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 O ‐acetylserine. 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 ≈300 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. Protein−protein 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 O ‐acetylserine 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.