Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction

Serine acetyltransferase (SAT) catalyzes the rate-limitingstep of cysteine biosynthesis in bacteria and plants andfunctions in association with O-acetylserine (thiol) lyase(OAS-TL) in the cysteine synthase complex. Very little isknown about the structure and catalysis of SATs except thatthey share a characteristic C-terminal hexapeptide-repeatdomain with a number of enzymatically unrelated acyl-transferases. Computational modeling of this domain wasperformed for the mitochondrial SAT isoform fromArabidopsis thaliana, based on crystal structures ofbacterial acyltransferases. The results indicate a left-handedparallel b-helix consisting of b-sheets alternating withturns, resulting in a prism-like structure. This model waschallenged by site-directed mutagenesis and tested for asuspected dual function of this domain in catalysis andhetero-oligomerization. The bifunctionality of the SATC-terminus in transferase activity and interaction withOAS-TL is demonstrated and discussed with respect to theputative role of the cysteine synthase complex in regulationof cysteine biosynthesis.Keywords: amino acid; catalysis; modeling; protein–proteininteraction; structure.Cysteine biosynthesis in plants and bacteria proceeds via atwo-step process which marks the connection betweennitrogen and sulfur assimilation [1–3]. First, serine isactivated by acetyl-CoA to form O-acetylserine (OAS),catalyzed by serine acetyltransferase (SAT). Secondly,O-acetylserine (thiol) lyase (OAS-TL) inserts sulfide intoOAS to yield cysteine. In plants as well as bacteria,OAS-TL activity is much higher than SATactivity, which istherefore considered to be rate-limiting. Whereas mostbacteria carry one gene that encodes SAT (cysE) and twogenes for OAS-TL (cysK, cysM), plants contain severalnuclear genes that encode cytosolic, plastid and mitochon-drial isoforms of SAT and OAS-TL. At least three cDNAsfor each enzyme have been isolated from Arabidopsisthaliana, the most extensively investigated plant withrespect to sulfur metabolism [4,5].SAT activity is exclusively found in association withOAS-TL, forming the cysteine synthase complex, whereasOAS-TL also exists as a free dimer [6–8]. The interactionof SAT and OAS-TL from A. thaliana, spinach andwatermelon has been investigated in vitro and in vivo[5,9–12]. According to these reports, SAT consists of twoprotein-interaction domains, a central SAT–SAT domainfor homomerization and a C-terminal SAT–OAS-TLdomain for heteromerization. On association in thecomplex, SAT is activated to yield a higher V