The missing enzymatic link in syntrophic methane formation from fatty acids.

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H2, CO2, or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO2 to methane by electrons derived from ß-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop-driven reduction of CO2 by acyl-coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of ß-oxidation in (methyl)menaquinone-containing organisms.

Sequence analysis and specificity of distinct types of menaquinone methyltransferases indicate the widespread potential of methylmenaquinone production in bacteria and archaea.

Menaquinone (MK) serves as an essential membranous redox mediator in various electron transport chains of aerobic and anaerobic respiration. In addition, the composition of the quinone/quinol pool has been widely used as a biomarker in microbial taxonomy. The HemN-like class C radical SAM methyltransferases (RSMTs) MqnK, MenK and MenK2 have recently been shown to facilitate specific menaquinone methylation reactions at position C-8 (MqnK/MenK) or C-7 (MenK2) to synthesize 8-methylmenaquinone, 7-methylmenaquinone and 7,8-dimethylmenaquinone. However, the vast majority of protein sequences from the MqnK/MenK/MenK2 family belong to organisms, whose capacity to produce methylated menaquinones has not been investigated biochemically. Here, representative putative menK and menK2 genes from Collinsella tanakaei and Ferrimonas marina were individually expressed in Escherichia coli (wild-type or ubiE deletion mutant) and the corresponding cells were found to produce methylated derivatives of the endogenous MK and 2-demethylmenaquinone. Cluster and phylogenetic analyses of 828 (methyl)menaquinone methyltransferase sequences revealed signature motifs that allowed to discriminate enzymes of the MqnK/MenK/MenK2 family from other radical SAM enzymes and to identify C-7-specific menaquinone methyltransferases of the MenK2 subfamily. This study will help to predict the methylation status of the quinone/quinol pool of a microbial species (or even a microbial community) from its (meta)genome and contribute to the future design of microbial quinone/quinol pools in a Synthetic Biology approach.

Biosynthesis and function of microbial methylmenaquinones.

The membranous quinone/quinol pool is essential for the majority of life forms and its composition has been widely used as a biomarker in microbial taxonomy. The most abundant quinone is menaquinone (MK), which serves as an essential redox mediator in various electron transport chains of aerobic and anaerobic respiration. Several methylated derivatives of MK, designated methylmenaquinones (MMKs), have been reported to be present in members of various microbial phyla possessing either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). Due to their low redox midpoint potentials, MMKs have been proposed to be specifically involved in appropriate electron transport chains of anaerobic respiration. The class C radical SAM methyltransferases MqnK, MenK and MenK2 have recently been shown to catalyse specific MK methylation reactions at position C-8 (MqnK/MenK) or C-7 (MenK2) to synthesise 8-MMK, 7-MMK and 7,8-dimethylmenaquinone (DMMK). MqnK, MenK and MenK2 from organisms such as Wolinella succinogenes, Adlercreutzia equolifaciens, Collinsella tanakaei, Ferrimonas marina and Syntrophus aciditrophicus have been functionally produced in Escherichia coli, enabling extensive quinone/quinol pool engineering of the native MK and 2-demethylmenaquinone (DMK). Cluster and phylogenetic analyses of available MK and MMK methyltransferase sequences revealed signature motifs that allowed the discrimination of MenK/MqnK/MenK2 family enzymes from other radical SAM enzymes and the identification of C-7-specific menaquinone methyltransferases of the MenK2 subfamily. It is envisaged that this knowledge will help to predict the methylation status of the menaquinone/menaquinol pool of any microbial species (or even a microbial community) from its (meta)genome.

FAM83A and FAM83B as Prognostic Biomarkers and Potential New Therapeutic Targets in NSCLC.

Although targeted therapy has improved the survival rates in the last decade, non-small-cell lung cancer (NSCLC) is still the most common cause of cancer-related death. The challenge of identifying new targets for further effective therapies still remains. The FAMily with sequence similarity 83 (FAM83) members have recently been described as novel oncogenes in numerous human cancer specimens and shown to be involved in epidermal growth factor receptor (EGFR) signaling. Here, gene expression of FAM83A and B was analyzed in a cohort of 362 NSCLC patients using qPCR. We further investigated relations in expression and their prognostic value. Functional assays in NSCLC cell lines were performed to evaluate FAM83A and B involvement in proliferation, anchorage-independent growth, migration, and the EGFR pathway. We observed a highly increased gene expression level of FAM83A (ø = 68-fold) and FAM83B (ø = 20-fold) which resulted in poor survival prognosis (p < 0.0001 and p = 0.002). Their expression was influenced by EGFR levels, pathway signaling, and mutation status. Both genes affected cell proliferation, and FAM83A depletion resulted in reduced migration and anchorage-independent growth. The results support the hypothesis that FAM83A and B have different functions in different histological subtypes of NSCLC and might be new therapeutic targets.

Globotriaosylceramide isoform profiles in human plasma by liquid chromatography-tandem mass spectrometry.

Globotriaosylceramide (GL3) is a heterogeneous glycosphingolipid that is elevated in the blood plasma of patients diagnosed with Fabry disease. GL3 consists of numerous isoforms, some of which are distinctly specific to human plasma. An electrospray-ionization LC/MS/MS method has been developed that has the capacity to monitor the GL3 isoform profiles in plasma extracts. Total GL3 is extracted from human plasma via chloroform/methanol liquid-liquid extraction, purified by C(18) solid-phase extraction and analyzed by multiple reaction monitoring LC/MS/MS. The relative responses of eight selected isoforms are calculated on the basis of the total GL3 response and the isoform responses are subsequently utilized to construct isoform profile plots.