The glutamyl tail length of the cofactor F420 in the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus.

The cofactor F420 is synthesized by many different organisms and as a redox cofactor, it plays a crucial role in the redox reactions of catabolic and biosynthetic metabolic pathways. It consists of a deazaflavin structure, which is linked via lactate to an oligoglutamate chain, that can vary in length. In the present study, the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus were cultivated on different carbon sources and their coenzyme F420 composition has been assayed by reversed-phase ion-pair high-performance liquid chromatography with fluorometric detection regarding both, overall cofactor F420 production and distribution of F420 glutamyl tail length. In Methanosarcina thermophila cultivated on methanol, acetate, and a mixture of acetate and methanol, the most abundant cofactors were F420-5 and F420-4, whereby the last digit refers to the number of expressed glutamyl rests. By contrast, in the obligate CO2 reducing Methanoculleus thermophilus the most abundant cofactors were F420-3 and F420-4. In Methanosarcina thermophila, the relative proportions of the expressed F420 tail length changed during batch growth on all three carbon sources. Over time F420-3 and F420-4 decreased while F420-5 and F420-6 increased in their relative proportion in comparison to total F420 content. In contrast, in Methanoculleus thermophilus the relative abundance of the different F420 cofactors remained stable. It was also possible to differentiate the two methanogenic Archaea based on the glutamyl tail length of the cofactor F420. The cofactor F420-5 in concentrations >2% could only be assigned to Methanosarcina thermophila. In all four variants a trend for a positive correlation between the DNA concentration and the total concentration of the cofactor could be shown. Except for the variant Methanosarcinathermophila with acetate as sole carbon source the same could be shown between the concentration of the mcrA gene copy number and the total concentration of the cofactor.

Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life.

Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.

Facile Stereoselective Reduction of Prochiral Ketones by using an F420 -dependent Alcohol Dehydrogenase.

Effective procedures for the synthesis of optically pure alcohols are highly valuable. A commonly employed method involves the biocatalytic reduction of prochiral ketones. This is typically achieved by using nicotinamide cofactor-dependent reductases. In this work, we demonstrate that a rather unexplored class of enzymes can also be used for this. We used an F420 -dependent alcohol dehydrogenase (ADF) from Methanoculleus thermophilicus that was found to reduce various ketones to enantiopure alcohols. The respective (S) alcohols were obtained in excellent enantiopurity (>99 % ee). Furthermore, we discovered that the deazaflavoenzyme can be used as a self-sufficient system by merely using a sacrificial cosubstrate (isopropanol) and a catalytic amount of cofactor F420 or the unnatural cofactor FOP to achieve full conversion. This study reveals that deazaflavoenzymes complement the biocatalytic toolbox for enantioselective ketone reductions.

Comparative genomic analyses reveal trehalose synthase genes as the signature in genus Methanoculleus.

To date, the only methanoarchaea isolated directly from methane hydrate bearing sediments were Methanoculleus submarinus Nankai-1T and Methanoculleus sp. MH98A. Here, we provide the genome of Methanoculleus taiwanensis CYW4T isolated from the deep-sea subseafloor sediment at the Deformation Front offshore southwestern Taiwan, where methane hydrate deposits are likely located. Through comparative genomics analyses of nine Methanoculleus strains from various habitats, 2-3 coding genes for trehalose synthases were found in all nine Methanoculleus genomes, which were not detected in other methanogens and are therefore suggested as a signature of genus Methanoculleus among methane-producing archaea. In addition, the structural genes adjacent to trehalose synthase genes are comprised of the signaling module of Per-Arnt-Sim (PAS) domain-containing proteins, Hsp20 family proteins, arabinose efflux permeases and multiple surface proteins with fasciclin-like (FAS) repeat. This indicates that trehalose synthase gene clusters in Methanoculleus might play roles in the response to various stresses and regulate carbon storage and modification of surface proteins through accumulation of trehalose. The non-gas hydrate-associated Methanoculleus strains harbor carbon-monoxide dehydrogenase (cooS/acsA) genes, which are important for the conversion of acetate to methane at the step of CO oxidation/CO2 reduction in acetoclastic methanogens and further implies that these strains may be able to utilize CO for methanogenesis in their natural habitats. In addition, both genomes of M. bourgensis strains MS2T and MAB1 harbor highly abundant transposase genes, which may be disseminated from microbial communities in their habitats, sewage treatment plants and biogas reactors, which are breeding grounds for antibiotic resistance. Through comparative genomic analyses, we gained insight into understanding the life of strictly anaerobic methane-producing archaea in various habitats, especially in methane-based deep-sea ecosystems.

Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay.

Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from Methanoculleus marisnigri JR1 in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min(-1) × 10(-3)), affinity and Michaelis constant (Km) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (Ki) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail.

Structure of a presenilin family intramembrane aspartate protease.

Presenilin and signal peptide peptidase (SPP) are intramembrane aspartyl proteases that regulate important biological functions in eukaryotes. Mechanistic understanding of presenilin and SPP has been hampered by lack of relevant structural information. Here we report the crystal structure of a presenilin/SPP homologue (PSH) from the archaeon Methanoculleus marisnigri JR1. The protease, comprising nine transmembrane segments (TMs), adopts a previously unreported protein fold. The amino-terminal domain, consisting of TM1-6, forms a horseshoe-shaped structure, surrounding TM7-9 of the carboxy-terminal domain. The two catalytic aspartate residues are located on the cytoplasmic side of TM6 and TM7, spatially close to each other and approximately 8 Å into the lipid membrane surface. Water molecules gain constant access to the catalytic aspartates through a large cavity between the amino- and carboxy-terminal domains. Structural analysis reveals insights into the presenilin/SPP family of intramembrane proteases.

Identification of an archaeal presenilin-like intramembrane protease.

BACKGROUND: The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of ß-amyloid peptides (Aß) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. METHODOLOGY AND PRINCIPAL FINDINGS: We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the ß-amyloid precursor protein (APP) without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aß40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. CONCLUSIONS AND SIGNIFICANCE: Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

Temporal evolution of methane cycling and phylogenetic diversity of archaea in sediments from a deep-sea whale-fall in Monterey Canyon, California.

Whale-falls represent localized areas of extreme organic enrichment in an otherwise oligotrophic deep-sea environment. Anaerobic remineralization within these habitats is typically portrayed as sulfidogenic; however, we demonstrate that these systems are also favorable for diverse methane-producing archaeal assemblages, representing up to 40% of total cell counts. Chemical analyses revealed elevated methane and depleted sulfate concentrations in sediments under the whale-fall, as compared to surrounding sediments. Carbon was enriched (up to 3.5%) in whale-fall sediments, as well as the surrounding sea floor to at least 10 m, forming a 'bulls eye' of elevated carbon. The diversity of sedimentary archaea associated with the 2893 m whale-fall in Monterey Canyon (California) varied both spatially and temporally. 16S rRNA diversity, determined by both sequencing and terminal restriction fragment length polymorphism analysis, as well as quantitative PCR of the methyl-coenzyme M reductase gene, revealed that methanogens, including members of the Methanomicrobiales and Methanosarcinales, were the dominant archaea (up to 98%) in sediments immediately beneath the whale-fall. Temporal changes in this archaeal community included the early establishment of methylotrophic methanogens followed by development of methanogens thought to be hydrogenotrophic, as well as members related to the newly described methanotrophic lineage, ANME-3. In comparison, archaeal assemblages in 'reference' sediments collected 10 m from the whale-fall primarily consisted of Crenarchaeota affiliated with marine group I and marine benthic group B. Overall, these results indicate that whale-falls can favor the establishment of metabolically and phylogenetically diverse methanogen assemblages, resulting in an active near-seafloor methane cycle in the deep sea.

Coenzyme binding in F420-dependent secondary alcohol dehydrogenase, a member of the bacterial luciferase family.

F(420)-dependent secondary alcohol dehydrogenase (Adf) from methanogenic archaea is a member of the growing bacterial luciferase family which are all TIM barrel enzymes, most of which with an unusual nonprolyl cis peptide bond. We report here on the crystal structure of Adf from Methanoculleus thermophilicus at 1.8 A resolution in complex with a F(420)-acetone adduct. The knowledge of the F(420) binding mode in Adf provides the molecular basis for modeling F(420) and FMN into the other enzymes of the family. A nonprolyl cis peptide bond was identified as an essential part of a bulge that serves as backstop at the Re-face of F(420) to keep it in a bent conformation. The acetone moiety of the F(420)-acetone adduct is positioned at the Si-face of F(420) deeply buried inside the protein. Isopropanol can be reliably modeled and a hydrogen transfer mechanism postulated. His39 and Glu108 can be identified as key players for binding of the acetone or isopropanol oxygens and for catalysis.