Femtomolar Transition State Analogue Inhibitors of 5′-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Escherichia coli

Singh, Vipender; Evans, Gary B.; Lenz, Dirk H.; Mason, Jennifer M.; Clinch, Keith; Mee, Simon; Painter, Gavin F.; Tyler, Peter C.; Furneaux, Richard H.; Lee, Jeffrey E.; Howell, P. Lynne; Schramm, Vern L.

doi:10.1074/jbc.m414472200

Cited by 133 publications

(213 citation statements)

References 44 publications

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“…Furthermore, very potent inhibitors of MTAN are known that contain a phenyl ring positioned in a very similar manner to that of futalosine (e.g. inhibitor 1), indicating that the active site can accommodate a substrate with this functionality (20).…”

Section: Resultsmentioning

confidence: 99%

“…This suggests that the DHFL required for menaquinone biosynthesis in C. jejuni could be provided by the action of MTAN on 6-amino-6-deoxyfutalosine. To assess the catalytic efficiency of this process, we employed a coupled kinetic assay for adenine release that had previously been used in monitoring MTAN kinetics with MTA (20). The nucleosidase activity with 6-amino-6-deoxyfutalosine was found to follow Michaelis-Menten kinetics, and the kinetic constants obtained were k cat ϭ 0.53 Ϯ 0.03 s Ϫ1 and K m ϭ 1.03 Ϯ 0.12 M (see supplemental "Materials and General Methods").…”

Section: Resultsmentioning

confidence: 99%

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5′-Methylthioadenosine Nucleosidase Is Implicated in Playing a Key Role in a Modified Futalosine Pathway for Menaquinone Biosynthesis in Campylobacter jejuni

Apel

Gaynor

et al. 2011

Journal of Biological Chemistry

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Menaquinone (vitamin K 2 ) serves as an electron carrier in the electron transport chain required for respiration in many pathogenic bacteria. Most bacteria utilize a common menaquinone biosynthetic pathway as exemplified by Escherichia coli. Recently, a novel biosynthetic pathway, the futalosine pathway, was discovered in Streptomyces. Bioinformatic analysis strongly suggests that this pathway is also operative in the human pathogens Campylobacter jejuni and Helicobacter pylori. Here, we provide compelling evidence that a modified futalosine pathway is operative in C. jejuni and that it utilizes 6-amino-6-deoxyfutalosine instead of futalosine. A key step in the Streptomyces pathway involves a nucleosidase called futalosine hydrolase. The closest homolog in C. jejuni has been annotated as a 5-methylthioadenosine nucleosidase (MTAN). We have shown that this C. jejuni enzyme has MTAN activity but negligible futalosine hydrolase activity. However, the C. jejuni MTAN is able to hydrolyze 6-amino-6-deoxyfutalosine at a rate comparable with that of its known substrates. This suggests that the adeninecontaining version of futalosine is the true biosynthetic intermediate in this organism. To demonstrate this in vivo, we constructed a C. jejuni mutant strain deleted for mqnA2, which is predicted to encode for the enzyme required to synthesize 6-amino-6-deoxyfutalosine. Growth of this mutant was readily rescued by the addition of 6-amino-6-deoxyfutalosine, but not futalosine. This provides the first direct evidence that a modified futalosine pathway is operative in C. jejuni. It also highlights the tremendous versatility of the C. jejuni MTAN, which plays key roles in S-adenosylmethionine recycling, the biosynthesis of autoinducer molecules, and the biosynthesis of menaquinone.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

5′-Methylthioadenosine Nucleosidase Is Implicated in Playing a Key Role in a Modified Futalosine Pathway for Menaquinone Biosynthesis in Campylobacter jejuni

Apel

Gaynor

et al. 2011

Journal of Biological Chemistry

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“…Studies utilizing kinetic isotope effects (KIEs) and crystal structures of MTANs in complex with known transition state analogues have allowed for transition state modeling of MTANs from E. coli, S. pneumoniae, Neisseria meningitidis, S. aureus, and V. cholerae and the subsequent design of analogue inhibitors (120)(121)(122)(149)(150)(151)(152)(153). Thus far, it appears that most MTANs share a fully dissociated S N 1 transition state characterized by a ribooxacarbenium ion (121,122,149,152), with the exception of MTANs from (149).…”

Section: Inhibition Of Mtansmentioning

confidence: 99%

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

LaSarre

Federle

2013

Microbiol Mol Biol Rev

677

556

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SUMMARY Cell-cell communication, or quorum sensing, is a widespread phenomenon in bacteria that is used to coordinate gene expression among local populations. Its use by bacterial pathogens to regulate genes that promote invasion, defense, and spread has been particularly well documented. With the ongoing emergence of antibiotic-resistant pathogens, there is a current need for development of alternative therapeutic strategies. An antivirulence approach by which quorum sensing is impeded has caught on as a viable means to manipulate bacterial processes, especially pathogenic traits that are harmful to human and animal health and agricultural productivity. The identification and development of chemical compounds and enzymes that facilitate quorum-sensing inhibition (QSI) by targeting signaling molecules, signal biogenesis, or signal detection are reviewed here. Overall, the evidence suggests that QSI therapy may be efficacious against some, but not necessarily all, bacterial pathogens, and several failures and ongoing concerns that may steer future studies in productive directions are discussed. Nevertheless, various QSI successes have rightfully perpetuated excitement surrounding new potential therapies, and this review highlights promising QSI leads in disrupting pathogenesis in both plants and animals.

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“…In most cases the inhibitor concentration was at least ten-fold greater than the enzyme concentration, as required for simple analysis of slow-onset tight-binding inhibition (22). When this condition could not be satisfied, corrections were made to compensate for the concentration of bound inhibitor (23).…”

Section: Steady-state Kinetic Assays and Inhibition Studiesmentioning

confidence: 99%

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

et al. 2007

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The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition state analogues show His 257 within hydrogen-bonding distance to the 5′-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (K m /K i ) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His 257 serves an important role in the early stages of transition state formation. Whereas mutation of His 257 resulted in little variation in the PNP·DADMe-ImmH·SO 4 structures, His257Phe·ImmH·PO 4 showed distortion at the 5′-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies on the remote 5′-3 H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5′-3 H intrinsic KIE to −3%, −14%, and 7%, respectively, while the BIEs contributed 2%, 2%, and −2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state. Keywordspurine nucleoside phosphorylase; mutagenesis; binding isotope effect; kinetic isotope effect; transition state; binding distortion; transition state geometry; dynamics Determination of the transition state structure of enzymatic reactions allows for the design of tight binding transition state analogue inhibitors. These transition state mimics can subvert the energetics that govern formation of the enzymatic transition state to achieve binding orders of magnitude stronger than that of substrate. The transition state structures of enzymatic reactions have frequently been established by measuring kinetic isotope effects (KIEs 1 ) from the competitive reaction of isotopically labeled substrates. These isotope effects on k cat /K m , often † Supported by NIH Research Grant GM41916.* To whom correspondence should be addressed. vern@aecom.yu.edu; Telephone, (718) 430-2813; Fax, (718) . ‡ Current address: Department of Chemistry, Barnard College, 3009 Broadway, New York, NY 10027. § Current address: Center for Synchrotron Bioscience, Brookhaven National Laboratory, Upton, NY 11973.1 Abbreviations: KIE, kinetic isotope effect; V/K KIE, kinetic isotope effect on the second-order rate constant, k cat /K m ; BIE, binding isotope effect; HsPNP, human purine nucleoside phosphorylase; PDB, Protein Data Bank; ImmH, Immucillin-H; DADMe-ImmH, 4′-deaza-1′-aza-2′-deoxy-1′-(9-methylene)-Immucillin-H; RMS, root mea...

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Femtomolar Transition State Analogue Inhibitors of 5′-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Escherichia coli

Cited by 133 publications

References 44 publications

5′-Methylthioadenosine Nucleosidase Is Implicated in Playing a Key Role in a Modified Futalosine Pathway for Menaquinone Biosynthesis in Campylobacter jejuni

5′-Methylthioadenosine Nucleosidase Is Implicated in Playing a Key Role in a Modified Futalosine Pathway for Menaquinone Biosynthesis in Campylobacter jejuni

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

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