Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. The function of Trx in archaea is, however, unexplored. To help fill this gap, we have investigated this aspect in methanarchaea-strict anaerobes that produce methane, a fuel and greenhouse gas. Bioinformatic analyses suggested that Trx is nearly universal in methanogens. Ancient methanogens that produce methane almost exclusively from H 2 plus CO 2 carried approximately two Trx homologs, whereas nutritionally versatile members possessed four to eight. Due to its simplicity, we studied the Trx system of Methanocaldococcus jannaschii-a deeply rooted hyperthermophilic methanogen growing only on H 2 plus CO 2 . The organism carried two Trx homologs, canonical Trx1 that reduced insulin and accepted electrons from Escherichia coli thioredoxin reductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets representing a range of processes-including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated two selected targets following partial deactivation by O 2 , validating proteomics observations: methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite reductase, a detoxification enzyme. The results suggest that Trx assists methanogens in combating oxidative stress and synchronizing metabolic activities with availability of reductant, making it a critical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it seems possible that Trx functioned originally in metabolic regulation independently of O 2 , thus raising the question whether a complex biological system of this type evolved at least 2.5 billion years ago. methanogenic archaea | redox regulation | hydrothermal vent | early Earth | evolution T hioredoxins (Trxs) are small (∼12-kDa) redox proteins typically bearing a characteristic Cys-Gly-Pro-Cys motif that reduce specific disulfide bonds of selected proteins (1). Reduction alters the biochemical properties of the proteins targeted-e.g., by increasing their activity or solubility (1). Trxs are found in the three domains of life: bacteria, eukarya, and archaea (2). In eukarya and bacteria, the regulatory role of Trx has been shown to span the major aspects of metabolism, including photosynthesis, biosynthesis, replication, transcription, translation, and stress response (1). Trx also acts as an electron donor for enzymes, notably ribonucleotide reductase, phosphoadenosinephosphosulfate reductase, methionine sulfoxide reductase, and peroxiredoxins (1). However, in contrast to the wealth of information for bacteria and eukaryotes, our understanding of archaeal Trx is limited to its biochemical and structural properties (3-9). Its physiological role remains a mystery.To help fill this gap, we have investigated the role of Trx in a group of a...

A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavincontaining NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F 420 (F 420 H 2 ), a stronger reductant with a mid-point redox potential (E 0 ) of ؊360 mV; E 0 of NAD(P)H is ؊320 mV. Because F 420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F 420 H 2 to thioredoxin via proteinbound flavin; K m values for thioredoxin and F 420 H 2 were 6.3 and 28.6 M, respectively. The E 0 of DFTR-bound flavin was approximately ؊389 mV, making electron transfer from NAD(P)H or F 420 H 2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F 420 /F 420 H 2 pair could be as low as ؊425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F 420 -dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F 420 systems involved in microbial pathogenesis and antibiotic production.

Flavin-containing Trx reductase (TrxR) of Thermoplasma acidophilum (Ta), a thermoacidophilic facultative anaerobic archaeon, lacks the structural features for the binding of 2′-phosphate of nicotinamide adenine dinucleotide phosphate (NADPH), and this feature has justified the observed lack of activity with NADPH; NADH has also been reported to be ineffective. Our recent phylogenetic analysis identified Ta-TrxR as closely related to the NADH-dependent enzymes of Thermotoga maritima and Desulfovibrio vulgaris, both being anaerobic bacteria. This observation instigated a reexamination of the activity of the enzyme, which showed that Ta-TrxR is NADH dependent; the apparent Km for NADH was 3.1 μM, a physiologically relevant value. This finding is consistent with the observation that NADH:TrxR has thus far been found primarily in anaerobic bacteria and archaea.

Coenzyme F420 plays a key role in the redox metabolisms of various archaea and bacteria, including Mycobacterium tuberculosis. In M. tuberculosis, F420-dependent reactions have been linked to several virulence factors. F420 carries multiple glutamate residues in the side chain, forming F420-n species (n, number of glutamate residues), and the length of this side chain impacts cellular physiology. M. tuberculosis strains with F420 species carrying shorter side chains exhibit resistance to delamanid and pretomanid, two new tuberculosis (TB) drugs. Thus, the process of polyglutamylation of F420 is of great interest. It has been known from genetic analysis that in mycobacteria an F420-0 γ-glutamyl ligase (FbiB) introduces up to seven glutamate residues into F420. However, purified FbiB of M. tuberculosis (MtbFbiB) is either inefficient or incapable of incorporating more than two glutamates. We found that, in vitro, MtbFbiB synthesized side chains containing up to seven glutamate residues if F420 was presented to the enzyme in a two-electron reduced state (F420H2). Our genetic analysis in Mycobacterium bovis BCG and Mycobacterium smegmatis and an analysis of literature data on M. tuberculosis revealed that in these mycobacteria the polyglutamylation process requires the assistance of F420-dependent glucose-6-phosphate dehydrogenase (Fgd) which reduces F420 to F420H2. We hypothesize that, starting with F420-0H2, the amino-terminal domain of FbiB builds F420-2H2, which is then transferred to the carboxy-terminal domain for further glutamylation; F420-2H2 modifies the carboxy-terminal domain structurally to accommodate longer glutamyl chains. This system is analogous to folylpolyglutamate synthase, which introduces more than one glutamate residue into folate only after this vitamin is reduced to tetrahydrofolate. IMPORTANCE Coenzyme F420-dependent reactions of Mycobacterium tuberculosis, which causes tuberculosis, potentially contributes to the virulence of this bacterium. The coenzyme carries a glutamic acid-derived tail, the length of which influences the metabolism of M. tuberculosis. Mutations that eliminate the production of F420 with longer tails make M. tuberculosis resistant to two new tuberculosis drugs. This report describes that the synthesis of longer glutamyl tails of F420 requires concerted actions of two enzymes, one of which reduces the coenzyme prior to the action of the other, which catalyzes polyglutamylation. This knowledge will help to develop more effective tuberculosis (TB) drugs. Remarkably, the introduction of multiple glutamate residues into the sidechain of folate (vitamin B9) requires similar concerted actions, where one enzyme reduces the vitamin to tetrahydrofolate and the other catalyzes polyglutamylation; folate is required for DNA and amino acid synthesis. Thus, the reported research has also revealed a key similarity between two important cellular systems.

Tall fescue KY-31 is an important primary forage for beef cattle. It carries a fungal endophyte that produces ergovaline, the main cause of tall fescue toxicosis that leads to major revenue loss for livestock producers. The MaxQ, an engineered cultivar, hosts an ergovaline nonproducing strain of the fungus and consequently is nontoxic. However, it is less attractive economically. It is not known how rumen microbiome processes these two forages towards nutrient generation and ergovaline transformation. We have analysed the rumen microbiome compositions of cattle that grazed MaxQ with an intervening KY-31 grazing period using the 16S rRNA-V4 element as an identifier and found that KY-31 remodelled the microbiome substantially, encompassing both cellulolytic and saccharolytic functions. The effect was not evident at the whole microbiome levels but was identified by analysing the sessile and planktonic fractions separately. A move from MaxQ to KY-31 lowered the Firmicutes abundance in the sessile fraction and increased it in planktonic part and caused an opposite effect for Bacteroidetes, although the total abundances of these dominant rumen organisms remained unchanged. The abundances of Fibrobacter , which degrades less degradable fibres, and certain cellulolytic Firmicutes such as Pseudobutyrivibrio and Butyrivibrio 2, dropped in the sessile fraction, and these losses were apparently compensated by increased occurrences of Eubacterium and specific Ruminococcaceae and Lachnospiraceae . A return to MaxQ restored the original Firmicutes and Bacteroidetes distributions. However, several KY-31 induced changes, such as the low abundance of Fibrobacter and Butyrivibrio two remained in place, and their substitutes maintained significant presence. The rumen microbiome was distinct from previously reported faecal microbiomes. In summary, KY-31 and MaxQ were digested in the cattle rumen with distinct consortia and the KY-31-specific features were dominant. The study also identified candidate ergovaline transforming bacteria. It highlighted the importance of analysing sessile and planktonic fractions separately.