration in response to sulphate deprivation. The observed fluctuations of intracellular OAS concentrations, combined with the OAS dissociation constant and the catalytic properties of OAS-TL, support the model of a dynamic cysteine synthesis system with regulatory function as can be expected from the position of the reaction in the sulphur assimilation pathway.
Wirtz
Markus
M
Heidelberg Institute of Plant Sciences, University of Heidelberg, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany.
Droux
Michel
M
Hell
Rüdiger
R
D017360:Arabidopsis / Q000201:enzymology*; D019757:Carbon-Oxygen Lyases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003545:Cysteine / Q000096:biosynthesis*; D003600:Cytosol / Q000201:enzymology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D007527:Isoenzymes; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D010802:Phylogeny; D018087:Plastids / Q000201:enzymology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D011994:Recombinant Proteins; C018725:O-acetylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases; D003545:Cysteine