Functional analysis of the cysteine synthase protein complex from plants: Structural, biochemical and regulatory properties

Summary

Cysteine synthesis in plants represents the final step of assimilatory sulfate reduction and the almost exclusive entry reaction of reduced sulfur into metabolism not only of plants, but also the human food chain in general. It is accomplished by the sequential reaction of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they form the hetero-oligomeric cysteine synthase complex (CSC). Recent evidence is reviewed that identifies the dual function of the CSC as a sensor and as part of a regulatory circuit that controls cellular sulfur homeostasis. Computational modeling of three-dimensional structures of plant SAT and OAS-TL based on the crystal structure of the corresponding bacterial enzymes supports quaternary conformations of SAT as a dimer of trimers and OAS-TL as a homodimer. These findings suggest an overall α₆β₄ structure of the subunits of the plant CSC. Kinetic measurements of CSC dissociation triggered by the reaction intermediate O-acetylserine as well as CSC stabilization by sulfide indicate quantitative reactions that are suited to fine-tune the equilibrium between free and associated CSC subunits. In addition, in vitro data show that SAT requires binding to OAS-TL for full activity, while at the same time bound OAS-TL becomes inactivated. Since OAS concentrations inside cells increase upon sulfate deficiency, whereas sulfide concentrations most likely decrease, these data suggest the dissociation of the CSC in vivo, accompanied by inactivation of SAT and activation of OAS-TL function in their free homo-oligomer states. Biochemical evidence describes this protein-interaction based mechanism as reversible, thus closing the regulatory circuit. The properties of the CSC and its subunits are therefore consistent with models of positive regulation of sulfate uptake and reduction in plants by OAS as well as a demand-driven repression/de-repression by a sulfur intermediate, such as sulfide.

Introduction

Reduced sulfur in phototrophic organisms enters metabolism exclusively via cysteine. The importance of cysteine formation in plants is therefore comparable to the fixation of ammonia into glutamine with its consequences for protein biosynthesis, biomass production and human nutrition. The biosynthesis of cysteine marks the transition between reduction in the assimilatory sulfate reduction pathway of plants and actual metabolization. The importance of reduced sulfur is further illustrated by the multitude of functions that are directly or indirectly mediated by the major sulfur metabolites cysteine, methionine and glutathione (GSH). These include redox and structural control in proteins, S-donation in iron sulfur cluster and vitamin biosynthesis and detoxification of oxygen radicals and xenobiotics (Leustek et al., 2000; Droux, 2003, Droux, 2004; Hell, 2003; Wirtz and Droux, 2005). Following sulfate reduction in plastids, cysteine synthesis uses free sulfide and O-acetylserine (OAS) to form the first stable and non-toxic reduced sulfur compound. This step also marks the convergence of sulfur and nitrogen metabolism, giving rise to regulatory interactions (Kopriva et al., 2002). Under non-stressed conditions cysteine levels are in the order of only 10–30 μM but undergo a high turnover, while GSH ranges between 0.2 and 0.5 mM (Blaszczyk et al., 1999; Harms et al., 2000; Buchner et al., 2004; Wirtz et al., 2004). These values refer to bulk measurements that average potential differences between subcellular compartments. First the reaction intermediate OAS is synthesized by serine acetyltransferase (SAT; EC 2.2.1.30; gene acronym Serat; Kawashima et al., 2005) from serine and acetyl-coenzyme A (Fig. 1). O-acetylserine (thiol) lyase (OAS-TL, also named cysteine synthase; EC 2.5.1.47), subsequently catalyses the insertion of sulfide into the carbon skeleton by an elimination reaction that yields the β-substituted alanine cysteine (hence the gene acronym β-substituted alanine synthase, Bsas; Hatzfeld et al., 2000). The two enzymes form the hetero-oligomeric cysteine synthase complex (CSC). SAT and OAS-TL are located not only in plastids but also in mitochondria and the cytosol (Lunn et al., 1990; Ruffet et al., 1995).

Reactions at branching points in cellular pathways are generally prime targets of regulatory mechanisms to sense the status of substrates and to control fluxes towards end products. Cysteine synthesis represents such a step, but the function of the CSC has remained enigmatic since its discovery through the groundbreaking work of Nicolas Kredich and coworkers on enterobacteria (Kredich et al., 1969). The cysteine regulon of Salmonella typhimurium and Escherichia coli is believed to be representative for the regulation of prokaryotic sulfate assimilation and cysteine biosynthesis (see Kredich, 1996 for review). The cysE gene encodes SAT in bacteria and is the only constitutively expressed gene of the cys-regulon. Under sulfur limiting conditions, the regulon is activated and the entire SAT protein is associated with about 5% of the much more abundant OAS-TL protein (encoded by cysK and cysM genes) in the bacterial CSC. Bacterial SAT is highly sensitive to feedback inhibition by cysteine (K_i=1.1 μM). If cysteine concentrations in bacterial cells run low because of limiting sulfide availability, the inhibition is released and SAT produces OAS. OAS isomerizes to N-acetylserine in a purely chemical and irreversible process. N-acetylserine acts as an inducer of the CysB protein, enabling its binding to several operators of genes within the cys-regulon. This induces expression of genes encoding the sulfate permease to enhance sulfate uptake and of sulfate reduction to provide sulfide for cysteine synthesis (Kredich, 1996). A regulatory function of the bacterial CSC by itself has not been reported. However, it was noted that OAS is able to dissociate and that sulfide stabilizes the CSC of S. typhimurium in vitro (Kredich et al., 1969). Furthermore, the BB1 mutant strain of S. typhimurium was identified as a cysteine auxotroph and biochemically characterized as being altered in the interaction between SAT and OAS-TL (Becker and Tomkins, 1969). The molecular identity of this mutation later on revealed an amino acid exchange within the C-terminal protein interaction domain of SAT (Wirtz et al., 2001). This evidence strongly suggests that the ability to assemble the CSC is required in bacteria to carry out proper cysteine synthesis.

While enteric bacteria initiate cysteine synthesis only in situations of metabolic emergency, plants as primary producers face a completely different situation. The challenges for plant metabolism may be best illustrated by the fact that each subcellular compartment with the capability of protein biosynthesis carries out cysteine synthesis (Lunn et al., 1990) and that most plants have several nuclear genes that encode for different isoforms of SAT and OAS-TL for each of these three compartments (Hell et al., 2002). In this contribution the structure of the involved enzymes, their functional properties and the regulation of cysteine synthesis with emphasis on the CSC as well as cellular compartmentation in Arabidopsis thaliana are reviewed with reference to other plant species and bacteria.