Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyl-transferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel b-helix consisting of b-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis. Cysteine biosynthesis in plants and bacteria proceeds via a two-step process which marks the connection between nitrogen and sulfur assimilation [1±3]. First, serine is activated by acetyl-CoA to form O-acetylserine (OAS), catalyzed by serine acetyltransferase (SAT). Secondly, O-acetylserine (thiol) lyase (OAS-TL) inserts sulfide into OAS to yield cysteine. In plants as well as bacteria, OAS-TL activity is much higher than SAT activity, which is therefore considered to be rate-limiting. Whereas most bacteria carry one gene that encodes SAT (cysE) and two genes for OAS-TL (cysK, cysM), plants contain several nuclear genes that encode cytosolic, plastid and mitochon-drial isoforms of SAT and OAS-TL. At least three cDNAs for each enzyme have been isolated from Arabidopsis thaliana, the most extensively investigated plant with respect to sulfur metabolism [4,5]. SAT activity is exclusively found in association with OAS-TL, forming the cysteine synthase complex, whereas OAS-TL also exists as a free dimer [6±8]. The interaction of SAT and OAS-TL from A. thaliana, spinach and watermelon has been investigated in vitro and in vivo [5,9±12]. According to these reports, SAT consists of two protein-interaction domains, a central SAT±SAT domain for homomerization and a C-terminal SAT±OAS-TL domain for heteromerization. On association in the complex, SAT is activated to yield a higher V max and substrate affinities for OAS production, whereas OAS-TL is essentially inactivated and apparently acts as a regulator of SAT activity in the complex. Consequently, the intermediate OAS cannot be channeled within the complex but diffuses into the surrounding solution, where it reacts with free OAS-TL dimers and sulfide to form cysteine. As, at least in vitro, OAS dissociates and sulfide stabilizes the two components, a regulatory function of the cysteine synthase complex in the rate of cysteine synthesis has been suggested which is based on the asso...
cDNAs encoding for two isoforms of O-acetylserine (thiol) lyase (OAS-TL), which catalyzes the synthesis of cysteine, have been isolated from Arabidopsis thaliana. Secondary structure together with expression patterns derived during photomorphogenesis indicate cellular localizations in the cytosol and plastids, thus allowing a direct comparison of compartment-specific forms within one species. The cytosolic OAS-TL complemented an E. coli auxotrophic mutant lacking cysteine synthesis. Both isoforms are represented by small gene families. They are expressed under all conditions investigated and were observed to increase in expression in plants grown with limited sulfate supply.
A cDNA encoding for serine acetyltransferase which catalyzes the committing step of cysteine biosynthesis has been cloned from Arabidopsis rhafiana. The plant protein has a predicted molecular weight of 32.8 kDa and shows up to 43% of amino acid homology to bacterial serine acetyltransferases. It complements a serine acetyltransferase negative E. coli mutant and can be enzymatically determined in the heterologous host. The corresponding mRNA is predominantely expressed in light exposed tissue and represents one of at least two related genes.
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