Is reduced ferredoxin the physiological electron donor for MetVF-type methylenetetrahydrofolate reductases in acetogenesis? A hypothesis.
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ABSTRACT:
SUBMITTER: Oppinger C
PROVIDER: S-EPMC8760232 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Linear tetrapyrroles (bilins) are produced from heme by heme oxygenase, usually forming biliverdin IXα (BV). Fungi and bacteria use BV as chromophore for phytochrome photoreceptors. Oxygenic photosynthetic organisms use BV as a substrate for ferredoxin-dependent bilin reductases (FDBRs), enzymes that produce diverse reduced bilins used as light-harvesting pigments in phycobiliproteins and as photoactive photoreceptor chromophores. Bilin biosynthesis is essential for phototrophic growth in Chlamydomonas reinhardtii despite the absence of phytochromes or phycobiliproteins in this organism, raising the possibility that bilins are more generally required for phototrophic growth by algae. We here leverage the recent expansion in available algal transcriptomes, cyanobacterial genomes, and environmental metagenomes to analyze the distribution and diversification of FDBRs. With the possible exception of euglenids, FDBRs are present in all photosynthetic eukaryotic lineages. Phylogenetic analysis demonstrates that algal FDBRs belong to the three previously recognized FDBR lineages. Our studies provide new insights into FDBR evolution and diversification.
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Project description: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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Project description:Ferredoxin-dependent thioredoxin reductase was identified 35 y ago in the fermentative bacterium Clostridium pasteurianum [Hammel KE, Cornwell KL, Buchanan BB (1983) Proc Natl Acad Sci USA 80:3681-3685]. The enzyme, a flavoprotein, was strictly dependent on ferredoxin as reductant and was inactive with either NADPH or NADH. This early work has not been further pursued. We have recently reinvestigated the problem and confirmed that the enzyme, here designated ferredoxin-dependent flavin thioredoxin reductase (FFTR), is a flavoprotein. The enzyme differs from ferredoxin-thioredoxin reductase (FTR), which has a signature [4Fe-4S] cluster, but shows structural similarities to NADP-dependent thioredoxin reductase (NTR). Comparative amino acid sequence analysis showed that FFTR is present in a number of clostridial species, some of which lack both FTR and an archetypal NTR. We have isolated, crystallized, and determined the structural properties of FFTR from a member of this group, Clostridium acetobutylicum, both alone and in complex with Trx. The structures showed an elongated FFTR homodimer, each monomer comprising two Rossmann domains and a noncovalently bound FAD cofactor that exposes the isoalloxazine ring to the solvent. The FFTR structures revealed an alternative domain organization compared with NTR that enables the enzyme to accommodate Fdx rather than NADPH. The results suggest that FFTR exists in a range of conformations with varying degrees of domain separation in solution and that the stacking between the two redox-active groups for the transfer of reducing equivalents results in a profound structural reorganization. A mechanism in accord with the findings is proposed.
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