Interactions between serine acetyltransferase and O‐acetylserine (thiol) lyase in higher plants

Abstract

The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form Oacetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass Ϸ300 kDa) called cysteine synthase containing 4 mol pyridoxal 5′-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. ProteinϪprotein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase : there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of Oacetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O-acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O-acetylserine.Keywords : cysteine synthase; serine acetyltransferase; O-acetylserine (thiol) lyase; proteinϪprotein interactions; cysteine synthesis in plants.In metabolic networks, a fine tuning of enzyme activities is verted into the final reaction product (P). Here, accumulation of I might be prevented through properly adjusted concentrations required to maintain the rates at which useful end-products are produced with regard to those at which they are needed, but also of E 2 over E 1 , so that, at steady state, the net rate of I transformation through E 2 at least equals that of synthesis through E 1 . This to avoid accumulation of toxic internal metabolites and/or to achieve efficient trapping of highly unstable reaction intermedi-situation is analogous to that encountered in enzyme assays with auxiliary enzymes [1]. ates [1Ϫ3]. Considering the simple case of two enzymes ...

Characterization of the Gene Encoding Serine Acetyltransferase, a Regulated Enzyme of Cysteine Biosynthesis from the Protist ParasitesEntamoeba histolyticaand Entamoeba dispar

“…Thus, the fact that we cloned the amoebic SAT cDNA by functional rescue using the E. coli SAT-deficient mutant implies that CS-SAT interaction is not essential either in the amoeba or in E. coli. Fourth, the overproduction of EhSAT did not result in the induction of CS, unlike in ␥-proteobacterial and plant counterparts (54,56). This implies that coordinated induction of CS expression by the accumulation of O-acetylserine or N-acetylserine, which is the isomeric product converted from O-acetylserine, is probably absent in amoebae.…”

“…The mixture was incubated to let any protein interaction occur and was then applied to the glutathione-Sepharose 4B column. If EhCS1 and/or the E. coli CS interacts with the GST-EhSAT fusion protein, they should be retained on the column, and eluted with either O-acetylserine or glutathione as shown for plant enzymes (54). However, SDS-PAGE analysis and CS assay of the eluents revealed that neither EhCS1 nor the E. coli CS was retained in the column (data not shown; also see Fig.…”

Arabidopsis Photorespiratory Serine Hydroxymethyltransferase Activity Requires the Mitochondrial Accumulation of Ferredoxin-Dependent Glutamate Synthase

“…O-acetylserine(thiol)-lyase has been shown to play such a regulatory role in the Cys synthase complex with serine acetyltransferase (SAT). Although it was proposed that this complex promotes metabolic channeling (Winkel, 2004), it was demonstrated using recombinantly produced enzymes that O-acetylserine(thiol)-lyase binds to SAT as a catalytically inactive enhancer of SAT activity (Ruffet et al, 1994;Droux et al, 1998). There is precedent for Fd-GOGAT playing a role independent of catalytic activity.…”

Increased Cysteine Biosynthesis Capacity of Transgenic Tobacco Overexpressing an O-Acetylserine(thiol) Lyase Modifies Plant Responses to Oxidative Stress

“…However, based on these findings and the observation that endogenous plant OASTL levels are far in excess of the levels of SAT (Ruffet et al, 1994), the enzyme that catalyzes the formation of OAS required for Cys biosynthesis, it was recently suggested that OASTL does not play an important role in regulating plant Cys biosynthesis (Harms et al, 2000). In contrast, analyses of the bi-enzyme complex that forms between OASTL and SAT in chloroplasts not only have clearly demonstrated that OASTL can act as a regulatory subunit of SAT activity, and hence Cys biosynthesis, but that because OASTL in this complex is almost inactive, a 400-fold excess of unbound auxiliary OASTL over SAT levels is required to convert the accumulating OAS and provide full Cys synthesis capacity (Ruffet et al, 1994, Droux et al, 1998. As cytosolic OASTL and SAT form similar complexes (Bogdanova and Hell, 1997), we consider it most likely that the wheat OASTL in the cys1 transgenic plants could augment the cytosolic tobacco OAST levels and thus provide an increased capacity for Cys biosynthesis.…”

“…For example, transcriptional and posttranscriptional control of several key enzymes are known be regulated by GSH and Cys, which appear to function as negative regulators of sulfur assimilation (Herschbach and Rennenberg, 1994;Bolchi et al, 1999), whereas the availability of OAS, synthesized by Ser acetyl transferase (SAT), is generally regarded as a limiting factor and a positive signal for sulfur assimilation and Cys biosynthesis (Rennenberg, 1984). A further level of control is also provided by the formation of a bi-enzyme complex between OASTL and SAT, in which the properties of the two enzymes are drastically modified, and the stability of which is dependent on the availability of the OASTL substrates, OAS and sulfide (Ruffet et al, 1994;Droux et al, 1998;Leustek and Saito, 1999).…”