MTHFS Research Articles

Functional study on candidate regulators mediating the expression of CYP6B7 induced by fenvalerate in a susceptible strain of Helicoverpa armigera

Transcription factors play an important role in regulating the expression of detoxification genes (e.g. P450s) that confer insecticide resistance. Our previous study identified a series of candidate transcription factors (CYP6B7-fenvalerate association proteins, CAPs) that may be related to fenvalerate-induced expression of CYP6B7 in a field HDTJ strain of H. armigera. Whether these CAPs can mediate the transcript of CYP6B7 induced by fenvalerate in a susceptible HDS strain of H. armigera remains unknown. Further study showed that the expression levels of multiple CAPs were significantly induced by fenvalerate in HDS strain. Knockdown of CAP19 [fatty acid synthase-like (FAS)], CAP22 [polysaccharide biosynthesis domain-containing protein 1 (PBDC1)], CAP24 [5-formyltetrahydrofolate cycloligase (5-FCL)], CAP30 [peptidoglycan recognition protein LB-like (PGRP)] and CAP33 [NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 (NDUFA11)] resulted in significant inhibition of CYP6B7 and some other P450 genes expression; meanwhile, the sensitivity of HDS strain larvae to fenvalerate was significantly increased. In addition, PBDC1, PGRP and NDUFA11, either alone or in combination, could significantly enhance the activity of CYP6B7 promoter in HDS strain, as well as the expression level of CYP6B7 gene in Sf9 cells line. These results suggested that PBDC1, PGRP and NDUFA11 may be involved in the transcript regulation of key detoxifying genes in response to fenvalerate in HDS strain of H. armigera.

A genome-wide association study of folates in sweet corn kernels.

Folate is commonly synthesized in natural plants and is an essential water-soluble vitamin of great importance inhuman health. Although the key genes involved in folate biosynthesis and transformation pathways have been identified in plants, the genetic architecture of folate in sweet corn kernels remain largely unclear. In this study, an association panel of 295 inbred lines of sweet corn was constructed. Six folate derivatives were quantified in sweet corn kernels at 20 days after pollination and a total of 95 loci were identified for eight folate traits using a genome-wide association study. A peak GWAS signal revealed that natural variation in ZmFCL, encoding a 5-formyltetrahydrofolate cyclo-ligase, accounted for 30.12% of phenotypic variation in 5-FTHF content. Further analysis revealed that two adjacent SNPs on the second exon resulting in an AA-to-GG in the gene and an Asn-to-Gly change in the protein could be the causative variant influencing 5-FTHF content. Meanwhile, 5-FTHF content was negatively correlated with ZmFCL expression levels in the population. These results extend our knowledge regarding the genetic basis of folate and provide molecular markers for the optimization of folate levels in sweet corn kernels.

Engineering of Biosynthesis Pathway and NADPH Supply for Improved L-5-Methyltetrahydrofolate Production by Lactococcus lactis.

L-5-methyltetrahydrofolate (5-MTHF) is one of the biological active forms of folate, which is widely used as a nutraceutical. However, low yield and serious pollution associated with the chemical synthesis of 5-MTHF hampers its sustainable supply. In this study, 5-MTHF production was improved by engineering the 5-MTHF biosynthesis pathway and NADPH supply in Lactococcus lactis for developing a green and sustainable biosynthesis approach. Specifically, overexpressing the key rate-limiting enzyme methylenetetrahydrofolate reductase led to intracellular 5-MTHF accumulation, reaching 18 μg/l. Next, 5-MTHF synthesis was further enhanced by combinatorial overexpression of 5-MTHF synthesis pathway enzymes with methylenetetrahydrofolate reductase, resulting in 1.7-fold enhancement. The folate supply pathway was strengthened by expressing folE encoding GTP cyclohydrolase I, which increased 5-MTHF production 2.4-fold to 72 μg/l. Furthermore, glucose-6-phosphate dehydrogenase was overexpressed to improve the redox cofactor NADPH supply for 5-MTHF biosynthesis, which led to a 60% increase in intracellular NADPH and a 35% increase in 5-MTHF production (97 μg/l). To reduce formation of the by-product 5-formyltetrahydrofolate, overexpression of 5-formyltetrahydrofolate cyclo-ligase converted 5-formyltetrahydrofolate to 5,10-methyltetrahydrofolate, which enhanced the 5-MTHF titer to 132 μg/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 μg/l. To the best of our knowledge, this titer is the highest achieved by L. lactis. This study lays the foundation for further engineering of L. lactis for efficient 5-MTHF biosynthesis.

Hepatopancreas transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with white hepatopancreas syndrome

White hepatopancreas is a syndrome that has recently emerged in aquaculture of Chinese mitten crab (Eriocheir sinensis). High lethality of the disease caused large economic loss, which drew considerable attention of fish farmers and scientific researchers. In this study, hepatopancreas reference transcriptome was de novo assembled and differential expression analysis was conducted between white hepatopancreas and normal (yellow) hepatopancreas of E. sinensis. A total of 90,687 transcripts were assembled, and 27,387 were annotated. Transcriptomic comparison revealed 69 differentially expressed genes between individuals featuring white hepatopancreas and yellow hepatopancreas. Genes associated with immune response and cell death, include thioredoxin-related transmembrane protein 1, hemocytin, methuselah-like 1, and E3 ubiquitin-protein ligase, and they were up-regulated, whereas titin and 5-formyltetrahydrofolate cyclo-ligase, which are genes related to cell proliferation, were down-regulated in E. sinensis with white hepatopancreas syndrome. Our study provides novel insights into genetic causes of formation and novel gene markers for detection of white hepatopancreas syndrome in aquaculture of E. sinensis.

The structure of Enterococcus faecalis thymidylate synthase provides clues about folate bacterial metabolism.

Drug resistance to therapeutic antibiotics poses a challenge to the identification of novel targets and drugs for the treatment of infectious diseases. Infections caused by Enterococcus faecalis are a major health problem. Thymidylate synthase (TS) from E. faecalis is a potential target for antibacterial therapy. The X-ray crystallographic structure of E. faecalis thymidylate synthase (EfTS), which was obtained as a native binary complex composed of EfTS and 5-formyltetrahydrofolate (5-FTHF), has been determined. The structure provides evidence that EfTS is a half-of-the-sites reactive enzyme, as 5-FTHF is bound to two of the four independent subunits present in the crystal asymmetric unit. 5-FTHF is a metabolite of the one-carbon transfer reaction catalysed by 5-formyltetrahydrofolate cyclo-ligase. Kinetic studies show that 5-FTHF is a weak inhibitor of EfTS, suggesting that the EfTS-5-FTHF complex may function as a source of folates and/or may regulate one-carbon metabolism. The structure represents the first example of endogenous 5-FTHF bound to a protein involved in folate metabolism.

A 5-formyltetrahydrofolate cycloligase paralog from all domains of life: comparative genomic and experimental evidence for a cryptic role in thiamin metabolism.

A paralog (here termed COG0212) of the ATP-dependent folate salvage enzyme 5-formyltetrahydrofolate cycloligase (5-FCL) occurs in all domains of life and, although typically annotated as 5-FCL in pro- and eukaryotic genomes, is of unknown function. COG0212 is similar in overall structure to 5-FCL, particularly in the substrate binding region, and has distant similarity to other kinases. The Arabidopsis thaliana COG0212 protein was shown to be targeted to chloroplasts and to be required for embryo viability. Comparative genomic analysis revealed that a high proportion (19%) of archaeal and bacterial COG0212 genes are clustered on the chromosome with various genes implicated in thiamin metabolism or transport but showed no such association between COG0212 and folate metabolism. Consistent with the bioinformatic evidence for a role in thiamin metabolism, ablating COG0212 in the archaeon Haloferax volcanii caused accumulation of thiamin monophosphate. Biochemical and functional complementation tests of several known and hypothetical thiamin-related activities (involving thiamin, its breakdown products, and their phosphates) were, however, negative. Also consistent with the bioinformatic evidence, the COG0212 proteins from A. thaliana and prokaryote sources lacked 5-FCL activity in vitro and did not complement the growth defect or the characteristic 5-formyltetrahydrofolate accumulation of a 5-FCL-deficient (ΔygfA) Escherichia coli strain. We therefore propose (a) that COG0212 has an unrecognized yet sometimes crucial role in thiamin metabolism, most probably in salvage or detoxification, and (b) that is not a 5-FCL and should no longer be so annotated.

Bacterial Conversion of Folinic Acid Is Required for Antifolate Resistance

Antifolates, which are among the first antimicrobial agents invented, inhibit cell growth by creating an intracellular state of folate deficiency. Clinical resistance to antifolates has been mainly attributed to mutations that alter structure or expression of enzymes involved in de novo folate synthesis. We identified a Mycobacterium smegmatis mutant, named FUEL (which stands for folate utilization enzyme for leucovorin), that is hypersusceptible to antifolates. Chemical complementation indicated that FUEL is unable to metabolize folinic acid (also known as leucovorin or 5-formyltetrahydrofolate), whose metabolic function remains unknown. Targeted mutagenesis, genetic complementation, and biochemical studies showed that FUEL lacks 5,10-methenyltetrahydrofolate synthase (MTHFS; also called 5-formyltetrahydrofolate cyclo-ligase; EC 6.3.3.2) activity responsible for the only ATP-dependent, irreversible conversion of folinic acid to 5,10-methenyltetrahydrofolate. In trans expression of active MTHFS proteins from bacteria or human restored both antifolate resistance and folinic acid utilization to FUEL. Absence of MTHFS resulted in marked cellular accumulation of polyglutamylated species of folinic acid. Importantly, MTHFS also affected M. smegmatis utilization of monoglutamylated 5-methyltetrahydrofolate exogenously added to the medium. Likewise, Escherichia coli mutants lacking MTHFS became susceptible to antifolates. These results indicate that folinic acid conversion by MTHFS is required for bacterial intrinsic antifolate resistance and folate homeostatic control. This novel mechanism of antimicrobial antifolate resistance might be targeted to sensitize bacterial pathogens to classical antifolates.

Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489).

Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 A resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted.

Structural and functional characterization of a 5,10‐methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087)

Mycoplasma pneumoniae 5,10-methenyltetrahydrofolate synthetase [MTHFS; also known as 5-formyltetrahydrofolate cycloligase; Enzyme Commission (EC) 6.3.3.2] belongs to a large cycloligase protein family with 97 sequence homologues from bacteria to human. To help define the molecular (biochemical and biophysical) function of the M. pneumoniae MTHFS, we have previously determined its crystal structure at 2.2 A resolution (Chen et al., Proteins 2004;56:839-843). In this current study, activity assays confirmed the functionality of the recombinant protein, with K(m) = 165 microM for 5-formyltetrahydrofolate (5-FTHF) and K(m) = 166 microM for MgATP. The methenyltetrahydrofolate activity of M. pneumoniae MTHFS has a requirement for divalent metal ions with Mg2+ being most effective, and an absolute requirement for nucleoside 5'-triphosphates with adenosine triphosphate (ATP) being most effective. Crystallization in the presence of substrates (MgATP, with or without 5-FTHF) produced the complex structures of the protein with adenosine diphosphate (ADP) and phosphate at 2.2 A resolution; with ADP, phosphate, and 5-FTHF at 2.5 A resolution. These structures directly demonstrated that the role of Mg2+ in the reaction is to form the ATP--Mg2+-enzyme complex.

5-Formyltetrahydrofolate Is an Inhibitory but Well Tolerated Metabolite in Arabidopsis Leaves

5-Formyltetrahydrofolate (5-CHO-THF) is formed via a second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 micromol of CO2 mol(-1)) and non-photorespiratory (3200 micromol of CO2 mol(-1)) conditions. The mutation had only mild visible effects at 370 micromol of CO2 mol(-1), reducing growth rate by approximately 20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 micromol of CO2 mol(-1), the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants.

Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog

By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its γ-proteobacterial homologs (∼185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the γ-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the γ-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

Cloning and Characterization of Mitochondrial 5-Formyltetrahydrofolate Cycloligase from Higher Plants

5-Formyltetrahydrofolate cycloligase (5-FCL) catalyzes the conversion of 5-formyltetrahydrofolate (5-CHO-H(4)PteGlu(n)) to 5,10-methenyltetrahydrofolate and is considered to be the main means whereby 5-CHO-H(4)PteGlu(n) is metabolized in mammals, yeast, and bacteria. 5-CHO-H(4)PteGlu(n) is known to occur in plants and to be highly abundant in leaf mitochondria. Genomics-based approaches identified Arabidopsis and tomato cDNAs encoding proteins homologous to 5-FCLs of other organisms but containing N-terminal extensions with the features of mitochondrial targeting peptides. These homologs were shown to have 5-FCL activity by characterizing recombinant enzymes produced in Escherichia coli and by functional complementation of a yeast fau1 mutation with the Arabidopsis 5-FCL cDNA. The recombinant Arabidopsis enzyme is active as a monomer, prefers the penta- to the monoglutamyl form of 5-CHO-H(4)PteGlu(n), and has kinetic properties broadly similar to those of 5-FCLs from other organisms. Enzyme assays and immunoblot analyses indicated that 5-FCL is located predominantly if not exclusively in plant mitochondria and that the mature, active enzyme lacks the putative targeting sequence. Serine hydroxymethyltransferase (SHMT) from plant mitochondria was shown to be inhibited by 5-CHO-H(4)PteGlu(n) as are SHMTs from other organisms. Since mitochondrial SHMT is crucial to photorespiration, 5-FCL may help prevent 5-CHO-H(4)PteGlu(n) from reaching levels that would inhibit this process. Consistent with this possibility, 5-FCL activity was far higher in leaf mitochondria than root mitochondria.