Decreased Methylenetetrahydrofolate Reductase Activity Leads to Increased Sensitivity to para -Aminosalicylic Acid in Mycobacterium tuberculosis

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis ( M. tuberculosis ), is one of the most fatal diseases in the world. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the production of 5-methyltetrahydrofolate (5-CH 3 -THF), which is required for the de novo biosynthesis of methionine in bacteria. In this study, we identified Rv2172c as an MTHFR in M. tuberculosis through in vitro and in vivo analyses and determined that the protein was essential for the in vitro growth of the bacterium. Subsequently, we constructed rv2172c R159N and L214A mutants in M. tuberculosis , and found that these mutants were more sensitive to the antifolates para -aminosalicylic acid (PAS) and sulfamethoxazole (SMX). Combining biochemical and genetic methods, we found that rv2172c R159N or L214A mutation impaired methionine production, leading to increased susceptibility of M. tuberculosis to PAS, which was largely restored by adding exogenous methionine. Moreover, overexpression of rv2172c in M. tuberculosis could increase methionine production and lead to PAS resistance. This research was the first to identify an MTHFR in M. tuberculosis and revealed that the activity of this enzyme was associated with susceptibility to antifolates. These findings have particular value for anti-tubercular drugs design for the treatment of drug-resistant TB.

Crystal structure of FAD‐independent methylene‐tetrahydrofolate reductase from Mycobacterium hassiacum

“…The heterologously over‐produced protein was purified to homogeneity (Table 1 and Figure 2A). The UV‐visible spectrum of hMfr revealed only one peak at 280 nm, confirming the absence of FAD (Figure 2B), as reported for Mfr from M. smegmatis and M. tuberculosis 17,18 . For crystallization, the purified enzyme fraction was further purified by size‐exclusion chromatography.…”

Crystal structure of FAD-independent methylene-tetrahydrofolate reductase fromMycobacterium hassiacum

“…The UV-visible spectrum of hMfr revealed only one peak at 280 nm, which confirms the absence of FAD (Figure 2B) as reported for Mfr from M. smegmatis and M. tuberclosis . 17 , 18 For crystallization, the purified enzyme fraction was further purified by size exclusion chromatography. hMfr was eluted as a single symmetric peak at an elution volume corresponding to a molecular mass of approximately 30 kDa (Figure 2C), which indicates that the purified hMfr is in a monomeric form because the genome-deduced molecular mass of hMfr is 32 kDa.…”

“…In the previous studies, Mfr of M. tuberculosis was investigated by in vivo mutation experiments. 18 In these experiments, the Arg159Asn and Leu214Ala mutations were introduced into an M. tuberculosis strain . 18 Both strains showed impaired methionine biosynthesis and higher sensitivity to antifolates.…”

“…18 In these experiments, the Arg159Asn and Leu214Ala mutations were introduced into an M. tuberculosis strain . 18 Both strains showed impaired methionine biosynthesis and higher sensitivity to antifolates. Leu214 of Mfr from M. tuberculosis is equivalent to Leu221 of hMfr, which is most likely involved in fixing the phenyl ring of H 4 F. Arg159 from M. tuberculosis corresponds to Arg148 in hMfr.…”

“…Gene deletion experiments revealed the Rv2172c-locus is essential for the growth of M. tuberculosis . 18 Apparently, the non-canonical MTHFR is the only enzyme catalyzing the reduction of methylene-H 4 F in M. tuberculosis. Since the non-canonical MTHFR found in mycobacteria is a new type of methylene-H 4 F reductase, 17 this enzyme is referred to as m ethylene-tetrahydro f olate r eductase (Mfr) in this paper to distinguish it from canonical MTHFR.…”