Structural characterization of the mitomycin 7‐ O ‐methyltransferase

Mitomycins are quinone‐containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin‐7‐ O ‐methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae , catalyzes the 7‐ O ‐methylation of both C9β‐ and C9α‐configured 7‐hydroxymitomycins. We have determined the crystal structures of the MmcR– S ‐adenosylhomocysteine (SAH) binary complex and MmcR–SAH–mitomycin A (MMA) ternary complex at resolutions of 1.9and 2.3 Å, respectively. The study revealed MmcR to adopt a common S ‐adenosyl‐ L ‐methionine‐dependent O ‐methyltransferase fold and the presence of a structurally conserved active site general acid–base pair is consistent with a proton‐assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins. Proteins 2011. © 2011 Wiley‐Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87038/1/PROT_23040_sm_suppinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/87038/2/23040_ftp.pd

Control of cyclic oligoadenylate synthesis in a type III CRISPR system

This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (REF: BB/M000400 /1 to MFW), and a Royal Society Challenge Grant (REF: CH160014 to MFW).The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3' end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The 'RNA shredding' activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence.Publisher PDFPeer reviewe

Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

Royal Society Challenge Grant [REF: CH160014 to M.F.W.]; Biotechnology and Biological Sciences Research Council [REF: BB/S000313/1 to M.F.W.]. Funding for open access charge: Institutional Block Grant.The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.Publisher PDFPeer reviewe

An unusual flavin-dependent halogenase from the metagenome of the marine sponge Theonella swinhoei WA

The authors thank EU BlueGenics (Seventh Framework Programme, Collaborative project “BlueGenics”, Grant no. 311848 RJMG and JP), the SNF (Grant no.205321_165695 to JP), the Helmut Horten Foundation (JP), and ERAIB (Grant no. 031A338A KHVP and RJMG) for funding.Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin dependent halogenase with a broad substrate specificity, and with potential as a useful new biocatalytic tool is described.PostprintPeer reviewe

Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

This work was funded by grants from the Biotechnology and Biological Sciences Research Council (REF BB/M000400/1 and BB/M021017/1). MFW is a Wolfson Research Merit Award holder.The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids1,2,3. Type III effector complexes were recently demonstrated to synthesize a  novel second messenger, cyclic oligoadenylate, on binding target RNA4,5. Cyclic oligoadenylate, in turn, binds to and activates ribonucleases  and other factors—via a CRISPR-associated Rossman-fold domain—and thereby induces in the cell an antiviral state that is important for immunity. The mechanism of the ‘off-switch’ that resets the system is not understood. Here we identify the nuclease that degrades these cyclic oligoadenylate ring molecules. This ‘ring nuclease’ is itself a protein of the CRISPR-associated Rossman-fold family, and has a metal-independent mechanism that cleaves cyclic tetraadenylate rings to generate linear diadenylate species and switches off the antiviral state. The identification of ring nucleases adds an important insight tothe CRISPR system.PostprintPeer reviewe

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

Funding: This work was supported by grants from the Biotechnology and Biological Sciences Research Council (Grant BB/T004789/1 to MFW), Medical Research Scotland (Grant CVG-1719-2020 to MFW) and The University of St Andrews Restarting Research Funding Scheme (SARRF), funded through the Scottish Funding Council (grant reference SFC/AN/08/020) to MFW and CSA.Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.Publisher PDFPeer reviewe

Nucleic acid analogs with restricted conformational flexibility in the sugar-phosphate backbone (Bicyclo-DNA). Part 7. Synthesis and properties of oligodeoxynucleotides containing [(3'S,5'S,6'R)-6'-amino-2'-deoxy-3',5'-ethano-beta-D-ribofuranosyl]thymine (6'-amino-bicyclothymidine)

Further investigations of the synthesis of endothio analogues of the segment 1-10 (8) of an apolar analogon of zervamicin IIA are described. The endothiodecapeptide Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu- Aib-Y(CS)-Pro-OMe (10) has been prepared in good yield by our novel methodology. On the other hand, all attempts to prepare endothio analogues of 8 with the thioamide group at position 3 (Alat) gave not the expected linear endothiopeptides but led to epimerized 1,3-thiazol-5(4H)-imine derivatives as the main products. The mixture of epimers of the thiazolimines 27, 30, and 31 have been separated by means of preparative HPLC, and their structures have been established by 2D-NMR experiments

Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo

Control of cyclic oligoadenylate synthesis in a type III CRISPR system

The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3' end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The 'RNA shredding' activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence

Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids1,2,3. Type III effector complexes were recently demonstrated to synthesize a  novel second messenger, cyclic oligoadenylate, on binding target RNA4,5. Cyclic oligoadenylate, in turn, binds to and activates ribonucleases  and other factors—via a CRISPR-associated Rossman-fold domain—and thereby induces in the cell an antiviral state that is important for immunity. The mechanism of the ‘off-switch’ that resets the system is not understood. Here we identify the nuclease that degrades these cyclic oligoadenylate ring molecules. This ‘ring nuclease’ is itself a protein of the CRISPR-associated Rossman-fold family, and has a metal-independent mechanism that cleaves cyclic tetraadenylate rings to generate linear diadenylate species and switches off the antiviral state. The identification of ring nucleases adds an important insight tothe CRISPR system