Structure-based mutational studies of O-acetylserine sulfhydrylase reveal the reason for the loss of cysteine synthase complex formation in Brucella abortus

2017 ◽  
Vol 474 (7) ◽  
pp. 1221-1239 ◽  
Author(s):  
Sudhaker Dharavath ◽  
Isha Raj ◽  
Samudrala Gourinath
Keyword(s):  
Surface Plasmon Resonance ◽  
Complex Formation ◽  
Surface Plasmon ◽  
Brucella Abortus ◽  
Active Site ◽  
Cysteine Biosynthesis ◽  
Protozoan Parasites ◽  
Serine Acetyltransferase ◽  
Cysteine Synthase ◽  
Active Site Pocket

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 of O-acetylserine sulfydrylase (OASS), playing a role in the regulation of this pathway. Here, we show that OASS from Brucella 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 μM Kd) 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 in B. abortus.

scholarly journals Structure ofLeishmania majorcysteine synthase

2012 ◽  
Vol 68 (7) ◽  
pp. 738-743 ◽  
Author(s):  
Paul K. Fyfe ◽  
Gareth D. Westrop ◽  
Tania Ramos ◽  
Sylke Müller ◽  
Graham H. Coombs ◽  
...  
Keyword(s):  
Active Site ◽  
Pyridoxal Phosphate ◽  
Biochemical Study ◽  
Molecular Replacement ◽  
Cysteine Biosynthesis ◽  
Asymmetric Unit ◽  
Serine Acetyltransferase ◽  
Cysteine Synthase ◽  
Enzyme Active Site ◽  
C Terminus

Cysteine biosynthesis is a potential target for drug development against parasiticLeishmaniaspecies; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect ofLeishmaniabiology, a crystallographic and biochemical study ofL. majorcysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-D-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a D-glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (Ki= 4 µM) by DYVI, a peptide based on the C-terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization.

scholarly journals The Active Site of O-Acetylserine Sulfhydrylase Is the Anchor Point for Bienzyme Complex Formation with Serine Acetyltransferase

2005 ◽  
Vol 187 (9) ◽  
pp. 3201-3205 ◽  
Author(s):  
Bin Huang ◽  
Matthew W. Vetting ◽  
Steven L. Roderick
Keyword(s):  
Crystal Structure ◽  
Complex Formation ◽  
Binding Site ◽  
Active Site ◽  
Binding Pocket ◽  
Anion Binding ◽  
Carboxyl Group ◽  
Serine Acetyltransferase ◽  
Cysteine Synthase

ABSTRACT The biosynthesis of cysteine in bacteria and plants is carried out by a two-step pathway, catalyzed by serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS; O-acetylserine [thiol] lyase). The aerobic form of OASS forms a tight bienzyme complex with SAT in vivo, termed cysteine synthase. We have determined the crystal structure of OASS in complex with a C-terminal peptide of SAT required for bienzyme complex formation. The binding site of the peptide is at the active site of OASS, and its C-terminal carboxyl group occupies the same anion binding pocket as the α-carboxylate of the O-acetylserine substrate of OASS. These results explain the partial inhibition of OASS by SAT on complex formation as well as the competitive dissociation of the complex by O-acetylserine.

Entamoeba histolytica: identification of thioredoxin-targeted proteins and analysis of serine acetyltransferase-1 as a prototype example

2013 ◽  
Vol 451 (2) ◽  
pp. 277-288 ◽  
Author(s):  
Sarah Schlosser ◽  
David Leitsch ◽  
Michael Duchêne
Keyword(s):  
Entamoeba Histolytica ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Biosynthetic Pathway ◽  
Interacting Proteins ◽  
Protozoan Parasites ◽  
Serine Acetyltransferase ◽  
Two Dimensional Gel Electrophoresis ◽  
Mixed Disulfide ◽  
Far Western Blot

Entamoeba histolytica, the causative agent of amoebiasis, possesses the dithiol-containing redox proteins Trx (thioredoxin) and TrxR (Trx reductase). Both proteins were found to be covalently modified and inactivated by metronidazole, a 5-nitroimidazole drug that is commonly used to treat infections with microaerophilic protozoan parasites in humans. Currently, very little is known about enzymes and other proteins participating in the Trx-dependent redox network of the parasite that could be indirectly affected by metronidazole treatment. On the basis of the disulfide/dithiol-exchange mechanism we constructed an active-site mutant of Trx, capable of binding interacting proteins as a stable mixed disulfide intermediate to screen the target proteome of Trx in E. histolytica. By applying Trx affinity chromatography, two-dimensional gel electrophoresis and MS, peroxiredoxin and 15 further potentially redox-regulated proteins were identified. Among them, EhSat1 (E. histolytica serine acetyltransferase-1), an enzyme involved in the L-cysteine biosynthetic pathway, was selected for detailed analysis. Binding of Trx to EhSat1 was verified by Far-Western blot analysis. Trx was able to restore the activity of the oxidatively damaged EhSat1 suggesting that the TrxR/Trx system protects sensitive proteins against oxidative stress in E. histolytica. Furthermore, the activity of peroxiredoxin, which is dependent on a functioning TrxR/Trx system, was strongly reduced in metronidazole-treated parasites.

Thermodynamic and kinetic study of epigallocatechin-3-gallate-bovine lactoferrin complex formation determined by surface plasmon resonance (SPR): A comparative study with fluorescence spectroscopy

2019 ◽  
Vol 95 ◽  
pp. 526-532 ◽  
Author(s):  
Jaqueline de Paula Rezende ◽  
Eliara Acipreste Hudson ◽  
Hauster Maximiler Campos de Paula ◽  
Yara Luiza Coelho ◽  
Luis Henrique Mendes da Silva ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Complex Formation ◽  
Fluorescence Spectroscopy ◽  
Comparative Study ◽  
Kinetic Study ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Bovine Lactoferrin

scholarly journals Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

2004 ◽  
Vol 78 (6) ◽  
pp. 3123-3132 ◽  
Author(s):  
Bradley C. Logsdon ◽  
John F. Vickrey ◽  
Philip Martin ◽  
Gheorghe Proteasa ◽  
Jay I. Koepke ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Surface Plasmon Resonance ◽  
Protease Inhibitors ◽  
Surface Plasmon ◽  
Active Site ◽  
Multidrug Resistant ◽  
Immunodeficiency Virus ◽  
Active Site Cavity ◽  
Hiv 1

ABSTRACT The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (Å) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease “flaps” stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 Å. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1′, S3, and S3′ pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k off rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k on and k off data (Kd = k off/k on) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

Complex Formation of Amphotericin B in Sterol-Containing Membranes As Evidenced by Surface Plasmon Resonance†

Biochemistry ◽  
2008 ◽  
Vol 47 (30) ◽  
pp. 7807-7815 ◽  
Author(s):  
Ryota Mouri ◽  
Keiichi Konoki ◽  
Nobuaki Matsumori ◽  
Tohru Oishi ◽  
Michio Murata
Keyword(s):  
Surface Plasmon Resonance ◽  
Complex Formation ◽  
Amphotericin B ◽  
Surface Plasmon ◽  
Plasmon Resonance

scholarly journals Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action

2019 ◽  
Vol 20 (20) ◽  
pp. 5219
Author(s):  
Brenda Rosa ◽  
Marialaura Marchetti ◽  
Gianluca Paredi ◽  
Heinz Amenitsch ◽  
Nina Franko ◽  
...  
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Solution Structure ◽  
Three Dimensional ◽  
Direct Interaction ◽  
Site Directed Mutagenesis ◽  
Fine Tuning ◽  
Bacterial Cells ◽  
Saxs Data ◽  
Cysteine Synthase

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein–protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS.

scholarly journals Study on complex formation between recombinant human thrombomodulin fragment and thrombin using surface plasmon resonance

2000 ◽  
Vol 63 (3) ◽  
pp. 136-140 ◽  
Author(s):  
Akio Kishida ◽  
Mihoko Nakashima ◽  
Nobuyuki Sakamoto ◽  
Takeshi Serizawa ◽  
Ikuro Maruyama ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Complex Formation ◽  
Surface Plasmon ◽  
Plasmon Resonance
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