Transition-State Analogues of Campylobacter jejuni 5'-Methylthioadenosine Nucleosidase.

Campylobacter jejuni is a Gram-negative bacterium responsible for food-borne gastroenteritis and associated with Guillain-Barré, Reiter, and irritable bowel syndromes. Antibiotic resistance in C. jejuni is common, creating a need for antibiotics with novel mechanisms of action. Menaquinone biosynthesis in C. jejuni uses the rare futalosine pathway, where 5'-methylthioadenosine nucleosidase ( CjMTAN) is proposed to catalyze the essential hydrolysis of adenine from 6-amino-6-deoxyfutalosine to form dehypoxanthinylfutalosine, a menaquinone precursor. The substrate specificity of CjMTAN is demonstrated to include 6-amino-6-deoxyfutalosine, 5'-methylthioadenosine, S-adenosylhomocysteine, adenosine, and 5'-deoxyadenosine. These activities span the catalytic specificities for the role of bacterial MTANs in menaquinone synthesis, quorum sensing, and S-adenosylmethionine recycling. We determined inhibition constants for potential transition-state analogues of CjMTAN. The best of these compounds have picomolar dissociation constants and were slow-onset tight-binding inhibitors. The most potent CjMTAN transition-state analogue inhibitors inhibited C. jejuni growth in culture at low micromolar concentrations, similar to gentamicin. The crystal structure of apoenzyme C. jejuni MTAN was solved at 1.25 Å, and five CjMTAN complexes with transition-state analogues were solved at 1.42 to 1.95 Å resolution. Inhibitor binding induces a loop movement to create a closed catalytic site with Asp196 and Ile152 providing purine leaving group activation and Arg192 and Glu12 activating the water nucleophile. With inhibitors bound, the interactions of the 4'-alkylthio or 4'-alkyl groups of this inhibitor family differ from the Escherichia coli MTAN structure by altered protein interactions near the hydrophobic pocket that stabilizes 4'-substituents of transition-state analogues. These CjMTAN inhibitors have potential as specific antibiotic candidates against C. jejuni.

[1]  V. Schramm,et al.  Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum , 2018, Proceedings of the National Academy of Sciences.

[2]  G. Evans,et al.  New Antibiotic Candidates against Helicobacter pylori. , 2015, Journal of the American Chemical Society.

[3]  V. Schramm,et al.  Active site and remote contributions to catalysis in methylthioadenosine nucleosidases. , 2015, Biochemistry.

[4]  V. Schramm,et al.  Conformational freedom in tight binding enzymatic transition-state analogues. , 2013, The journal of physical chemistry. B.

[5]  G. Evans,et al.  Salmonella enterica MTAN at 1.36 Å resolution: a structure-based design of tailored transition state analogs. , 2013, Structure.

[6]  N. Iovine,et al.  Resistance mechanisms in Campylobacter jejuni , 2013, Virulence.

[7]  G. Evans,et al.  Transition state analogue inhibitors of human methylthioadenosine phosphorylase and bacterial methylthioadenosine/S-adenosylhomocysteine nucleosidase incorporating acyclic ribooxacarbenium ion mimics. , 2012, Bioorganic & medicinal chemistry.

[8]  M. Tanner,et al.  5′-Methylthioadenosine Nucleosidase Is Implicated in Playing a Key Role in a Modified Futalosine Pathway for Menaquinone Biosynthesis in Campylobacter jejuni* , 2011, The Journal of Biological Chemistry.

[9]  Owen Johnson,et al.  iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM , 2011, Acta crystallographica. Section D, Biological crystallography.

[10]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[11]  Nikhat Parveen,et al.  Methylthioadenosine/S‐adenosylhomocysteine nucleosidase, a critical enzyme for bacterial metabolism , 2011, Molecular microbiology.

[12]  D. Ronning,et al.  Enzyme–ligand interactions that drive active site rearrangements in the Helicobacter pylori 5′‐methylthioadenosine/S‐adenosylhomocysteine nucleosidase , 2010, Protein science : a publication of the Protein Society.

[13]  G. Evans,et al.  Design and synthesis of potent "sulfur-free" transition state analogue inhibitors of 5'-methylthioadenosine nucleosidase and 5'-methylthioadenosine phosphorylase. , 2010, Journal of medicinal chemistry.

[14]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[15]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[16]  T. Dairi.  An alternative menaquinone biosynthetic pathway operating in microorganisms: an attractive target for drug discovery to pathogenic Helicobacter and Chlamydia strains , 2009, The Journal of Antibiotics.

[17]  Qijing Zhang,et al.  Antibiotic resistance in Campylobacter: emergence, transmission and persistence. , 2009, Future microbiology.

[18]  V. Schramm,et al.  Transition state analogues of 5′-methylthioadenosine nucleosidase disrupt quorum sensing , 2009, Nature chemical biology.

[19]  V. DiRita,et al.  Growth and Laboratory Maintenance of Campylobacter jejuni , 2008, Current protocols in microbiology.

[20]  G. Evans,et al.  Picomolar inhibitors as transition-state probes of 5'-methylthioadenosine nucleosidases. , 2007, ACS chemical biology.

[21]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[22]  S. O'Brien,et al.  Campylobacters as zoonotic pathogens: a food production perspective. , 2007, International journal of food microbiology.

[23]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[24]  G. Ruiz-Palacios.  The health burden of Campylobacter infection and the impact of antimicrobial resistance: playing chicken. , 2007, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[25]  C. Walsh,et al.  The behavior and significance of slow-binding enzyme inhibitors. , 2006, Advances in enzymology and related areas of molecular biology.

[26]  G. Evans,et al.  Structure and inhibition of a quorum sensing target from Streptococcus pneumoniae. , 2006, Biochemistry.

[27]  V. Schramm,et al.  Enzymatic transition states and transition state analogues. , 2005, Current opinion in structural biology.

[28]  P. Howell,et al.  Structural snapshots of MTA/AdoHcy nucleosidase along the reaction coordinate provide insights into enzyme and nucleoside flexibility during catalysis. , 2005, Journal of molecular biology.

[29]  V. Schramm,et al.  Transition state structure of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues. , 2005, Biochemistry.

[30]  G. Evans,et al.  Second generation transition state analogue inhibitors of human 5'-methylthioadenosine phosphorylase. , 2005, Journal of medicinal chemistry.

[31]  G. Evans,et al.  Femtomolar Transition State Analogue Inhibitors of 5′-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Escherichia coli* , 2005, Journal of Biological Chemistry.

[32]  J. Cronan,et al.  A nucleosidase required for in vivo function of the S-adenosyl-L-methionine radical enzyme, biotin synthase. , 2005, Chemistry & biology.

[33]  J. Butzler,et al.  Campylobacter, from obscurity to celebrity. , 2004, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[34]  G. Evans,et al.  Targeting the polyamine pathway with transition-state analogue inhibitors of 5'-methylthioadenosine phosphorylase. , 2004, Journal of medicinal chemistry.

[35]  G. Evans,et al.  Synthesis of a transition state analogue inhibitor of purine nucleoside phosphorylase via the Mannich reaction. , 2003, Organic letters.

[36]  S. Altekruse,et al.  Human campylobacteriosis: a challenge for the veterinary profession. , 2003, Journal of the American Veterinary Medical Association.

[37]  G. Evans,et al.  Exploring structure-activity relationships of transition state analogues of human purine nucleoside phosphorylase. , 2003, Journal of medicinal chemistry.

[38]  R. Wolfenden,et al.  The depth of chemical time and the power of enzymes as catalysts. , 2001, Accounts of chemical research.

[39]  P. Howell,et al.  Structure of E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase reveals similarity to the purine nucleoside phosphorylases. , 2001, Structure.

[40]  G. Evans,et al.  Synthesis of transition state analogue inhibitors for purine nucleoside phosphorylase and N-riboside hydrolases , 2000 .

[41]  E. Greenberg,et al.  Acyl homoserine-lactone quorum-sensing signal generation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[42]  B. Allart,et al.  The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli--chemical evidence for a transition state with a substantial oxocarbenium character. , 1998, European journal of biochemistry.

[43]  C. O'Morain,et al.  Treatment of Helicobacter pylori , 2005, Helicobacter.

[44]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[45]  L. Ng,et al.  Comparison of basal media for culturing Campylobacter jejuni and Campylobacter coli , 1985, Journal of clinical microbiology.

[46]  G. Cacciapuoti,et al.  Substrate specificity of 5'-methylthioadenosine phosphorylase from human prostate. , 1978, The Biochemical journal.

[47]  A. Pegg,et al.  Phosphate-stimulated breakdown of 5'-methylthioadenosine by rat ventral prostate. , 1969, The Biochemical journal.

[48]  R. Wolfenden,et al.  Transition State Analogues for Enzyme Catalysis , 1969, Nature.

[49]  J. A. Duerre.  A Hydrolytic Nucleosidase Acting on S-Adenosylhomocysteine and on 5'-Methylthioadenosine , 1962 .

[50]  G. Cantoni,et al.  The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine. , 1959, The Journal of biological chemistry.

[51]  S. K. Shapiro,et al.  The enzymatic decomposition of S-adenosyl-L-methionine. , 1958, The Journal of biological chemistry.

[52]  R. Wolfenden,et al.  Transition state analog inhibitors and enzyme catalysis. , 1976, Annual review of biophysics and bioengineering.