, a widespread and important protozoan pathogen of humans and animals, requires cysteine for protein biosynthesis and as a precursor of trypanothione, a glutathione–spermidine conjugate unique to trypanosomatids with an essential role in redox metabolism and antioxidant defence (Krauth-Siegel & Comini, 2008
). Cysteine is also the source of reduced sulfur for the biosynthesis of important metabolites such as coenzyme A, enzyme cofactors and iron–sulfur clusters (Nozaki et al.
). The vital role of cysteine raises the questions of how Leishmania
obtains the amino acid, how cysteine metabolism in Leishmania
might differ from that in the mammalian host and whether such differences might be targeted in drug-discovery research. L. major
does not have a high-affinity transporter for the uptake of cysteine, but it can acquire methionine and, like the mammalian host, it has the enzymes required to convert methionine to cysteine by transsulfuration (Williams et al.
). The parasite can also produce cysteine from serine in a two-step process (Williams et al.
). Firstly, serine acetyltransferase (SAT) generates O
-acetylserine (OAS) to supply the substrate for the second stage, which is catalyzed by the pyridoxal phosphate (PLP)-dependent cysteine synthase (CS; EC 220.127.116.11). This de novo
pathway for cysteine biosynthesis is found in plants, bacteria and some protozoa, but is absent from mammals. In principle, L. major
CS) may represent a drug target, and an improved understanding of the enzyme might usefully inform on its potential in this respect. In particular, knowledge of the structure can support the development of reagents to chemically validate the target or to provide early-stage information on inhibitors (Hunter, 2009
Some types of CS, including bacterial O
-acetylserine sulfhydrylase type A (OASS-A) and plant O
-acetylserine thiol-lyase (OAS-TL), combine reversibly with SAT to form a bi-enzyme complex in which SAT is active and CS is strongly inhibited (Campanini et al.
). The substrates of CS are effectors of complex formation; the complex is dissociated by elevated levels of OAS but is stabilized by sulfide. The complexes formed in plants and bacteria have distinctive features that indicate different regulatory functions (Salsi, Campanini et al.
; Wirtz et al.
). It has been established that the C-terminal end of SAT is critical for its interaction with CS and, in particular, all SATs possess a C-terminal isoleucine which is essential for CS binding. Peptides corresponding to the C-terminus of SAT bind to the active site of CS and structural data have revealed that the carboxylate group of the C-terminal isoleucine occupies the same space and makes the same interactions as the carboxylate of the α-aminoacrylate catalytic intermediate formed after β-elimination of acetate from the substrate OAS (Rabeh & Cook, 2004
; Huang et al.
; Francois et al.
; Schnell et al.
; Salsi, Bayden et al.
). A four-amino-acid SAT peptide has been shown to be a competitive inhibitor of Mycobacterium tuberculosis
CS with a K
of 5 µM
, providing a simple mechanism for complex formation and its dissociation in the presence of elevated levels of OAS (Schnell et al.
). Sequence alignments indicate that Lm
CS contains a SAT-binding motif that was originally identified in Arabidopsis thaliana
OAS-TL; Bonner et. al.
) and the enzyme can also bind SAT when the proteins are co-expressed in Escherichia coli
(Williams et al.
We undertook a crystallographic and biochemical study of Lm
CS to investigate the interactions of the enzyme with ligands, including potential inhibitors. Our overall aim was to improve understanding of the enzyme in Leishmania
and to provide information that might help to assess the potential of CS as a target for structure-based approaches to develop inhibitors with suitable chemical properties to underpin early-stage drug discovery (Hunter, 2009