30 results on '"Clarissa M. Czekster"'
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2. Catalytic Cycle of the Bifunctional Enzyme Phosphoribosyl-ATP Pyrophosphohydrolase/Phosphoribosyl-AMP Cyclohydrolase
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Gemma Fisher, Ennio Pečaver, Benjamin J. Read, Susannah K. Leese, Erin Laing, Alison L. Dickson, Clarissa M. Czekster, Rafael G. da Silva, University of St Andrews. School of Biology, University of St Andrews. School of Medicine, and University of St Andrews. Biomedical Sciences Research Complex
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Acinetobacter baumannii ,Substrate channeling ,MCC ,Enzyme kinetics ,Kinetic isotope effects ,NDAS ,QD ,Phosphoribosyl-ATP pyrophosphohydrolase ,General Chemistry ,QD Chemistry ,Histidine biosynthesis ,Catalysis ,Phosphoribosyl-AMP cyclohydrolase - Abstract
Funding: This study was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant BB/M010996/1) via EASTBIO Doctoral Training Partnership studentships to G.F. and B.J.R., and by the University of St Andrews via a StARIS summer research bursary to S.K.L. The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N1-(5-phospho-β-D-ribosyl)-ATP (PRATP) to N1-(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N-(5′-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV–VIS spectroscopy and LC–MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall kcat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on kcat and kcat/KM ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N1-(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel. Publisher PDF
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- 2023
3. The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent
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Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Fiona G. McKissock, Peter Mullen, Peijun Tang, Greice M. Zickuhr, Clarissa M. Czekster, David J. Harrison, Innovate UK, University of St Andrews. School of Biology, University of St Andrews. School of Medicine, University of St Andrews. Biomedical Sciences Research Complex, University of St Andrews. Sir James Mackenzie Institute for Early Diagnosis, and University of St Andrews. Cellular Medicine Division
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MCC ,Pharmacology ,Cancer Research ,RC0254 Neoplasms. Tumors. Oncology (including Cancer) ,NDAS ,Toxicology ,Colorectal cancer ,RC0254 ,NUC-3373 ,SDG 3 - Good Health and Well-being ,Oncology ,Thymidylate synthase ,DNA damage ,Pharmacology (medical) ,Fluoropyrimidine - Abstract
Introduction Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. Methods Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC–MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. Results We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. Conclusion Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.
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- 2023
4. The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling
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Januka S Athukoralage, Shirley Graham, Christophe Rouillon, Sabine Grüschow, Clarissa M Czekster, and Malcolm F White
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CRISPR ,Sulfolobus solfataricus ,cyclic oligoadenylate ,ribonuclease ,ring nuclease ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an ‘off-switch’ for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.
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- 2020
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5. Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK
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Pamela Sweeney, Ashleigh Galliford, Abhishek Kumar, Dinesh Raju, Naveen B. Krishna, Emmajay Sutherland, Caitlin J. Leo, Gemma Fisher, Roopa Lalitha, Likith Muthuraj, Gladstone Sigamani, Verena Oehler, Silvia Synowsky, Sally L. Shirran, Tracey M. Gloster, Clarissa M. Czekster, Pravin Kumar, Rafael G. da Silva, The Wellcome Trust, University of St Andrews. School of Biology, University of St Andrews. Institute of Behavioural and Neural Sciences, University of St Andrews. School of Chemistry, and University of St Andrews. Biomedical Sciences Research Complex
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Staphylococcus aureus ,RM ,DAS ,Cell Biology ,S-adenosylmethionine (SAM) ,QR Microbiology ,Biochemistry ,Transfer RNA (tRNA) ,RM Therapeutics. Pharmacology ,QR ,RNA methyltransferase ,TrmK ,Molecular Biology ,X-ray crystallography - Abstract
This work was supported by a Wellcome Trust Seed Award in Science [208980/Z/17/Z] to RGdS; a University of St Andrews/Scottish Funding Council St Andrews Restarting Research Fund to RGdS; and a Wellcome Trust Institutional Strategic Support Fund [204821/Z/16/Z] to the University of St Andrews. ES is the recipient of a Cunningham Trust PhD studentship (PhD-CT-18-41). The enzyme m1A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery. Publisher PDF
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- 2022
6. Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m
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Pamela, Sweeney, Ashleigh, Galliford, Abhishek, Kumar, Dinesh, Raju, Naveen B, Krishna, Emmajay, Sutherland, Caitlin J, Leo, Gemma, Fisher, Roopa, Lalitha, Likith, Muthuraj, Gladstone, Sigamani, Verena, Oehler, Silvia, Synowsky, Sally L, Shirran, Tracey M, Gloster, Clarissa M, Czekster, Pravin, Kumar, and Rafael G, da Silva
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S-Adenosylmethionine ,Staphylococcus aureus ,tRNA Methyltransferases ,Bacterial Proteins ,RNA, Transfer ,Protein Conformation ,Adenine ,Crystallography, X-Ray - Abstract
The enzyme m
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- 2021
7. Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase
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Pamela Sweeney, Ashleigh Crowe, Abhishek Kumar, Dinesh Raju, Naveen B. Krishna, Emmajay Sutherland, Caitlin J. Leo, Gemma Fisher, Roopa Lalitha, Likith Muthuraj, Gladstone Sigamani, Verena Oehler, Silvia Synowsky, Sally L. Shirran, Tracey M. Gloster, Clarissa M. Czekster, Pravin Kumar, and Rafael G. da Silva
- Abstract
The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.
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- 2021
8. Structural-based engineering expands the substrate scope of a cyclodipeptide synthase
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Christopher J. Harding, Emmajay Sutherland, and Clarissa M. Czekster
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chemistry.chemical_classification ,Residue (chemistry) ,Enzyme ,chemistry ,Stereochemistry ,Transfer RNA ,Chemical biology ,Substrate (chemistry) ,Protein engineering ,Histidine ,Amino acid - Abstract
Cyclodipeptide synthases (CDPSs) are a growing family of enzymes capable of producing a large variety of cyclodipetide products using aminoacylated tRNA. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate. Here, we studied the activities of both Para-CDPS from Parabacteroides sp. 20_3 and Parcu-CDPS from Parcubacteria bacterium RAAC4_OD1_1 which synthesise cyclo(His-Phe) and cyclo(His-Pro) respectively. Both enzymes accepted canonical and non-canonical amino acids as substrates to generate a library of novel molecules. In order to understand the substrate selectivity of these CDPSs, the crystal structure of Parcu-CDPS was solved (alongside a number of mutants) and the role of residues important for catalysis and histidine recognition were probed using mutagenesis. Three successive generations of mutants containing both single and double residue substitutions were generated leading to a change in substrate selectivity from histidine to phenylalanine and leucine. The research detailed herein is the first instance of successful engineering of a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.
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- 2021
9. Abstract 1835: NUC-3373 targets the DNA-directed pathway more effectively than 5-FU
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Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Clarissa M. Czekster, and David J. Harrison
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Cancer Research ,Oncology - Abstract
Background: Over the past 60 years, numerous strategies have been employed to enhance the generation of the anticancer metabolite FUDR-MP (FdUMP) by 5-FU and to reduce the generation of toxic metabolites. FUDR-MP, the primary anticancer metabolite of 5-FU, exerts its activity via TS inhibition which results in increased dUMP, depletion of dTMP and DNA damage. 5-FU can also generate FdUTP, which is incorporated into DNA, resulting in DNA damage. 5-FU can also be metabolized to FUTP, which results in the RNA mediated dose-limiting toxicities of myelosuppression and GI tract inflammation. NUC-3373 is a phosphoramidate transformation of FUDR designed specifically to inhibit TS. It generates higher intracellular levels of FUDR-MP, lower levels of toxic metabolites and through its favorable PK profile can be administered via a short infusion. NUC-3373 is a more potent inhibitor of TS than 5-FU and we tested the hypothesis that it more effectively targets DNA than 5-FU. Methods: CRC cells (HCT116 & SW480) were exposed to sub-IC50 doses of NUC-3373 or 5-FU for 6 or 24h. Cells were harvested and analyzed as follows: metabolite levels and incorporation of FdUTP into DNA and FUTP into RNA (using FdUr and FUr as surrogates) by LC-MS and LC-MS/MS. Gene expression of dUTPase was knocked down by siRNA and protein assessed by western blot. Cytotoxicity of NUC-3373 and 5-FU was determined by IC50, measured with sulforhodamine B. Results: NUC-3373 generated significantly higher levels of FUDR-MP compared to 5-FU (AUC >45x greater) resulting in a pronounced increase in dUMP levels (AUC >162x compared to 5-FU). NUC-3373 treated cells incorporated FdUTP into DNA, while incorporation by 5-FU treated cells was below the LLOQ (0.1 nM). NUC-3373 was effective in the presence of dUTPase, but its potency increased after dUTPase knockdown demonstrating that FdUTP incorporation contributes to NUC-3373’s cytotoxicity. At equimolar doses, 42x more FUTP was incorporated into RNA in 5-FU treated cells compared to NUC-3373. Conclusion: NUC-3373 generates higher intracellular levels of the anticancer metabolite FUDR-MP and is a more potent inhibitor of TS than 5-FU resulting in markedly greater accumulation of intracellular dUMP. Additionally, in contrast to 5-FU, NUC-3373 treatment results in incorporation of FdUTP into DNA which indicates that NUC-3373 is a more efficient DNA damaging agent. Through bypassing the FUTP-generation pathway, NUC-3373 avoids the RNA damage that is known to be associated with dose-limiting toxicities such as myelosuppression and GI tract inflammation. This is consistent with the very low rates of FUTP-related toxicities observed in patients treated with NUC-3373 (NCT03428958). By exploiting a more DNA-directed approach, NUC-3373 provides a potentially more effective, safer and convenient therapeutic option than 5-FU for patients with cancer. Citation Format: Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Clarissa M. Czekster, David J. Harrison. NUC-3373 targets the DNA-directed pathway more effectively than 5-FU [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1835.
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- 2022
10. Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme
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Douglas R. Houston, Clarissa M. Czekster, Emmajay Sutherland, Christopher J. Harding, Jane G. Hanna, Cunningham Trust, University of St Andrews. School of Biology, and University of St Andrews. Biomedical Sciences Research Complex
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0301 basic medicine ,QH301 Biology ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,QH301 ,Molecular Biology ,chemistry.chemical_classification ,Aminoacyl-tRNA ,Dipeptide ,030102 biochemistry & molecular biology ,ATP synthase ,biology ,Amino esters ,Substrate (chemistry) ,DAS ,Combinatorial chemistry ,Small molecule ,Chemistry ,030104 developmental biology ,Enzyme ,chemistry ,Chemistry (miscellaneous) ,Transfer RNA ,biology.protein - Abstract
Cyclodipeptide synthases (CDPSs) produce a variety of cyclic dipeptide products by utilising two aminoacylated tRNA substrates. We sought to investigate the minimal requirements for substrate usage in this class of enzymes as the relationship between CDPSs and their substrates remains elusive. Here, we investigated the Bacillus thermoamylovorans enzyme, BtCDPS, which synthesises cyclo(l-Leu–l-Leu). We systematically tested where specificity arises and, in the process, uncovered small molecules (activated amino esters) that will suffice as substrates, although catalytically poor. We solved the structure of BtCDPS to 1.7 Å and combining crystallography, enzymatic assays and substrate docking experiments propose a model for how the minimal substrates interact with the enzyme. This work is the first report of a CDPS enzyme utilizing a molecule other than aa-tRNA as a substrate; providing insights into substrate requirements and setting the stage for the design of improved simpler substrates., Cyclodipeptide synthases recognize a minimalistic substrate to produce cyclic dipeptides in a tRNA-independent manner.
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- 2021
11. The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling
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Clarissa M. Czekster, Shirley Graham, Malcolm F. White, Januka S Athukoralage, Sabine Grüschow, Christophe Rouillon, BBSRC, University of St Andrews. School of Biology, and University of St Andrews. Biomedical Sciences Research Complex
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QH301 Biology ,ved/biology.organism_classification_rank.species ,chemistry.chemical_compound ,0302 clinical medicine ,CRISPR ,Biology (General) ,R2C ,0303 health sciences ,biology ,Chemistry ,General Neuroscience ,Sulfolobus solfataricus ,General Medicine ,Cell biology ,Viruses ,Second messenger system ,Medicine ,Nucleotides, Cyclic ,ribonuclease ,BDC ,Signal Transduction ,QH301-705.5 ,Science ,Neuroscience(all) ,General Biochemistry, Genetics and Molecular Biology ,QH301 ,03 medical and health sciences ,Cyclic nucleotide ,Biochemistry and Chemical Biology ,Immunology and Microbiology(all) ,Escherichia coli ,Ribonuclease ,030304 developmental biology ,Nuclease ,Host Microbial Interactions ,General Immunology and Microbiology ,Biochemistry, Genetics and Molecular Biology(all) ,ved/biology ,E. coli ,RNA ,DAS ,cyclic oligoadenylate ,biology.protein ,CRISPR-Cas Systems ,ring nuclease ,Research Advance ,Cyclase activity ,030217 neurology & neurosurgery - Abstract
This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (Grant REF BB/S000313/1 to MFW) and the Wellcome Trust (Grant 210486/Z/18/Z to CMC). Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an ‘off-switch’ for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict. Publisher PDF
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- 2020
12. Author response: The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling
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Clarissa M. Czekster, Sabine Grüschow, Christophe Rouillon, Shirley Graham, Januka S Athukoralage, and Malcolm F. White
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chemistry.chemical_classification ,Cyclic nucleotide ,chemistry.chemical_compound ,Signalling ,Enzyme ,chemistry ,Host (biology) ,CRISPR ,Biology ,Cell biology - Published
- 2020
13. Abstract P025: NUC-3373 is a more potent inhibitor of thymidylate synthase than 5-FU and reduces generation of toxic metabolites
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Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Peter Mullen, Clarissa M. Czekster, and David J. Harrison
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Cancer Research ,Oncology - Abstract
Background NUC-3373 is a novel anti-cancer agent designed to replace 5-FU, one of the most widely used therapies across a broad range of tumors, including colorectal cancer (CRC). 5-FU exerts its main anti-cancer activity via the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds and inhibits thymidylate synthase (TS), preventing the conversion of dUMP into dTMP and disrupting DNA synthesis and repair. NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, and reduce generation of toxic metabolites including FUTP, which is incorporated into RNA causing myelosuppression and gastrointestinal toxicity, and FBAL which causes hand-foot syndrome. Thymidine supplementation rescues cells from NUC-3373 induced cytotoxicity, supporting a DNA targeted mode of action. The aim of this study was to further evaluate the differences between NUC-3373 and 5-FU. Methods CRC cell lines HCT116 and SW480 were exposed to sub-IC50 doses of NUC-3373 or 5-FU (0.1-25 μM) for 6h. At specific time-points, cells were harvested and analyzed as follows: TS inhibition by western blot, metabolite levels by mass spectrometry (LC-MS & LC-MS/MS) and cell cycle by flow cytometry. In rescue experiments, NUC-3373 and 5-FU were supplemented with thymidine (8 μg/mL) for 24h and cell survival assessed at 96h post-treatment. Results In both cell lines, NUC-3373 was a more potent inhibitor of TS than 5-FU with a higher proportion of TS protein bound to FUDR-MP at low concentrations. At 24h, 10 µM 5-FU was required to achieve the same level of TS binding as 0.1 µM NUC-3373 in HTC116 cells and as 0.5 µM NUC-3373 in SW480 cells. TS inhibition by NUC-3373 was almost maximal by 6 hours and was sustained for at least 48 hours. NUC-3373 generated significantly higher levels (50x) of free FUDR-MP compared to 5-FU, resulting in a more pronounced increase in dUMP levels (5x compared to 5-FU). At 48h, NUC-3373 treated cells remained arrested in S phase, while 5-FU treated cells had reverted to a normal cell cycle. FUTP was present in cells exposed to low doses of 5-FU (0.5 μM) but was not detectable in NUC-3373 treated cells. Finally, thymidine supplementation did not alter cell sensitivity to 5-FU but rescued cells treated with NUC-3373. Conclusion NUC-3373 generates higher intracellular levels of FUDR-MP and is a more potent inhibitor of TS than 5-FU, leading to more pronounced effects on cell cycle arrest and perturbation of the nucleotide pool that can result in misincorporation of uracil into DNA. Furthermore, NUC-3373 did not generate FUTP, consistent with the observation that patients treated with NUC-3373 in clinical studies have experienced much lower rates of FUTP-related toxicities. NUC-3373 is a potent TS inhibitor with a favorable safety profile. Citation Format: Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Peter Mullen, Clarissa M. Czekster, David J. Harrison. NUC-3373 is a more potent inhibitor of thymidylate synthase than 5-FU and reduces generation of toxic metabolites [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P025.
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- 2021
14. Correction: Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme
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Emmajay Sutherland, Christopher J. Harding, Douglas R. Houston, Jane G. Hanna, and Clarissa M. Czekster
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chemistry.chemical_classification ,Aminoacyl-tRNA ,biology ,ATP synthase ,Chemistry ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,chemistry.chemical_compound ,Enzyme ,Chemistry (miscellaneous) ,biology.protein ,Chromatin structure remodeling (RSC) complex ,Molecular Biology - Abstract
Correction for ‘Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme’ by Christopher J. Harding et al., RSC Chem. Biol., 2021, 2, 230–240, DOI: 10.1039/D0CB00142B.
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- 2021
15. Abstract 1003: NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells
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Jennifer Bré, Oliver J. Read, Clarissa M. Czekster, David J. Harrison, Essam Ghazaly, Alison L. Dickson, and Dillum Patel
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Cancer Research ,chemistry.chemical_compound ,Biliary tract cancer ,Oncology ,Chemistry ,Cancer research ,Deoxycytidine ,DNA - Abstract
Background: NUC-1031 is a ProTide transformation of the nucleoside analog gemcitabine, designed to overcome key cancer resistance mechanisms and generate significantly higher levels of the active metabolite, dFdCTP. Its incorporation into DNA interrupts cell replication and induces DNA damage, leading to cell death. NUC-1031 has shown broad clinical activity across a range of solid tumors as both a single agent and in combination with platinum agents. Increasing evidence in the clinical setting in both ovarian and biliary tract cancer populations suggests potential synergy between NUC-1031 and platinum agents, which may lead to increased anti-tumor activity. The aim of this study was to investigate the dose and time relationships between the incorporation of dFdCTP into DNA, the effect on the cell cycle and DNA damage response in biliary tract cancer cells treated with NUC-1031. Methods: Human intrahepatic cholangiocarcinoma HuCCT1 cells were treated with 500 nM (half-IC50 dose) or 1 µM (IC50 dose) NUC-1031 for 24h and samples were collected every 24h up to 96h. The intracellular conversion of NUC-1031 to its active metabolite dFdCTP was assessed by liquid chromatography mass spectrometry (LC-Q-TOF). This technique was also used to determine the incorporation of dFdCTP into DNA, using the dFdC signature as a surrogate and deoxyguanosine (2dG) for normalization. Flow cytometry was used to measure cell cycle populations and to analyse histone variant H2AX phosphorylation (γH2AX) as a marker for response to DNA damage, where increased signal is related to increase in response. Results: NUC-1031 generated dFdCTP in HuCCT1 cells. The incorporation of this fluorinated deoxycytidine into DNA was dose-dependent over time. At 48h post-treatment, the ratio of dFdC:2dG was 0.70 at half-IC50 dose, with comparable levels at IC50 dose. After 48h, the ratio continued to rise for the 1 µM dose but decreased with 500 nM. The incorporation of the active metabolite was accompanied by an increase in cells in S phase, up to 46% for 500 nM and 64% for 1 µM. This dose-dependent increase coincides with H2AX phosphorylation over time up to 48h. Active metabolite incorporation, S phase population and yH2AX signals reduced towards the 96h time point. Conclusion: NUC-1031 is a potent cytotoxic agent that causes DNA damage through the incorporation of its active metabolite into DNA, in a cell cycle dependant manner. This incorporation induces S phase arrest and evokes the DNA damage response via generation of double-strand breaks. The cytotoxic effect of NUC-1031 is prolonged post-treatment. Ongoing studies are investigating NUC-1031 in combination with cisplatin, where relative levels of dFdCTP incorporated in tumor DNA may act as a pharmacodynamic biomarker to determine synergy in patients who receive the combination treatment. Citation Format: Dillum Patel, Alison L. Dickson, Oliver J. Read, Clarissa M. Czekster, Essam A. Ghazaly, David J. Harrison, Jennifer Bré. NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1003.
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- 2021
16. The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family
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James H. Naismith, Clark L. Grieve, Indu Murugathasan, Cassandra S. Carroll, Margo M. Moore, Andrew J. Bennet, Clarissa M. Czekster, Huanting Liu, University of St Andrews. School of Chemistry, University of St Andrews. Biomedical Sciences Research Complex, and University of St Andrews. EaSTCHEM
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0301 basic medicine ,Mucorales ,Siderophore ,QH301 Biology ,Iron ,030106 microbiology ,NDAS ,Siderophores ,Virulence ,Ferric Compounds ,Biochemistry ,Microbiology ,Fungal Proteins ,QH301 ,03 medical and health sciences ,chemistry.chemical_compound ,Biosynthesis ,Siderophore biosynthesis ,NRPS-independent siderophore (NIS) ,Gene Expression Regulation, Fungal ,Mucormycosis ,Gene family ,QD ,Rhizopus delemar ,Gene ,chemistry.chemical_classification ,biology ,Computational Biology ,Cell Biology ,QD Chemistry ,biology.organism_classification ,Kinetics ,030104 developmental biology ,Enzyme ,chemistry ,Mutagenesis, Site-Directed ,Rhizopus ,Bacteria - Abstract
This work was supported by the Natural Sciences and Engineering Research Council of Canada award to MM (grant number 611181). C. Carroll thanks Simon Fraser University for a travel and research award. Iron is essential for growth and in low iron environments such as serum many bacteria and fungi secrete ferric iron-chelating molecules called siderophores. All fungi produce hydroxamate siderophores with the exception of Mucorales fungi, which secrete rhizoferrin, a polycarboxylate siderophore. Here we investigated the biosynthesis of rhizoferrin by the opportunistic human pathogen, Rhizopus delemar. We searched the genome of R. delemar 99–880 for a homologue of the bacterial NRPS-independent siderophore (NIS) protein, SfnaD that is involved in biosynthesis of staphyloferrin A in Staphylococcus aureus. A protein was identified in R. delemar with 22% identity and 37% similarity with SfnaD, containing an N-terminal IucA/IucC family domain, and a C-terminal conserved ferric iron reductase FhuF-like transporter domain. Expression of the putative fungal rhizoferrin synthetase (rfs) gene was repressed by iron. The rfs gene was cloned and expressed in E.coli and siderophore biosynthesis from citrate and diaminobutane was confirmed using high resolution LC–MS. Substrate specificity was investigated showing that Rfs produced AMP when oxaloacetic acid, tricarballylic acid, ornithine, hydroxylamine, diaminopentane and diaminopropane were employed as substrates. Based on the production of AMP and the presence of a mono-substituted rhizoferrin, we suggest that Rfs is a member of the superfamily of adenylating enzymes. We used site-directed mutagenesis to mutate selected conserved residues predicted to be in the Rfs active site. These studies revealed that H484 is essential for Rfs activity and L544 may play a role in amine recognition by the enzyme. This study on Rfs is the first characterization of a fungal NIS enzyme. Future work will determine if rhizoferrin biosynthesis is required for virulence in Mucorales fungi. Postprint
- Published
- 2017
17. Insights into the Mechanism of the Cyanobactin Heterocyclase Enzyme
- Author
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Ying Ge, Clarissa M. Czekster, James H. Naismith, Catherine H. Botting, Ulrich Schwarz-Linek, Ona K Miller, BBSRC, University of St Andrews. School of Biology, University of St Andrews. Biomedical Sciences Research Complex, University of St Andrews. EaSTCHEM, and University of St Andrews. School of Chemistry
- Subjects
Signal peptide ,Threonine ,Stereochemistry ,Peptide ,Biochemistry ,Pyrophosphate ,Peptides, Cyclic ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,Bacterial Proteins ,Animals ,Nucleotide ,QD ,Amino Acid Sequence ,Cysteine ,Urochordata ,Peptide sequence ,chemistry.chemical_classification ,0303 health sciences ,Molecular Structure ,Chemistry ,030302 biochemistry & molecular biology ,Substrate (chemistry) ,DAS ,QD Chemistry ,Adenosine Monophosphate ,Adenosine Diphosphate ,Diphosphates ,Enzyme ,Models, Chemical ,Cyclization ,Prochloron ,Adenylyl Cyclases - Abstract
The work is supported by the European Research Council NCB-TNT (339367), Biotechnology and Biological Sciences Research Council (BB/K015508/1 and BB/M001679/1). Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the order of reaction for threonines is different and depends in part on a leader peptide within the substrate. In contrast to other YcaO domains, which have been reported to exclusively break down ATP into ADP and inorganic phosphate, cyanobactin heterocyclases have been observed to produce AMP and inorganic pyrophosphate during catalysis. We dissect the nucleotide profiles associated with heterocyclization and propose a unifying mechanism, where the γ-phosphate of ATP is transferred in a kinase mechanism to the substrate to yield a phosphorylated intermediate common to all YcaO domains. In cyanobactin heterocyclases, this phosphorylated intermediate, in a proportion of turnovers, reacts with ADP to yield AMP and pyrophosphate. Publisher PDF
- Published
- 2019
18. Allosteric Activation Shifts the Rate-Limiting Step in a Short-Form ATP Phosphoribosyltransferase
- Author
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Gemma, Fisher, Catherine M, Thomson, Rozanne, Stroek, Clarissa M, Czekster, Jennifer S, Hirschi, and Rafael G, da Silva
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Models, Molecular ,Binding Sites ,Protein Conformation ,Moraxellaceae Infections ,Psychrobacter ,Phosphoribosyl Pyrophosphate ,ATP Phosphoribosyltransferase ,Adenosine Monophosphate ,Article ,Substrate Specificity ,Adenosine Diphosphate ,Kinetics ,Allosteric Regulation ,Catalytic Domain ,Protein Multimerization - Abstract
Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg2+-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisGS is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS’s catalytic rate, but decreases the hetero-octamer’s, indicating that chemistry and product release are rate-limiting for HisGS and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.
- Published
- 2018
19. Characterization of the fast and promiscuous macrocyclase from plant PCY1 enables the use of simple substrates
- Author
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Andrew F. Bent, Clarissa M. Czekster, Mohammed Arshad, Hannes Ludewig, Elizabeth S. Munday, James H. Naismith, Silvia Anna Synowsky, Emilia Oueis, BBSRC, European Research Council, University of St Andrews. School of Chemistry, University of St Andrews. School of Biology, University of St Andrews. EaSTCHEM, and University of St Andrews. Biomedical Sciences Research Complex
- Subjects
0301 basic medicine ,Macrocyclic Compounds ,QH301 Biology ,Peptide ,Peptides, Cyclic ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,Residue (chemistry) ,chemistry.chemical_compound ,QH301 ,Biosynthesis ,Drug Discovery ,Hydrolase ,QD ,chemistry.chemical_classification ,Serine Endopeptidases ,Substrate (chemistry) ,DAS ,Articles ,General Medicine ,Plants ,QD Chemistry ,Combinatorial chemistry ,Cyclic peptide ,030104 developmental biology ,Enzyme ,chemistry ,Drug development ,Biocatalysis ,Molecular Medicine ,Prolyl Oligopeptidases - Abstract
H.L. is funded by the George and Stella Lee Scholarship and EPSRC. This project was funded by the European Research Council project 339367 NCB-TNT and by the BBSRC (J.H.N.). E.S.M. and M.A. are funded by EPSRC. S.A.S. is funded by BSRC mass spec facility. Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1’s potential in biocatalysis. Postprint
- Published
- 2018
20. CHAPTER 2. The Biosynthesis of Cyclic Peptides – RiPPs – An Overview
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Clarissa M. Czekster and James H. Naismith
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chemistry.chemical_classification ,Molecular complexity ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biosynthesis ,Biochemistry ,Protease resistant ,Peptide synthesis ,food and beverages ,Peptide ,Small molecule ,Cyclic peptide - Abstract
Cyclic peptides are of considerable interest because they possess protease resistant, rigid scaffolds that can be almost infinitely diversified. Their size and molecular complexity means that they are able to target protein–protein interactions, a task that current small molecule drugs struggle to achieve. Macrocyclic peptides can be synthesized using non-ribosomal peptide synthesis machineries – NRPS for short (see next chapter) – or through extensive modification of ribosomally synthesized peptide precursors (ribosomally synthesized and post-translationally modified peptides – RiPPs). RiPPs are attractive because they are genetically encoded and can be easily diversified. The same peptide precursors can be utilized to generate a wide array of natural products by “mixing-and-matching” enzymes involved in their post-translational modification. Here, we discuss the biosynthetic machineries producing the main classes of cyclic RiPPs.
- Published
- 2017
21. Protein Mass-Modulated Effects in the Catalytic Mechanism of Dihydrofolate Reductase: Beyond Promoting Vibrations
- Author
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Zhen Wang, Amnon Kohen, Vern L. Schramm, Clarissa M. Czekster, Priyanka Singh, University of St Andrews. School of Biology, and University of St Andrews. Biomedical Sciences Research Complex
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Models, Molecular ,Protein Conformation ,Kinetics ,Ligands ,Biochemistry ,Vibration ,Article ,Catalysis ,Enzyme catalysis ,Colloid and Surface Chemistry ,Kinetic isotope effect ,Dihydrofolate reductase ,Escherichia coli ,QD ,Conformational ensembles ,R2C ,Protein Unfolding ,chemistry.chemical_classification ,Carbon Isotopes ,biology ,Nitrogen Isotopes ,Chemistry ,Protein dynamics ,Temperature ,General Chemistry ,QD Chemistry ,Molecular Weight ,Tetrahydrofolate Dehydrogenase ,Enzyme ,Models, Chemical ,biology.protein ,Biophysics ,BDC ,Hydrogen ,Protein Binding - Abstract
This work was supported by NIH research grants GM068036 (V.L.S.) and GM65368 (A.K.), and NSF grant CHE-0133117 (A.K.). The role of fast protein dynamics in enzyme catalysis has been of great interest in the past decade. Recent “heavy enzyme” studies demonstrate that protein mass-modulated vibrations are linked to the energy barrier for the chemical step of catalyzed reactions. However, the role of fast dynamics in the overall catalytic mechanism of an enzyme has not been addressed. Protein mass-modulated effects in the catalytic mechanism of Escherichia coli dihydrofolate reductase (ecDHFR) are explored by isotopic substitution (13C, 15N, and non-exchangeable 2H) of the wild-type ecDHFR (l-DHFR) to generate a vibrationally perturbed “heavy ecDHFR” (h-DHFR). Steady-state, pre-steady-state, and ligand binding kinetics, intrinsic kinetic isotope effects (KIEint) on the chemical step, and thermal unfolding experiments of both l- and h-DHFR show that the altered protein mass affects the conformational ensembles and protein–ligand interactions, but does not affect the hydride transfer at physiological temperatures (25–45 °C). Below 25 °C, h-DHFR shows altered transition state (TS) structure and increased barrier-crossing probability of the chemical step compared with l-DHFR, indicating temperature-dependent protein vibrational coupling to the chemical step. Protein mass-modulated vibrations in ecDHFR are involved in TS interactions at cold temperatures and are linked to dynamic motions involved in ligand binding at physiological temperatures. Thus, mass effects can affect enzymatic catalysis beyond alterations in promoting vibrations linked to chemistry. Publisher PDF
- Published
- 2014
22. One Substrate, Five Products: Reactions Catalyzed by the Dihydroneopterin Aldolase from Mycobacterium tuberculosis
- Author
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Clarissa M. Czekster and John S. Blanchard
- Subjects
Oxygenase ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Fructose-bisphosphate aldolase ,Dihydroneopterin aldolase ,Biochemistry ,Article ,Catalysis ,Cofactor ,Substrate Specificity ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Escherichia coli ,Cloning, Molecular ,Pterin ,Purine metabolism ,Chromatography, High Pressure Liquid ,Aldehyde-Lyases ,chemistry.chemical_classification ,Molecular Structure ,biology ,Chemistry ,Aldolase A ,Mycobacterium tuberculosis ,General Chemistry ,Hydrogen-Ion Concentration ,Kinetics ,Enzyme ,biology.protein ,Protein Binding - Abstract
Tetrahydrofolate cofactors are required for one carbon transfer reaction involved in the synthesis of purines, amino acids, and thymidine. Inhibition of tetrahydrofolate biosynthesis is a powerful therapeutic strategy in the treatment of several diseases, and the possibility of using antifolates to inhibit enzymes from Mycobacterium tuberculosis has been explored. This work focuses on the study of the first enzyme in tetrahydrofolate biosynthesis that is unique to bacteria, dihydroneopterin aldolase (MtDHNA). This enzyme requires no metals or cofactors and does not form a protein-mediated Schiff base with the substrate, unlike most aldolases. Here, we were able to demonstrate that the reaction catalyzed by MtDHNA generates three different pterin products, one of which is not produced by other wild-type DHNAs. The enzyme-substrate complex partitions 51% in the first turnover to form the aldolase products, 24% to the epimerase product and 25% to the oxygenase products. The aldolase reaction is strongly pH dependent, and apparent pK(a) values were obtained for the first time for this class of enzyme. Furthermore, chemistry is rate limiting for the aldolase reaction, and the analysis of solvent kinetic isotope effects in steady-state and pre-steady-state conditions, combined with proton inventory studies, revealed that two protons and a likely solvent contribution are involved in formation and breakage of a common intermediate. This study provides information about the plasticity required from a catalyst that possesses high substrate specificity while being capable of utilizing two distinct epimers with the same efficiency to generate five distinct products.
- Published
- 2012
23. Mechanisms of cyanobactin biosynthesis
- Author
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James H. Naismith, Clarissa M. Czekster, and Ying Ge
- Subjects
chemistry.chemical_classification ,Biological Products ,010405 organic chemistry ,Stereochemistry ,Protein engineering ,010402 general chemistry ,Protein Engineering ,01 natural sciences ,Biochemistry ,Peptides, Cyclic ,Article ,0104 chemical sciences ,Analytical Chemistry ,Amino acid ,Serine ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biosynthesis ,Prenylation ,Peptide bond ,Cysteine - Abstract
Cyanobactins are a diverse collection of natural products that originate from short peptides made on a ribosome. The amino acids are modified in a series of transformations catalyzed by multiple enzymes. The patellamide pathway is the most well studied and characterized example. Here we review the structures and mechanisms of the enzymes that cleave peptide bonds, macrocyclise peptides, heterocyclise cysteine (as well as threonine and serine) residues, oxidize five-membered heterocycles and attach prenyl groups. Some enzymes operate by novel mechanisms which is of interest and in addition the enzymes uncouple recognition from catalysis. The normally tight relationship between these factors hinders biotechnology. The cyanobactin pathway may be particularly suitable for exploitation, with progress observed with in vivo and in vitro approaches.
- Published
- 2016
24. In Vivo Biosynthesis of a β-Amino Acid-Containing Protein
- Author
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Alanna Schepartz, Clarissa M. Czekster, Allison S. Walker, Wesley E. Robertson, and Dieter Söll
- Subjects
0301 basic medicine ,Phenylalanine ,Mutant ,Molecular Dynamics Simulation ,Peptide Elongation Factor Tu ,medicine.disease_cause ,Protein Engineering ,Biochemistry ,Ribosome ,Catalysis ,Article ,Substrate Specificity ,Amino Acyl-tRNA Synthetases ,03 medical and health sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Biosynthesis ,medicine ,Escherichia coli ,chemistry.chemical_classification ,Chemistry ,Escherichia coli Proteins ,Translation (biology) ,General Chemistry ,Protein engineering ,Amino acid ,RNA, Ribosomal, 23S ,Tetrahydrofolate Dehydrogenase ,030104 developmental biology ,Transfer RNA ,Mutation ,Phenylalanine-tRNA Ligase - Abstract
It has recently been reported that ribosomes from erythromycin-resistant Escherichia coli strains, when isolated in S30 extracts and incubated with chemically mis-acylated tRNA, can incorporate certain β-amino acids into full length DHFR in vitro. Here we report that wild-type E. coli EF-Tu and phenylalanyl-tRNA synthetase collaborate with these mutant ribosomes and others to incorporate β(3)-Phe analogs into full length DHFR in vivo. E. coli harboring the most active mutant ribosomes are robust, with a doubling time only 14% longer than wild-type. These results reveal the unexpected tolerance of E. coli and its translation machinery to the β(3)-amino acid backbone and should embolden in vivo selections for orthogonal translational machinery components that incorporate diverse β-amino acids into proteins and peptides. E. coli harboring mutant ribosomes may possess the capacity to incorporate many non-natural, non-α-amino acids into proteins and other sequence-programmed polymeric materials.
- Published
- 2016
25. Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding
- Author
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Matthew Levy, Maria de Lourdes Borba Magalhães, Steven C. Almo, Vladimir N. Malashkevich, Clarissa M. Czekster, and Rong Guan
- Subjects
Streptavidin ,Chemistry ,Mutant ,Plasma protein binding ,Ligand (biochemistry) ,Biochemistry ,In vitro compartmentalization ,chemistry.chemical_compound ,Protein structure ,Biophysics ,Binding site ,Surface plasmon resonance ,Molecular Biology - Abstract
We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ∼10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.
- Published
- 2011
26. The catalytic mechanism of indole-3-glycerol phosphate synthase (IGPS) investigated by electrospray ionization (tandem) mass spectrometry
- Author
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Brenno A. D. Neto, Luiz Augusto Basso, Diógenes Santiago Santos, Jairton Dupont, Marcos N. Eberlin, Clarissa M. Czekster, Gustavo H.M.F. Souza, and Alexandre A. M. Lapis
- Subjects
chemistry.chemical_classification ,Chromatography ,ATP synthase ,biology ,Electrospray ionization ,Organic Chemistry ,Tryptophan ,Indole-3-Glycerol-Phosphate Synthase ,Tandem mass spectrometry ,Phosphate ,Mass spectrometry ,Biochemistry ,chemistry.chemical_compound ,Enzyme ,chemistry ,Drug Discovery ,biology.protein - Abstract
An enzymatic reaction has been monitored by on-line direct infusion electrospray ionization (tandem) mass spectrometry. Using this fast and sensitive technique, a key and transient intermediate of Mycobacterium tuberculosis indole-3-glycerol phosphate synthase (IGPS)-catalyzed reaction has been trapped. The reaction catalyzed by indole-3-glycerol phosphate synthase is part of the tryptophan biosynthetic pathway, and is not present in mammals, including humans. This peculiarity renders this enzyme a potential target for the development of biospecific agents with potential anti-TB activity. The present results indicate the presence of two intermediates in the mechanism of this enzymatic reaction.
- Published
- 2008
27. Molecular Models of Tryptophan Synthase From Mycobacterium tuberculosis Complexed With Inhibitors
- Author
-
Luis Augusto Basso, Nelson José Freitas da Silveira, Walter Filgueira de Azevedo, Clarissa M. Czekster, Fernanda Canduri, Diógenes Santiago Santos, Marcio Vinicius Bertacine Dias, and Mario Sergio Palma
- Subjects
Models, Molecular ,Tuberculosis ,Molecular model ,Protein Conformation ,Pharmacology toxicology ,Biophysics ,Tryptophan synthase ,Fagaceae ,Ligands ,Biochemistry ,Substrate Specificity ,Mycobacterium tuberculosis ,Tryptophan Synthase ,medicine ,Computer Simulation ,Rosaceae ,Plant Proteins ,Indole test ,chemistry.chemical_classification ,Binding Sites ,Molecular Structure ,biology ,Tryptophan ,Hydrogen Bonding ,Cell Biology ,General Medicine ,biology.organism_classification ,medicine.disease ,Enzyme ,chemistry ,Drug Design ,biology.protein ,Sequence Alignment ,Protein Binding - Abstract
The development of new therapies against infectious diseases is vital in developing countries. Among infectious diseases, tuberculosis is considered the leading cause of death. A target for development of new drugs is the tryptophan pathway. The last enzyme of this pathway, tryptophan synthase (TRPS), is responsible for conversion of the indole 3-glycerol phosphate into indol and the condensation of this molecule with serine-producing tryptophan. The present work describes the molecular models of TRPS from Mycobacterium tuberculosis (MtTRPS) complexed with six inhibitors, the indole 3-propanol phosphate and five arylthioalkyl-phosphonated analogs of substrate of the alpha-subunit. The molecular models of MtTRPS present good stereochemistry, and the binding of the inhibitors is favorable. Thus, the generated models can be used in the design of more specific drugs against tuberculosis and other infectious diseases.
- Published
- 2006
28. Two parallel pathways in the kinetic sequence of the dihydrofolate reductase from Mycobacterium tuberculosis
- Author
-
An Vandemeulebroucke, Clarissa M. Czekster, and John S. Blanchard
- Subjects
chemistry.chemical_classification ,biology ,Stereochemistry ,Mycobacterium tuberculosis ,Biochemistry ,Markov Chains ,Recombinant Proteins ,Article ,Turnover number ,Kinetics ,Tetrahydrofolate Dehydrogenase ,Reaction rate constant ,Enzyme ,chemistry ,Catalytic cycle ,Dihydrofolate reductase ,biology.protein ,Biocatalysis ,Escherichia coli ,Steady state (chemistry) ,NAD+ kinase ,Purine metabolism ,Protein Binding - Abstract
Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)H-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism can be maintained. Previously, steady-state studies revealed that the chemical step significantly contributes to the steady-state turnover number, but that a step after the chemical step was likely limiting the reaction rate. Here, we report the first pre-steady-state investigation of the kinetic sequence of the MtDHFR aiming to identify kinetic intermediates, and the identity of the rate-limiting steps. This kinetic analysis suggests a kinetic sequence comprising two parallel pathways with a rate-determining product release. Although product release is likely occurring in a random fashion, there is a slight preference for the release of THF first, a kinetic sequence never observed for a wild-type dihydrofolate reductase of any organism studied to date. Temperature studies were conducted to determine the magnitude of the energetic barrier posed by the chemical step, and the pH dependence of the chemical step was studied, demonstrating an acidic shift from the pK(a) observed at the steady state. The rate constants obtained here were combined with the activation energy for the chemical step to compare energy profiles for each kinetic sequence. The two parallel pathways are discussed, as well as their implications for the catalytic cycle of this enzyme.
- Published
- 2011
29. Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis
- Author
-
John S. Blanchard, Clarissa M. Czekster, and An Vandemeulebroucke
- Subjects
biology ,Stereochemistry ,Chemistry ,Hydride ,Protonation ,Mycobacterium tuberculosis ,Hydrogen-Ion Concentration ,Deuterium ,Biochemistry ,Article ,Enzyme catalysis ,Kinetics ,Tetrahydrofolate Dehydrogenase ,Models, Chemical ,Dihydrofolate reductase ,Kinetic isotope effect ,biology.protein ,NADPH binding ,NAD+ kinase ,Equilibrium constant ,NADP - Abstract
Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. In this work, we report the kinetic characterization of the MtDHFR. This enzyme has a sequential steady-state random kinetic mechanism, probably with a preferred pathway with NADPH binding first. A pK(a) value for an enzymic acid of approximately 7.0 was identified from the pH dependence of V, and the analysis of the primary kinetic isotope effects revealed that the hydride transfer step is at least partly rate-limiting throughout the pH range analyzed. Additionally, solvent and multiple kinetic isotope effects were determined and analyzed, and equilibrium isotope effects were measured on the equilibrium constant. (D(2)O)V and (D(2)O)V/K([4R-4-(2)H]NADH) were slightly inverse at pH 6.0, and inverse values for (D(2)O)V([4R-4-(2)H]NADH) and (D(2)O)V/K([4R-4-(2)H]NADH) suggested that a pre-equilibrium protonation is occurring before the hydride transfer step, indicating a stepwise mechanism for proton and hydride transfer. The same value was obtained for (D)k(H) at pH 5.5 and 7.5, reaffirming the rate-limiting nature of the hydride transfer step. A chemical mechanism is proposed on the basis of the results obtained here.
- Published
- 2010
30. Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis
- Author
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Jairton Dupont, Clarissa M. Czekster, Luiz Augusto Basso, Brenno A. D. Neto, Diógenes Santiago Santos, and Alexandre A. M. Lapis
- Subjects
DNA, Bacterial ,Spectrometry, Mass, Electrospray Ionization ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Decarboxylation ,Biophysics ,Indole-3-Glycerol-Phosphate Synthase ,Biochemistry ,Biophysical Phenomena ,chemistry.chemical_compound ,Ribulosephosphates ,Deprotonation ,Biosynthesis ,Kinetic isotope effect ,Organic chemistry ,Cloning, Molecular ,Molecular Biology ,DNA Primers ,ATP synthase ,biology ,Base Sequence ,Virulence ,Chemistry ,Tryptophan ,Mycobacterium tuberculosis ,Hydrogen-Ion Concentration ,Phosphate ,Kinetics ,Spectrometry, Fluorescence ,Models, Chemical ,Genes, Bacterial ,biology.protein ,Thermodynamics - Abstract
Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for MtIGPS-catalized chemical reaction is proposed.
- Published
- 2009
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