title: Structure-based mutational studies ofO-acetylserine sulfhydrylase reveal the reason for the loss of cysteine synthase complex formation inBrucella abortus.
abstract: Cysteine biosynthesis takes place via a two-step pathway in bacteria, fungi, plants and protozoan parasites, but not in humans, and hence, the machinery of cysteine biosynthesis is an opportune target for therapeutics. The decameric cysteine synthase complex (CSC) is formed when the C-terminal tail of serine acetyltransferase (SAT) binds in the active site ofO-acetylserine sulfydrylase (OASS), playing a role in the regulation of this pathway. Here, we show that OASS fromBrucella abortus(BaOASS) does not interact with its cognate SAT C-terminal tail. Crystal structures of native BaOASS showed that residues Gln96 and Tyr125 occupy the active-site pocket and interfere with the entry of the SAT C-terminal tail. The BaOASS (Q96A-Y125A) mutant showed relatively strong binding (Kd= 32.4 μM) to BaSAT C-terminal peptides in comparison with native BaOASS. The mutant structure looks similar except that the active-site pocket has enough space to bind the SAT C-terminal end. Surface plasmon resonance results showed a relatively strong (7.3 μMKd) interaction between BaSAT and the BaOASS (Q96A-Y125A), but no interaction with native BaOASS. Taken together, our observations suggest that the CSC does not form inB. abortus.
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi - 110067, India.
authors.forename: Sudhaker
authors.initials: S
authors.lastname: Dharavath
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi - 110067, India.
authors.forename: Isha
authors.initials: I
authors.lastname: Raj
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi - 110067, India sgourinath@mail.jnu.ac.in.
authors.forename: Samudrala
authors.initials: S
authors.lastname: Gourinath
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002003:Brucella abortus / Q000737:chemistry* / Q000201:enzymology; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000096:biosynthesis*; D003547:Cysteine Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D051050:Serine O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; D003545:Cysteine
keywords: O-acetyl serine sulfydrylase; active site; cysteine synthase complex; protein–protein interactions