Structural Insights into Mycobacterium tuberculosis Rv2671 Protein as a Dihydrofolate Reductase Functional Analogue Contributing to para-Aminosalicylic Acid Resistance.

Mycobacterium tuberculosis (Mtb) Rv2671 is annotated as a 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (AROPP) reductase (RibD) in the riboflavin biosynthetic pathway. Recently, a strain of Mtb with a mutation in the 5' untranslated region of Rv2671, which resulted in its overexpression, was found to be resistant to dihydrofolate reductase (DHFR) inhibitors including the anti-Mtb drug para-aminosalicylic acid (PAS). In this study, a biochemical analysis of Rv2671 showed that it was able to catalyze the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which explained why the overexpression of Rv2671 was sufficient to confer PAS resistance. We solved the structure of Rv2671 in complex with the NADP(+) and tetrahydrofolate (THF), which revealed the structural basis for the DHFR activity. The structures of Rv2671 complexed with two DHFR inhibitors, trimethoprim and trimetrexate, provided additional details of the substrate binding pocket and elucidated the differences between their inhibitory activities. Finally, Rv2671 was unable to catalyze the reduction of AROPP, which indicated that Rv2671 and its closely related orthologues are not involved in riboflavin biosynthesis.

[1]  P. Langan,et al.  Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography , 2014, Proceedings of the National Academy of Sciences.

[2]  Q. Jin,et al.  Genetic Determinants Involved in p-Aminosalicylic Acid Resistance in Clinical Isolates from Tuberculosis Patients in Northern China from 2006 to 2012 , 2014, Antimicrobial Agents and Chemotherapy.

[3]  T. Blundell,et al.  Mycobacterium tuberculosis dihydrofolate reductase reveals two conformational states and a possible low affinity mechanism to antifolate drugs. , 2014, Structure.

[4]  Tatiana A. Tatusova,et al.  RefSeq microbial genomes database: new representation and annotation strategy , 2013, Nucleic Acids Res..

[5]  Melissa J. Landrum,et al.  RefSeq: an update on mammalian reference sequences , 2013, Nucleic Acids Res..

[6]  Jichan Jang,et al.  para-Aminosalicylic Acid Is a Prodrug Targeting Dihydrofolate Reductase in Mycobacterium tuberculosis♦ , 2013, The Journal of Biological Chemistry.

[7]  Yingfang Liu,et al.  Structural and Functional Insights into Saccharomyces cerevisiae Riboflavin Biosynthesis Reductase RIB7 , 2013, PloS one.

[8]  S. Chakraborty,et al.  Para-Aminosalicylic Acid Acts as an Alternative Substrate of Folate Metabolism in Mycobacterium tuberculosis , 2013, Science.

[9]  Tatiana A. Tatusova,et al.  NCBI Reference Sequences (RefSeq): current status, new features and genome annotation policy , 2011, Nucleic Acids Res..

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

[11]  T. Alber,et al.  Depletion of antibiotic targets has widely varying effects on growth , 2011, Proceedings of the National Academy of Sciences.

[12]  J. Blanchard,et al.  Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis. , 2011, Biochemistry.

[13]  Liisa Holm,et al.  Dali server: conservation mapping in 3D , 2010, Nucleic Acids Res..

[14]  O. Gascuel,et al.  New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. , 2010, Systematic biology.

[15]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[16]  Xin Guo,et al.  2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors. , 2010, Bioorganic & medicinal chemistry.

[17]  Stephen J Benkovic,et al.  Kinetic and structural characterization of dihydrofolate reductase from Streptococcus pneumoniae. , 2010, Biochemistry.

[18]  J. Ayling,et al.  The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake , 2009, Proceedings of the National Academy of Sciences.

[19]  Roman G. Efremov,et al.  PLATINUM: a web tool for analysis of hydrophobic/hydrophilic organization of biomolecular complexes , 2009, Bioinform..

[20]  Yu-Hsin Lin,et al.  Complex Structure of Bacillus subtilis RibG , 2009, Journal of Biological Chemistry.

[21]  W. Eisenreich,et al.  2,5‐diamino‐6‐ribitylamino‐4(3H)‐pyrimidinone 5′‐phosphate synthases of fungi and archaea , 2008, The FEBS journal.

[22]  S. Cahill,et al.  Kinetic and mechanistic analysis of the Escherichia coli ribD-encoded bifunctional deaminase-reductase involved in riboflavin biosynthesis. , 2008, Biochemistry.

[23]  Jean-Michel Claverie,et al.  Phylogeny.fr: robust phylogenetic analysis for the non-specialist , 2008, Nucleic Acids Res..

[24]  P. Nordlund,et al.  The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism. , 2007, Journal of molecular biology.

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

[26]  J. Soulages,et al.  Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase , 2007, The FEBS journal.

[27]  Marie-Christine Brun,et al.  TreeDyn: towards dynamic graphics and annotations for analyses of trees , 2006, BMC Bioinformatics.

[28]  R. Huber,et al.  Biosynthesis of riboflavin: structure and properties of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase of Methanocaldococcus jannaschii. , 2006, Journal of molecular biology.

[29]  Matthew W Vetting,et al.  Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid , 2006, Nature Structural &Molecular Biology.

[30]  Yuan-Chih Chang,et al.  Crystal Structure of a Bifunctional Deaminase and Reductase from Bacillus subtilis Involved in Riboflavin Biosynthesis* , 2006, Journal of Biological Chemistry.

[31]  Christiane Branlant,et al.  Pseudouridylation at Position 32 of Mitochondrial and Cytoplasmic tRNAs Requires Two Distinct Enzymes in Saccharomyces cerevisiae* , 2004, Journal of Biological Chemistry.

[32]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[33]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[34]  V. Escuyer,et al.  Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase. , 2004, FEMS microbiology letters.

[35]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[36]  G. Maglia,et al.  Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima. , 2003, The Biochemical journal.

[37]  E. Rubin,et al.  Genes required for mycobacterial growth defined by high density mutagenesis , 2003, Molecular microbiology.

[38]  Robert H. White,et al.  The Pyrimidine Nucleotide Reductase Step in Riboflavin and F420 Biosynthesis in Archaea Proceeds by the Eukaryotic Route to Riboflavin , 2002, Journal of bacteriology.

[39]  N. Glansdorff,et al.  Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase. , 1998, European journal of biochemistry.

[40]  B. Barrell,et al.  Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence , 1998, Nature.

[41]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[42]  J. Kraut,et al.  Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications. , 1995, Biochemistry.

[43]  D. Santi,et al.  Dihydrofolate reductase from the pathogenic fungus Pneumocystis carinii: catalytic properties and interaction with antifolates. , 1993, Archives of biochemistry and biophysics.

[44]  S. Benkovic,et al.  The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases. , 1990, Biochemistry.

[45]  W. Beard,et al.  Unusual transient- and steady-state kinetic behavior is predicted by the kinetic scheme operational for recombinant human dihydrofolate reductase. , 1990, The Journal of biological chemistry.

[46]  K. Houk,et al.  Theoretical Transition Structures for Hydride Transfer to Methyleniminium Ion from Methylamine and Dihydropyridine. Nonlinearity of Hydride Transfers. , 1987 .

[47]  S. Benkovic,et al.  Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli. , 1987, Biochemistry.

[48]  Yun-Dong Wu,et al.  Theoretical transition structures for hydride transfer to methyleneiminium ion from methylamine and dihydropyridine. On the nonlinearity of hydride transfers , 1987 .

[49]  K. Houk,et al.  Transition structures for hydride transfers , 1987 .

[50]  J. Morrison,et al.  Kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli. , 1982, Biochemistry.

[51]  A. Bacher,et al.  Biosynthesis of vitamin b2 (riboflavin). , 2000, Annual review of nutrition.

[52]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[53]  D. W. Fry,et al.  Biochemical pharmacology of the lipophilic antifolate, trimetrexate. , 1984, Advances in enzyme regulation.