title: E84G mutation in dihydrofolate reductase from drug resistant strains of Mycobacterium tuberculosis (Mumbai, India) leads to increased interaction with Trimethoprim.
abstract: BACKGROUND
  Dihydrofolate reductase (DHFR) (dfrA gene) is an essential enzyme for cell survival and an unexplored target in Mycobacterium tuberculosis (Mtb). This study was carried out to analyze mutations in the dfrA gene amongst 20 clinical DNA samples from Mtb isolates obtained from Mumbai, India.
  
  
  METHODS
  Sequencing of the PCR amplified dfrA gene from these DNA isolates revealed a point mutation in one strain, leading to a glutamic acid to glycine change. In silico simulation studies revealed a surface alteration in the enzyme due to this E84G mutation. The amplified mutant gene was cloned and expressed. The mutant protein was assessed against known DHFR inhibitors: Methotrexate and Trimethoprim.
  
  
  RESULTS
  An increased affinity for inhibitor Trimethoprim and native substrate dihydrofolate was observed with the mutant. Methotrexate did not vary in its activity with both the enzyme forms.
  
  
  CONCLUSIONS
  The Glu84Gly point mutation may lead to a variation in the strain which may cause resistance in the future.
authors.affiliation: Institute of Chemical Technology, Department of Pharmaceutical Sciences and Technology, N.P. Marg, Matunga, Mumbai 400019, India.
authors.forename: Archana
authors.initials: A
authors.lastname: Raju
authors.affiliation: Radiation Medicine Centre - BARC, Tuberculosis Immunology & Immunoassay Development Section, Tata Memorial Hospital, Annexe Bldg, Parel, Mumbai 400012, India.
authors.forename: Savita
authors.initials: S
authors.lastname: Kulkarni
authors.affiliation: Radiation Medicine Centre - BARC, Tuberculosis Immunology & Immunoassay Development Section, Tata Memorial Hospital, Annexe Bldg, Parel, Mumbai 400012, India.
authors.forename: M K
authors.initials: MK
authors.lastname: Ray
authors.affiliation: Radiation Medicine Centre - BARC, Tuberculosis Immunology & Immunoassay Development Section, Tata Memorial Hospital, Annexe Bldg, Parel, Mumbai 400012, India.
authors.forename: M G R
authors.initials: MG
authors.lastname: Rajan
authors.affiliation: Institute of Chemical Technology, Department of Pharmaceutical Sciences and Technology, N.P. Marg, Matunga, Mumbai 400019, India. Electronic address: ms.degani@ictmumbai.edu.in.
authors.forename: Mariam S
authors.initials: MS
authors.lastname: Degani
mesh_terms: D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007194:India; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014295:Trimethoprim / Q000737:chemistry / Q000378:metabolism*; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase
keywords: Dihydrofolate reductase; Enzyme assay; Molecular docking; Mycobacterium tuberculosis; Point mutation; Trimethoprim