title: Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.
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 Kmof 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.
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India; Department of Biotechnology, National Institute of Pharmaceutical Education and Research, EPIP Complex, Hajipur, 844102, India.
authors.forename: Kuljit
authors.initials: K
authors.lastname: Singh
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India.
authors.forename: Krishn Pratap
authors.initials: KP
authors.lastname: Singh
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India.
authors.forename: Asif
authors.initials: A
authors.lastname: Equbal
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India.
authors.forename: Shashi S
authors.initials: SS
authors.lastname: Suman
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India.
authors.forename: Amir
authors.initials: A
authors.lastname: Zaidi
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India; Department of Biotechnology, National Institute of Pharmaceutical Education and Research, EPIP Complex, Hajipur, 844102, India.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Garg
authors.affiliation: Department of Clinical Medicine, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India.
authors.forename: Krishna
authors.initials: K
authors.lastname: Pandey
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, 800007, India; Department of Biotechnology, National Institute of Pharmaceutical Education and Research, EPIP Complex, Hajipur, 844102, India. Electronic address: vahab_ali@yahoo.com.
authors.forename: Vahab
authors.initials: V
authors.lastname: Ali
mesh_terms: D053898:Biosynthetic Pathways; D003001:Cloning, Molecular; D003545:Cysteine / Q000096:biosynthesis; D003547:Cysteine Synthase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008018:Life Cycle Stages; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D051050:Serine O-Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D013053:Spectrophotometry; D015854:Up-Regulation
chemical_list: D015800:Protozoan Proteins; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; D003545:Cysteine
keywords: Cysteine synthase; Enzyme kinetics; Leishmania; Serine O-acetyltransferase; Thiol metabolism; Trypanothione