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Potency boost of a Mycobacterium tuberculosis dihydrofolate reductase inhibitor by multienzyme F₄₂₀H₂-dependent reduction.

ABSTRACT: Triaza-coumarin (TA-C) is a Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor with an IC₅₀ (half maximal inhibitory concentration) of ∼1 µM against the enzyme. Despite this moderate target inhibition, TA-C shows exquisite antimycobacterial activity (MIC₅₀, concentration inhibiting growth by 50% = 10 to 20 nM). Here, we investigated the mechanism underlying this potency disconnect. To confirm that TA-C targets DHFR and investigate its unusual potency pattern, we focused on resistance mechanisms. In Mtb, resistance to DHFR inhibitors is frequently associated with mutations in thymidylate synthase thyA, which sensitizes Mtb to DHFR inhibition, rather than in DHFR itself. We observed thyA mutations, consistent with TA-C interfering with the folate pathway. A second resistance mechanism involved biosynthesis of the redox coenzyme F₄₂₀ Thus, we hypothesized that TA-C may be metabolized by Mtb F₄₂₀-dependent oxidoreductases (FDORs). By chemically blocking the putative site of FDOR-mediated reduction in TA-C, we reproduced the F₄₂₀-dependent resistance phenotype, suggesting that F₄₂₀H₂-dependent reduction is required for TA-C to exert its potent antibacterial activity. Indeed, chemically synthesized TA-C-Acid, the putative product of TA-C reduction, displayed a 100-fold lower IC₅₀ against DHFR. Screening seven recombinant Mtb FDORs revealed that at least two of these enzymes reduce TA-C. This redundancy in activation explains why no mutations in the activating enzymes were identified in the resistance screen. Analysis of the reaction products confirmed that FDORs reduce TA-C at the predicted site, yielding TA-C-Acid. This work demonstrates that intrabacterial metabolism converts TA-C, a moderately active "prodrug," into a 100-fold-more-potent DHFR inhibitor, thus explaining the disconnect between enzymatic and whole-cell activity.

SUBMITTER: Aragaw WW

PROVIDER: S-EPMC8237569 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Anaerobic methanotrophic archaea (ANME), which oxidize methane in marine sediments through syntrophic associations with sulfate-reducing bacteria, carry homologs of coenzyme F420-dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a hyperthermophilic methanogen from deep-sea hydrothermal vents. M. jannaschii Fsr (MjFsr) and ANME-Fsr belong to two phylogenetically distinct groups, FsrI and FsrII, respectively. MjFsrI reduces sulfite to sulfide with reduced F420 (F420H2), protecting methyl coenzyme M reductase (Mcr), an essential enzyme for methanogens, from sulfite inhibition. However, the function of FsrIIs in ANME, which also rely on Mcr and live in sulfidic environments, is unknown. We have determined the catalytic properties of FsrII from a member of ANME-2c. Since ANME remain to be isolated, we expressed ANME2c-FsrII in a closely related methanogen, Methanosarcina acetivorans. Purified recombinant FsrII contained siroheme, indicating that the methanogen, which lacks a native sulfite reductase, produced this coenzyme. Unexpectedly, FsrII could not reduce sulfite or thiosulfate with F420H2. Instead, it acted as an F420H2-dependent nitrite reductase (FNiR) with physiologically relevant Km values (nitrite, 5 μM; F420H2, 14 μM). From kinetic, thermodynamic, and structural analyses, we hypothesize that in FNiR, F420H2-derived electrons are delivered at the oxyanion reduction site at a redox potential that is suitable for reducing nitrite (E0' [standard potential], +440 mV) but not sulfite (E0', -116 mV). These findings and the known nitrite sensitivity of Mcr suggest that FNiR may protect nondenitrifying ANME from nitrite toxicity. Remarkably, by reorganizing the reductant processing system, Fsr transforms two analogous oxyanions in two distinct archaeal lineages with different physiologies and ecologies. IMPORTANCE Coenzyme F420-dependent sulfite reductase (Fsr) protects methanogenic archaea inhabiting deep-sea hydrothermal vents from the inactivation of methyl coenzyme M reductase (Mcr), one of their essential energy production enzymes. Anaerobic methanotrophic archaea (ANME) that oxidize methane and rely on Mcr, carry Fsr homologs that form a distinct clade. We show that a member of this clade from ANME-2c functions as F420-dependent nitrite reductase (FNiR) and lacks Fsr activity. This specialization arose from a distinct feature of the reductant processing system and not the substrate recognition element. We hypothesize FNiR may protect ANME Mcr from inactivation by nitrite. This is an example of functional specialization within a protein family that is induced by changes in electron transfer modules to fit an ecological need.

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Potency boost of a Mycobacterium tuberculosis dihydrofolate reductase inhibitor by multienzyme F₄₂₀H₂-dependent reduction.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Potency boost of a Mycobacterium tuberculosis dihydrofolate reductase inhibitor by multienzyme F420H2-dependent reduction.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Potency boost of a Mycobacterium tuberculosis dihydrofolate reductase inhibitor by multienzyme F₄₂₀H₂-dependent reduction.