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

Biochemistry. 2016 Feb 23;55(7):1107-19. doi: 10.1021/acs.biochem.5b00993. Epub 2016 Feb 5.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminosalicylic Acid / pharmacology
  • Antitubercular Agents / chemistry
  • Antitubercular Agents / metabolism
  • Antitubercular Agents / pharmacology
  • Bacterial Proteins / antagonists & inhibitors
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Catalytic Domain
  • Drug Resistance, Bacterial
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Folic Acid Antagonists / chemistry
  • Folic Acid Antagonists / metabolism
  • Folic Acid Antagonists / pharmacology
  • Kinetics
  • Ligands
  • Models, Molecular*
  • Molecular Conformation
  • Mycobacterium tuberculosis / drug effects
  • Mycobacterium tuberculosis / enzymology*
  • Mycobacterium tuberculosis / growth & development
  • NADP / chemistry*
  • NADP / metabolism
  • Nucleotide Deaminases / antagonists & inhibitors
  • Nucleotide Deaminases / chemistry*
  • Nucleotide Deaminases / genetics
  • Nucleotide Deaminases / metabolism
  • Phylogeny
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Tetrahydrofolate Dehydrogenase / chemistry*
  • Tetrahydrofolate Dehydrogenase / genetics
  • Tetrahydrofolate Dehydrogenase / metabolism
  • Tetrahydrofolates / chemistry*
  • Tetrahydrofolates / metabolism
  • Trimethoprim / chemistry
  • Trimethoprim / metabolism
  • Trimethoprim / pharmacology
  • Trimetrexate / chemistry
  • Trimetrexate / metabolism
  • Trimetrexate / pharmacology

Substances

  • Antitubercular Agents
  • Bacterial Proteins
  • Enzyme Inhibitors
  • Folic Acid Antagonists
  • Ligands
  • Recombinant Proteins
  • Tetrahydrofolates
  • 5,6,7,8-tetrahydrofolic acid
  • NADP
  • Aminosalicylic Acid
  • Trimethoprim
  • Tetrahydrofolate Dehydrogenase
  • Nucleotide Deaminases
  • Trimetrexate