Structure of mycobacterial ergothioneine-biosynthesis C-S lyase EgtE.

L-ergothioneine is widely distributed among various microbes to regulate their physiology and pathogenicity within complex environments. One of the key steps in the ergothioneine-biosynthesis pathway, the C-S bond cleavage reaction, uses the pyridoxal 5'-phosphate dependent C-S lyase to produce the final product L-ergothioneine. Here, we present the crystallographic structure of the ergothioneine-biosynthesis C-S lyase EgtE from Mycobacterium smegmatis (MsEgtE) represents the first published structure of ergothioneine-biosynthesis C-S lyases in bacteria and shows the effects of active site residues on the enzymatic reaction. The MsEgtE and the previously reported ergothioneine-biosynthesis C-S lyase Egt2 from Neurospora crassa (NcEgt2) fold similarly. However, discrepancies arise in terms of substrate recognition, as observed through sequence and structure comparison of MsEgtE and NcEgt2. The structural-based sequence alignment of the ergothioneine-biosynthesis C-S lyase from fungi and bacteria shows clear distinctions among the recognized substrate residues, but Arg348 is critical and an extremely conserved residue for substrate recognition. The α14 helix is exclusively found in the bacteria EgtE, which represent the most significant difference between bacteria EgtE and fungi Egt2, possibly resulting from the convergent evolution of bacteria and fungi.

Nanopore Signatures of Nucleoside Drugs.

Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of nucleoside drugs, repurposing of existing drugs, and combined use of multiple drug types have made the rapid sensing of nucleoside drugs urgently needed. Nanopores are emerging single-molecule sensors that have high resolution to resolve even minor structural differences between chemical compounds. Here, an engineered Mycobacterium smegmatis porin A hetero-octamer was used to perform general nucleoside drug analysis. Ten nucleoside drugs were simultaneously detected and fully discriminated. An accuracy of >99.9% was consequently reported. This sensing capacity was further demonstrated in direct nanopore analysis of ribavirin buccal tablets, confirming its sensing reliability against complex samples and environments. No sample separation is needed, however, significantly minimizing the complexity of the measurement. This technique may inspire nanopore applications in pharmaceutical production and pharmacokinetics measurements.

Site-Specific Introduction of Bioorthogonal Handles to Nanopores by Genetic Code Expansion.

Site-specific functionalization of natural amino acid-containing biological nanopores is pivotal in single molecule sensing. However, pore engineering methodologies are restricted to a limited choice and introduction of unnatural chemical components is extremely difficult. Herein we report the genetic code expansion (GCE) strategy to introduce unnatural amino acid (UAA) to an octameric Mycobacterium smegmatis porin A (MspA) nanopore. GCE allows for rapid and efficient introduction of bioorthogonal reactive site (i.e., azide) to the pore rim, and conjugation of single stranded DNA or lysozyme was demonstrated. The lysozyme-conjugated pore was further used for the discrimination of different oligosaccharides, demonstrating a sensing capacity that a bare MspA nanopore does not possess. GCE with bioorthogonal handles, which has never been previously applied in the preparation of nanopores, is a versatile strategy for pore engineering and may further expand the application scenarios of nanopores.

Production and Purification of Phosphatidylinositol Mannosides from Mycobacterium smegmatis Biomass.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The mycobacterial cell wall is vital for cell growth, virulence, and resistance to antibiotics, and thus constitutes a unique target for drug development. To characterize the enzymes catalyzing the synthesis of the cell wall components, considerable amounts of substrates are required. Since many mycobacterial cell wall lipids, particularly phosphatidylinositol mannosides (PIMs), are not commercially available, isolation from cell biomass is the most straightforward way to obtain these compounds. In this study, we optimized a protocol to extract and purify PIM species, in particular Ac1 PIM2 and Ac1 PIM4 , which can be further used for the identification and characterization of target enzymes. PIMs were extracted from Mycobacterium smegmatis mc2 155 ΔPimE using organic solvents, and purified through three consecutive chromatography steps. Thin-layer chromatography (TLC) was used in-between purification steps to evaluate the success of lipid separation, and nuclear magnetic resonance (NMR) was used for product quantification and to assess purity. Typically, from a 60 g batch of M. smegmatis biomass we were able to isolate approximately 9 mg of Ac1 PIM2 and 1.8 mg of Ac1 PIM4 . This is the first time the purification of phosphatidylinositol tetramannoside has been reported. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of M. smegmatis mc2 155 ∆PimE Basic Protocol 2: Extraction of lipids from M. smegmatis mc2 155 ∆PimE Basic Protocol 3: Treatment of the lipid extract for isolation of phospholipids Basic Protocol 4: Isolation of phosphatidylinositol mannosides Basic Protocol 5: Quantification of phosphatidylinositol mannosides.

Control of subunit stoichiometry in single-chain MspA nanopores.

Transmembrane protein channels enable fast and highly sensitive detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level compared with other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Different mutations in specific subunits of a pore protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, individual subunits of MspA and other oligomeric protein pores are randomly assembled in vivo and in vitro, preventing the efficient production of designed pores with different subunit mutations. In this study, we converted octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. Lipid bilayer experiments demonstrated that single-chain MspA formed membrane-spanning channels and discriminated all four nucleotides identical to MspA produced from monomers in DNA hairpin experiments. Single-chain constructs comprising three, five, six, and seven connected subunits assembled to functional channels, demonstrating a remarkable plasticity of MspA to different subunit stoichiometries. Thus, single-chain MspA constitutes a new milestone in the optimization of MspA as a biosensor for DNA sequencing and many other applications by enabling the production of pores with distinct subunit mutations and pore diameters.

MmpL3, the trehalose monomycolate transporter, is stable in solution in several detergents and can be reconstituted into peptidiscs.

Mycobacteria possess a complex and waxy cell wall comprising a large panel of glycolipids. Among these, trehalose monomycolate (TMM) represents abundant and crucial components for the elaboration of the mycomembrane. TMM is synthesized in the cytoplasmic compartment and translocated across the inner membrane by the MmpL3 transporter. Inhibitors impeding TMM transport by targeting MmpL3 show great promises as new antimycobacterials. The recent X-ray or Cryo-EM structures of MmpL3 complexed to TMM or its inhibitors have shed light on the mechanisms of TMM transport and inhibition. So far, purification procedures mainly involved the use of n-Dodecyl-ß-d-Maltopyranoside to solubilize and stabilize MmpL3 from Mycobacterium smegmatis (MmpL3Msm) or Lauryl Maltose Neopentyl Glycol for MmpL3 from Mycobacterium tuberculosis. Herein, we explored the possibility to solubilize and stabilize MmpL3 with other detergents. We demonstrate that several surfactants from the ionic, non-ionic and zwitterionic classes are prone to solubilize MmpL3Msm expressed in Escherichia coli. The capacity of these detergents to stabilize MmpL3Msm was evaluated by size-exclusion chromatography and thermal stability. This study unraveled three new detergents DM, LDAO and sodium cholate that favor solubilization and stabilization of MmpL3Msm in solution. In addition, we report a protocol that allows reconstitution of MmpL3Msm into peptidiscs.

MSMEG_3955 from Mycobacterium smegmatis is a FMN bounded homotrimeric NAD(P)H:Flavin mononucleotide (FMN) oxidoreductase.

BACKGROUND: Tuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb. RESULTS: In this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% ß-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways. CONCLUSION: MSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.

Inside a Shell-Organometallic Catalysis Inside Encapsulin Nanoreactors.

Compartmentalization of chemical reactions inside cells are a fundamental requirement for life. Encapsulins are self-assembling protein-based nanocompartments from the prokaryotic repertoire that present a highly attractive platform for intracellular compartmentalization of chemical reactions by design. Using single-molecule Förster resonance energy transfer and 3D-MINFLUX analysis, we analyze fluorescently labeled encapsulins on a single-molecule basis. Furthermore, by equipping these capsules with a synthetic ruthenium catalyst via covalent attachment to a non-native host protein, we are able to perform in vitro catalysis and go on to show that engineered encapsulins can be used as hosts for transition metal catalysis inside living cells in confined space.

Mass Spectrometry-Based Analysis of Mycobacterial Single-Colony Proteome.

Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."

Secondary structure and transmembrane topology analysis of the N-terminal domain of the inner membrane protein EccE1 from M. smegmatis using site-directed spin labeling EPR.

Protein EccE1 is an essential component of the mycobacterial ESX-1 secretion system, which plays a crucial part in the process of virulence factors secretion, especially for pathogenic mycobacteria such as Mycobacterium tuberculosis. While EccE1 was previously postulated to be the inner membrane pore-forming unit of a membrane complex through which substrates are transported, the structural properties of EccE1 remains to be explored. In the present study, systematic Site-Directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance (EPR) spectroscopic studies was carried out to reveal the secondary structure and transmembrane topology of the N-terminal Domain of EccE1 protein (EccE1-NTD) from M. smegmatis in detergent micelles. EPR-based mobility and accessibility analysis of the R1 side chain for 64 residue positions of EccE1-NTD indicates that the transmembrane domain adopts two α-helices spanning Phe7-Cys30 and Leu36-Ile54. A tentative structural topology model of EccE1-NTD embedded in membrane is also suggested based on EPR spectroscopic data in this study, which will provide further insights into this protein and the ESX secretion systems of mycobacteria.

A Single-Molecule Observation of Dichloroaurate(I) Binding to an Engineered Mycobacterium smegmatis porin A (MspA) Nanopore.

Gold(I) compounds are known to bind sulfur-containing proteins, forming the basis in the design of gold(I)-based drugs. However, the intrinsic molecular mechanism of the chemical reaction is easily hidden when monitored in ensemble. We have previously demonstrated that Mycobacterium smegmatis porin A (MspA) can be engineered (MspA-M) to contain a specialized nanoreactor to probe chemical reactions involving tetrachloroaurate(III). Here, we provide further investigations of coordination interactions between dichloroaurate(I) and MspA-M. Gold compounds of different coordination geometry and valence states are as well probed and evaluated, demonstrating the generality of MspA-M. With single-molecule evidence, MspA-M demonstrates a preference for dichloroaurate(I) than tetrachloroaurate(III), an observation in a single molecule that has never been reported. By counting the maximum number of simultaneous ion bindings, the narrowly confined pore restriction also efficiently distinguishes dichloroaurate(I) and tetrachloroaurate(III) according to their differences in geometry or size. The above demonstration complemented a previous study by demonstrating other possible gold-based single-molecule chemical reactions observable by MspA. These observations bring insights in the understanding of gold-based coordination chemistry in a nanoscale.

Cryo-EM structure of trimeric Mycobacterium smegmatis succinate dehydrogenase with a membrane-anchor SdhF.

Diheme-containing succinate:menaquinone oxidoreductases (Sdh) are widespread in Gram-positive bacteria but little is known about the catalytic mechanisms they employ for succinate oxidation by menaquinone. Here, we present the 2.8 Å cryo-electron microscopy structure of a Mycobacterium smegmatis Sdh, which forms a trimer. We identified the membrane-anchored SdhF as a subunit of the complex. The 3 kDa SdhF forms a single transmembrane helix and this helix plays a role in blocking the canonically proximal quinone-binding site. We also identified two distal quinone-binding sites with bound quinones. One distal binding site is formed by neighboring subunits of the complex. Our structure further reveals the electron/proton transfer pathway for succinate oxidation by menaquinone. Moreover, this study provides further structural insights into the physiological significance of a trimeric respiratory complex II. The structure of the menaquinone binding site could provide a framework for the development of Sdh-selective anti-mycobacterial drugs.

Liposomes derived from Mycobacterium smegmatis promote immune activation of mice bone marrow-derived dendritic cells.

Background: Tuberculosis (TB) is the leading cause of mortality due to infectious diseases. The development of new generation vaccines against TB is of paramount importance for the control of the disease. In previous studies, liposomes obtained from lipids of Mycobacterium smegmatis (LMs) demonstrated their immunogenicity and protective capacity against Mycobacterium tuberculosis in mice. To characterize the immunomodulatory capacity of this experimental vaccine candidate, in the current study, the stimulatory capacity of LMs was determined on bone marrow-derived dendritic cells (BMDCs) from mice. Methods: LMs were obtained and incubated with mature BMDCs. The internalization of LMs by BMDCs was studied by confocal microscopy, and the LMs immune-stimulatory capacity was determined by the expression of surface molecules (CD86 and MHCII) and the cytokine production (interleukin [IL]-12, interferon-Υ, tumor necrosis factor-α, and IL-10) 24 h after exposure to LMs. Results: The interaction of LMs with BMDCs and its internalization was demonstrated as well as the immune activation of BMDCs, characterized by the increased expression of CD86 and the production of IL-12. The LMs internalization and immune activation of BMDCs were blocked in the presence of cytochalasin, filipin III and chlorpromazine, which demonstrated that internalization of LMs by BMDCs is a key process for the LMs induced immune activation of BMDCs. Conclusions: The results obtained support the further evaluation of LMs as a mycobacterial vaccine, adjuvant, and in immunotherapy.

Rapid, direct detection of bacterial topoisomerase 1-DNA adducts by RADAR/ELISA.

Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by "RADAR fractionation", a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches.

Phosphorus NMR and Its Application to Metabolomics.

Stable isotopes are routinely employed by NMR metabolomics to highlight specific metabolic processes and to monitor pathway flux. 13C-carbon and 15N-nitrogen labeled nutrients are convenient sources of isotope tracers and are commonly added as supplements to a variety of biological systems ranging from cell cultures to animal models. Unlike 13C and 15N, 31P-phosphorus is a naturally abundant and NMR active isotope that does not require an external supplemental source. To date, 31P NMR has seen limited usage in metabolomics because of a lack of reference spectra, difficulties in sample preparation, and an absence of two-dimensional (2D) NMR experiments, but 31P NMR has the potential of expanding the coverage of the metabolome by detecting phosphorus-containing metabolites. Phosphorylated metabolites regulate key cellular processes, serve as a surrogate for intracellular pH conditions, and provide a measure of a cell's metabolic energy and redox state, among other processes. Thus, incorporating 31P NMR into a metabolomics investigation will enable the detection of these key cellular processes. To facilitate the application of 31P NMR in metabolomics, we present a unified protocol that allows for the simultaneous and efficient detection of 1H-, 13C-, 15N-, and 31P-labeled metabolites. The protocol includes the application of a 2D 1H-31P HSQC-TOCSY experiment to detect 31P-labeled metabolites from heterogeneous biological mixtures, methods for sample preparation to detect 1H-, 13C-, 15N-, and 31P-labeled metabolites from a single NMR sample, and a data set of one-dimensional (1D) 31P NMR and 2D 1H-31P HSQC-TOCSY spectra of 38 common phosphorus-containing metabolites to assist in metabolite assignments.

Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines.

Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.

Structure of subunit I of the anthranilate synthase complex of Mycolicibacterium smegmatis.

The tryptophan biosynthesis pathway, which does not exist in mammals, is highly conserved in Mycobacterium. Anthranilate synthase (AS) catalyzes the initial reactions in the tryptophan biosynthesis pathway in many microorganisms, catalyzing the conversion of glutamine and chorismate to form pyruvate and anthranilate. Here, the crystal structure of anthranilate synthase component I (AS I) from Mycolicibacterium smegmatis (MsTrpE) has been determined to 1.7 Å resolution. MsTrpE crystallizes in the space group P1 with two monomers in the asymmetric unit, which is consistent with the oligomeric state in solution as confirmed by analytical ultracentrifugation. Inspection of the active site shows that it is in the active form with a bound Mg2+ ion and a ligand that is modelled as benzoate. The position of benzoate mimics the position of the anthranilate product in the active site. The structure of MsTrpE will provide a starting point for the investigation of latent biotechnology and pharmaceutical applications of anthranilate synthase component I.

The ABC exporter IrtAB imports and reduces mycobacterial siderophores.

Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins1. M. tuberculosis produces two classes of siderophore, lipid-bound mycobactin and water-soluble carboxymycobactin2,3. Functional studies have revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ATP binding cassette (ABC) transporter IrtAB4, which features an additional cytoplasmic siderophore interaction domain5. However, the predicted ABC exporter fold of IrtAB is seemingly contradictory to its import function. Here we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the siderophore interaction domain to facilitate iron release. Structure determination by X-ray crystallography and cryo-electron microscopy not only confirms that IrtAB has an ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB that result in a partially collapsed inward-facing substrate-binding cavity. The siderophore interaction domain is positioned in close proximity to the inner membrane leaflet, enabling the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB has a preference for mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore-import machinery in mycobacteria.

MSMEG_2432 of Mycobacterium smegmatis mc2155 is a dual function enzyme that exhibits DD-carboxypeptidase and ß-lactamase activities.

Mycobacterial peptidoglycan (PG) is an unsolved puzzle due to its complex structure and involvement of multiple enzymes in the process of its remodelling. dd-Carboxypeptidases are low molecular mass penicillin-binding proteins (LMM-PBPs) that catalyzes the cleavage of terminal d-Ala of muramyl pentapeptide branches and thereby helps in the PG remodelling process. Here, we have assigned the function of a putative LMM-PBP, MSMEG_2432 of Mycobacterium smegmatis, by showing that it exhibits both dd-CPase and ß-lactamase activities. Like conventional dd-CPase (PBP5 from E. coli), upon ectopic complementation in a deformed seven PBP deletion mutant of E. coli, MSMEG_2432 has manifested its ability to restore ~75 % of the cell population to their normal rod shape. Further, in vitrodd-CPase assay has confirmed its ability to release terminal d-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending with d-Ala-d-Ala. Also, elevated resistance against penicillins and cephalosporins upon ectopic expression of MSMEG_2432 suggests the presence of ß-lactamase activity, which is further confirmed in vitro through nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG_2432 influences both of its in vivo and in vitrodd-CPase and ß-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses both dd-CPase and ß-lactamase activities.

Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases from Mycobacterium smegmatis Lack Flavin Coenzyme.

5,10-Methylenetetrahydrofolate reductase (MetF/MTHFR) is an essential enzyme in one-carbon metabolism for de novo biosynthesis of methionine. Our in vivo and in vitro analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR in Mycobacterium smegmatis, failed to reveal their function as MTHFRs. However, we identified two hypothetical proteins, MSMEG_6596 and MSMEG_6649, as noncanonical MTHFRs in the bacterium. MTHFRs are known to be oligomeric flavoproteins. Both MSMEG_6596 and MSMEG_6649 are monomeric proteins and lack flavin coenzymes. In vitro, the catalytic efficiency (kcat/Km ) of MSMEG_6596 (MTHFR1) for 5,10-CH2-THF and NADH was ∼13.5- and 15.3-fold higher than that of MSMEG_6649 (MTHFR2). Thus, MSMEG_6596 is the major MTHFR. This interpretation was further supported by better rescue of the E. coli Δmthfr strain by MTHFR1 than by MTHFR2. As identified by liquid chromatography-tandem mass spectrometry, the product of MTHFR1- or MTHFR2-catalyzed reactions was 5-CH3-THF. The M. smegmatis Δmsmeg_6596 strain was partially auxotrophic for methionine and grew only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the Δmsmeg_6596 strain was more sensitive to folate pathway inhibitors (sulfachloropyridazine, p-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are two noncanonical MTHFR proteins that are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.IMPORTANCE MTHFR/MetF is an essential enzyme in a one-carbon metabolic pathway for de novo biosynthesis of methionine. MTHFRs are known to be oligomeric flavoproteins. Our in vivo and in vitro analyses of Mycobacterium smegmatis MSMEG_6664/MSMEI_6484, annotated as putative MTHFR, failed to reveal their function as MTHFRs. However, we identified two of the hypothetical proteins, MSMEG_6596 and MSMEG_6649, as MTHFR1 and MTHFR2, respectively. Interestingly, both MTHFRs are monomeric and lack flavin coenzymes. M. smegmatis deleted for the major mthfr (mthfr1) was partially auxotroph for methionine and more sensitive to folate pathway inhibitors (sulfachloropyridazine, para-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are novel MTHFRs involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.