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Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani

Author:
Singh, Kuljit, Singh, Krishn Pratap, Equbal, Asif, Suman, Shashi S., Zaidi, Amir, Garg, Gaurav, Pandey, Krishna, Das, Pradeep, Ali, Vahab
Source:
Biochimie 2016 v.131 pp. 29-44
ISSN:
0300-9084
Subject:
Leishmania donovani, bioavailability, biosynthesis, cysteine, cysteine synthase, cytoplasm, digitonin, drug resistance, enzyme activity, enzyme kinetics, fluorescence microscopy, fractionation, gel chromatography, gene expression regulation, genes, homeostasis, mammals, pH, parasites, promastigotes, proteins, serine O-acetyltransferase, sulfides
Abstract:
Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a Km of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania.
Agid:
5522442