Experimental and computational snapshots of C-C bond formation in a C-nucleoside synthase

The biosynthetic enzyme, ForT, catalyses the formation of a C-C bond between 4-amino-1H-pyrazoledicarboxylic acid and MgPRPP to produce a C-nucleoside precursor of formycin A. The transformation catalysed by ForT is of chemical interest because it is one of only a few examples in which C-C bond formation takes place via an electrophilic substitution of a small, aromatic heterocycle. In addition, ForT is capable of discriminating between the aminopyrazoledicarboxylic acid and an analogue in which the amine is replaced by a hydroxyl group; a remarkable feat given the steric and electronic similarities of the two molecules. Here we report biophysical measurements, structural biology and quantum chemical calculations that provide a detailed molecular picture of ForT-catalysed C-C bond formation and the conformational changes that are coupled to catalysis. Our findings set the scene for employing engineered ForT variants in the biocatalytic production of novel, anti-viral C-nucleoside and C-nucleotide analogues.

[1]  Z. Deng,et al.  Biosynthesis of C-nucleoside antibiotics in actinobacteria: recent advances and future developments , 2022, Microbial Cell Factories.

[2]  Binju Wang,et al.  Molecular basis of enzymatic nitrogen-nitrogen formation by a family of zinc-binding cupin enzymes , 2021, Nature Communications.

[3]  C. Aldrich,et al.  Innovative Strategies for the Construction of Diverse 1'-Modified C-Nucleoside Derivatives. , 2021, The Journal of organic chemistry.

[4]  Hung‐wen Liu,et al.  Identification of a Pyrrole Intermediate Which Undergoes C-Glycosidation and Autoxidation to Yield the Final Product in Showdomycin Biosynthesis. , 2021, Angewandte Chemie.

[5]  Deepthi Ramesh,et al.  Advances in Nucleoside and Nucleotide Analogues in Tackling Human Immunodeficiency Virus and Hepatitis Virus Infections , 2021, ChemMedChem.

[6]  T. Schäberle,et al.  Heterologous Expression of Pseudouridimycin and Description of the Corresponding Minimal Biosynthetic Gene Cluster , 2021, Molecules.

[7]  W. Fessner,et al.  Biocatalytic routes to anti-viral agents and their synthetic intermediates. , 2020, Chemical Society reviews.

[8]  Z. Deng,et al.  Harnessing synthetic biology-based strategies for engineered biosynthesis of nucleoside natural products in actinobacteria. , 2020, Biotechnology advances.

[9]  S. van Calenbergh,et al.  Sydnone Ribosides as a Platform for the Synthesis of Pyrazole C-Nucleosides: A Unified Synthesis of Formycin B and Pyrazofurin. , 2020, Organic letters.

[10]  Louis-Charles Campeau,et al.  A short de novo synthesis of nucleoside analogs , 2020, Science.

[11]  Yong Qin,et al.  Improvement of the C-glycosylation Step for the Synthesis of Remdesivir , 2020, Organic process research & development.

[12]  V. de Crécy-Lagard,et al.  Uncovering the chemistry of C–C bond formation in C-nucleoside biosynthesis: crystal structure of a C-glycoside synthase/PRPP complex , 2020, Chemical communications.

[13]  S. Anzick,et al.  Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2 , 2020, Nature.

[14]  S. Anzick,et al.  Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2 , 2020, Nature.

[15]  V. de Crécy-Lagard,et al.  Whole-Genome Sequence of Streptomyces kaniharaensis Shomura and Niida SF-557 , 2020, Microbiology Resource Announcements.

[16]  J. Lian,et al.  The Biosynthetic Gene Cluster of Pyrazomycin—A C‐Nucleoside Antibiotic with a Rare Pyrazole Moiety , 2020, Chembiochem : a European journal of chemical biology.

[17]  W. Tao,et al.  Divergent Biosynthesis of C-Nucleoside Minimycin and Indigoidine in Bacteria , 2019, iScience.

[18]  Hung‐wen Liu,et al.  Identification of the C-Glycoside Synthases during Biosynthesis of the Pyrazole-C-Nucleosides Formycin and Pyrazofurin. , 2019, Angewandte Chemie.

[19]  Z. Deng,et al.  Comparative Investigation into Formycin A and Pyrazofurin A Biosynthesis Reveals Branch Pathways for the Construction of C-Nucleoside Scaffolds , 2019, Applied and Environmental Microbiology.

[20]  Hui Hong,et al.  C-Nucleoside Formation in the Biosynthesis of the Antifungal Malayamycin A. , 2019, Cell chemical biology.

[21]  Hung‐wen Liu,et al.  Identification of the Formycin A Biosynthetic Gene Cluster from Streptomyces kaniharaensis Illustrates the Interplay between Biological Pyrazolopyrimidine Formation and de Novo Purine Biosynthesis. , 2019, Journal of the American Chemical Society.

[22]  R. Ebright,et al.  Discovery, properties, and biosynthesis of pseudouridimycin, an antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase , 2019, Journal of Industrial Microbiology & Biotechnology.

[23]  R. Ebright,et al.  Analysis of the Pseudouridimycin Biosynthetic Pathway Provides Insights into the Formation of C-nucleoside Antibiotics. , 2018, Cell chemical biology.

[24]  Kartik W Temburnikar,et al.  Recent advances in synthetic approaches for medicinal chemistry of C-nucleosides , 2018, Beilstein journal of organic chemistry.

[25]  J. Balzarini,et al.  The ProTide Prodrug Technology: From the Concept to the Clinic , 2017, Journal of medicinal chemistry.

[26]  M. Maurel,et al.  Phosphoribosyl Pyrophosphate: A Molecular Vestige of the Origin of Life on Minerals. , 2017, Angewandte Chemie.

[27]  E. De Clercq,et al.  Antiviral activity of the adenosine analogue BCX4430 against West Nile virus and tick-borne flaviviruses. , 2017, Antiviral research.

[28]  B. Baral,et al.  Discovery of the Showdomycin Gene Cluster from Streptomyces showdoensis ATCC 15227 Yields Insight into the Biosynthetic Logic of C-Nucleoside Antibiotics. , 2017, ACS chemical biology.

[29]  Hung‐wen Liu,et al.  Identification and Characterization of Enzymes Catalyzing Pyrazolopyrimidine Formation in the Biosynthesis of Formycin A. , 2017, Organic letters.

[30]  Valentina Ferrari,et al.  Nucleoside Derived Antibiotics to Fight Microbial Drug Resistance: New Utilities for an Established Class of Drugs? , 2016, Journal of medicinal chemistry.

[31]  E. Clercq.  C-Nucleosides To Be Revisited. , 2016 .

[32]  William A. Lee,et al.  Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys , 2016, Nature.

[33]  A. Maiti,et al.  Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA. , 2014, Organic & biomolecular chemistry.

[34]  R. Schinazi,et al.  Synthesis of Nucleoside Phosphate and Phosphonate Prodrugs , 2014, Chemical reviews.

[35]  James J. P. Stewart,et al.  Optimization of parameters for semiempirical methods VI: more modifications to the NDDO approximations and re-optimization of parameters , 2012, Journal of Molecular Modeling.

[36]  T. Begley,et al.  Pseudouridine monophosphate glycosidase: a new glycosidase mechanism. , 2012, Biochemistry.

[37]  Michal Hocek,et al.  C-nucleosides: synthetic strategies and biological applications. , 2009, Chemical reviews.

[38]  F. Himo,et al.  Recent developments of the quantum chemical cluster approach for modeling enzyme reactions , 2009, JBIC Journal of Biological Inorganic Chemistry.

[39]  V. Davisson,et al.  Interrogating the mechanism of a tight binding inhibitor of AIR carboxylase. , 2009, Bioorganic & medicinal chemistry.

[40]  M. Walkinshaw,et al.  Engineering water to act as an active site acid catalyst in a soluble fumarate reductase. , 2002, Biochemistry.

[41]  A. Kuzin,et al.  Substrate deformation in a hypoxanthine-guanine phosphoribosyltransferase ternary complex: the structural basis for catalysis. , 2000, Structure.

[42]  N. Grishin,et al.  Structure and mechanism of homoserine kinase: prototype for the GHMP kinase superfamily. , 2000, Structure.

[43]  J. L. Smith,et al.  Coupled formation of an amidotransferase interdomain ammonia channel and a phosphoribosyltransferase active site. , 1997, Biochemistry.

[44]  J. Klinman,et al.  Calculation of substrate dissociation constants from steady-state isotope effects in enzyme-catalyzed reactions , 1985 .

[45]  K. Ochi,et al.  Biosynthesis of formycin. Incorporation and distribution of 13C-, 14C-, and 15N-labeled compounds into formycin. , 1979, The Journal of biological chemistry.

[46]  M. Maurel,et al.  A molecular vestige of the origin of life on minerals : phosphoribosyl-pyrophosphate , 2017 .

[47]  James J. P. Stewart,et al.  Application of localized molecular orbitals to the solution of semiempirical self‐consistent field equations , 1996 .

[48]  Michael R Hamblin,et al.  The biosynthesis of pyrazofurin and formycin , 1980 .