Utilisation of 10-formyldihydrofolate as substrate by dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) tranformylase/IMP cyclohydrolase (PurH) in Escherichia coli

Sah, Shivjee; Shah, Riyaz Ahmad; Govindan, Ashwin; Varada, Rajagopal; Rex, Kervin; Varshney, Umesh

doi:10.1099/mic.0.000671

Cited by 5 publications

(9 citation statements)

References 38 publications

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“…However, in the presence of IPTG, the growth of the strain harboring pET-msmeg_6596 showed a longer lag phase, indicating toxicity upon its overexpression. Thus, to further investigate the efficiency of the genetic complementation, we exploited an earlier observation (5) showing that the pET constructs afford low-level expression even in E. coli lacking the T7 RNA polymerase gene. Under these conditions, the E. coli KL16 ⌬mthfr strain harboring pET-msmeg_6596 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases fromMycobacterium smegmatisLack Flavin Coenzyme

Sah¹,

Lahry²,

Talwar³

et al. 2020

J Bacteriol

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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.

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Section: Resultsmentioning

confidence: 99%

“…catalyzing interconversions of the pathway intermediates are highly conserved in all life forms (1)(2)(3)(4)(5). Serine hydroxymethyltransferase (GlyA) catalyzes the reversible reaction of the conversion of serine and THF to glycine and 5,10-methylenetetrahydrofolate (5,10-CH 2 -THF) (6).…”

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confidence: 99%

Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases fromMycobacterium smegmatisLack Flavin Coenzyme

Sah¹,

Lahry²,

Talwar³

et al. 2020

J Bacteriol

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“…While it is known that 10-CHO-DHF can be utilized by DHFR and PurH [18], it is not known if 10-CHO-DHF is also a natural metabolite in bacteria. Thus, we asked if treatment of the ∆ purH strain (deleted for PurH) with TMP (to inhibit DHFR) would lead to accumulation of 10-CHO-DHF.…”

Section: Resultsmentioning

confidence: 99%

“…Engineering 10-CHo-dHF uptake in E. coli complements folate homeostasis and formylation of tRnA fMet DHFR and PurH can utilize 10-CHO-DHF as substrate (in vitro and in vivo) to form 10-CHO-THF and DHF, respectively [18]. Thus, to investigate for 10-CHO-DHF utilization by Fmt in vivo, we decided to make use of a plasmid-borne gene encoding a folate transporter (on p-FBT, Tet R ) to enable E. coli to obtain folates from the medium [30].…”

Section: Resultsmentioning

confidence: 99%

Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action

Sah

Varshney

2023

Microbiology

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Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNAfMet to fMet-tRNAfMet is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH2-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using in vivo and in vitro approaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNAfMet. Dihydrofolate (DHF) formed as a by-product in the in vitro assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆fmt strain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment to Escherichia coli showed a decrease in the reduced folate species (THF, 5,10-CH2-THF, 5-CH3-THF, 5,10-CH+-THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNAfMet.

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“…In addition, the changes in the intestinal microbiome diversity are also accompanied by changes in bacterial differential gene expression and metabolic pathways. According to a query of the KEGG database, the changes in differential genes and pathways are related to the bacterial stress response (Herrou et al, 2017), glycolysis-related energy metabolism (Davies et al, 2011), the formation of galactose (Gao et al, 2019), and the target of antibiotic sterilization on bacteria (Liu et al, 2009;Liu et al, 2011;Sah et al, 2018) and the production of bacterial resistance (Behmard et al, 2019). However, at present, we can only observe the changes in genes and metabolic pathways.…”

Section: Discussionmentioning

confidence: 99%

Effects of Sevoflurane Inhalation Anesthesia on the Intestinal Microbiome in Mice

Han

Zhang

Guo

et al. 2021

Front. Cell. Infect. Microbiol.

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In recent years, more and more attention has been paid to intestinal microbiome. Almost all operations will go through the anesthesia process, but it is not clear whether the intervention of anesthesia alone will affect the change in the intestinal microbiome. The purpose of this study was to verify the effect of sevoflurane inhalation anesthesia on the intestinal microbiome. The animal in the experimental group was used to provide sevoflurane inhalation anesthesia for 4 hours. The control group was not intervened. The feces of the experimental group and the control group were collected on the 1st, 3rd, 7th and 14th days after anesthesia. Sevoflurane inhalation anesthesia will cause changes in the intestinal microbiome of mice. It appears on the 1st day after anesthesia and is most obvious on the 7th day. The specific manifestation is that the abundance of microbiome and the diversity of the microbiome is reduced. At the same time, Untargeted metabonomics showed that compared with the control group, the experimental group had more increased metabolites related to the different microbiome, among which 5-methylthioadenosine was related to the central nervous system. Subsequently, the intestinal microbiome diversity of mice showed a trend of recovery on the 14th day. At the genus level, the fecal samples obtained on the 14th day after anesthesia exhibited significantly increased abundances of Bacteroides, Alloprevotella, and Akkermansia and significantly decreased abundances of Lactobacillus compared with the samples obtained on the 1st day after anesthesia. However, the abundance of differential bacteria did not recover with the changing trend of diversity. Therefore, we believe that sevoflurane inhalation anesthesia is associated with changes in the internal microbiome and metabolites, and this change may be completed through the brain-gut axis, while sevoflurane inhalation anesthesia may change the intestinal microbiome for as long as 14 days or longer.

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Utilisation of 10-formyldihydrofolate as substrate by dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) tranformylase/IMP cyclohydrolase (PurH) in Escherichia coli

Cited by 5 publications

References 38 publications

Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases fromMycobacterium smegmatisLack Flavin Coenzyme

Monomeric NADH-Oxidizing Methylenetetrahydrofolate Reductases fromMycobacterium smegmatisLack Flavin Coenzyme

Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action

Effects of Sevoflurane Inhalation Anesthesia on the Intestinal Microbiome in Mice

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