Understanding the Surface Characteristics of Biochar and Its Catalytic Activity for the Hydrodeoxygenation of Guaiacol

Biochar (BCR) was obtained from the pyrolysis of a palm-oil-empty fruit bunch at 773 K for 2 h and used as a catalyst for the hydrodeoxygenation (HDO) of guaiacol (GUA) as a bio-oil model compound. Brunauer–Emmet–Teller surface area analysis, NH3 and CO2-temperature-programmed desorption, scanning electron microscope–dispersive X-ray spectroscopy, CHN analysis and X-ray fluorescence spectroscopy suggested that macroporous and mesoporous structures were formed in BCR with a co-presence of hydrophilic and hydrophobic sites and acid–base behavior. A combination of infrared, Raman and inelastic neutron scattering (INS) was carried out to achieve a complete vibrational assignment of BCR. The CH–OH ratio in BCR is ~5, showing that the hydroxyl functional groups are a minority species. There was no evidence for any aromatic C–H stretch modes in the infrared, but they are clearly seen in the INS and are the majority species, with a ratio of sp3–CH:sp2–CH of 1:1.3. The hydrogen bound to sp2–C is largely present as isolated C–H bonds, rather than adjacent C–H bonds. The Raman spectrum shows the characteristic G band (ideal graphitic lattice) and three D bands (disordered graphitic lattice, amorphous carbon, and defective graphitic lattice) of sp2 carbons. Adsorbed water in BCR is present as disordered layers on the surface rather than trapped in voids in the material and could be removed easily by drying prior to catalysis. Catalytic testing demonstrated that BCR was able to catalyze the HDO of GUA, yielding phenol and cresols as the major products. Phenol was produced both from the direct demethoxylation of GUA, as well as through the demethylation pathway via the formation of catechol as the intermediate followed by deoxygenation.

Substrate specificity of the 3-methylmercaptopropionyl-CoA (DmdC1) dehydrogenase from Ruegeria pomeroyi DSS-3

The acyl-CoA dehydrogenase family enzyme DmdC catalyzes the third step in the dimethylsulfoniopropionate (DMSP) demethylation pathway, the oxidation of 3-methylmercaptopropionyl-CoA (MMPA-CoA) to 3-methylthioacryloyl-CoA (MTA-CoA). To study its substrate specificity, the recombinant DmdC1 from Ruegeria pomeroyi was characterized. In addition to MMPA-CoA, the enzyme was highly active with short chain acyl-CoAs, with K m values for MMPA-CoA, butyryl-CoA, valeryl-CoA, caproyl-CoA, heptanoyl-CoA, caprylyl-CoA and isobutyryl-CoA of 36, 19, 7, 11, 14, 10, and 149 μM, respectively, and k cat values of 1.48, 0.40, 0.48, 0.73, 0.46, 0.23 and 0.01 sec −1 , respectively. Among these compounds, MMPA-CoA was the best substrate. The high affinity of DmdC1 for its substrate supports the model for kinetic regulation of the demethylation pathway. In contrast to DmdB, which catalyzes the formation of MMPA-CoA from MMPA, CoA and ATP, DmdC1 was not affected by physiological concentrations of potential effectors, such as DMSP, MMPA, ATP and ADP. Thus, compared to the other enzymes of the DMSP demethylation pathway, DmdC1 has only minimal adaptations for DMSP metabolism compared to other enzymes in the same family with similar substrates, supporting the hypothesis that it evolved relatively recently from a short chain acyl-CoA dehydrogenase involved in fatty acid oxidation. Importance We report the kinetic properties of DmdC1 from the model organism R. pomeroyi and close an important gap in the literature. While the crystal structure of this enzyme was recently solved and its mechanism of action described (X. Shao, H. Y. Cao, F. Zhao, M. Peng, et al., Mol Microbiol 111:1057-1073, 2019, https://doi.org/10.1111/mmi.14211 ), its substrate specificity and sensitivity to potential effectors was never examined. We show that DmdC1 has a high affinity for other short chain acyl-CoAs in addition to MMPA-CoA, which is the natural substrate in DMSP metabolism and is not affected by the potential effectors tested. This evidence supports the hypothesis that DmdC1 possesses few adaptations to DMSP metabolism and likely evolved relatively recently from a short chain acyl-CoA dehydrogenase involved in fatty acid oxidation. This work is important because it expands our understanding about the adaptation of marine bacteria to the increased availability of DMSP about 250 million years ago.

Novel Insights into Dimethylsulfoniopropionate Catabolism by Cultivable Bacteria in the Arctic Kongsfjorden

Dimethylsulfoniopropionate (DMSP) is one of the most abundant organic sulfur compounds in the oceans, which is mainly degraded by bacteria through two pathways, a cleavage pathway and a demethylation pathway. Its volatile catabolites dimethyl sulfide (DMS) and methanethiol (MT) in these pathways play important roles in the global sulfur cycle and have potential influences on the global climate. Intense DMS/DMSP cycling occurs in the Arctic. However, little is known about the diversity of cultivable DMSP-catabolizing bacteria in the Arctic and how they catabolize DMSP. Here, we screened DMSP-catabolizing bacteria from Arctic samples and found that bacteria of four genera ( Psychrobacter , Pseudoalteromonas , Alteromonas and Vibrio ) could grow with DMSP as the sole carbon source, among which Psychrobacter and Pseudoalteromonas are predominant. Four representative strains ( Psychrobacter sp. K31L, Pseudoalteromonas sp. K222D, Alteromonas sp. K632G and Vibrio sp. G41H) from different genera were selected to probe their DMSP catabolic pathways. All these strains produce DMS and MT simultaneously during their growth on DMSP, indicating that all strains likely possess the two DMSP catabolic pathways. On the basis of genomic and biochemical analyses, the DMSP catabolic pathways in these strains were proposed. Bioinformatic analysis indicated that most bacteria of Psychrobacter and Vibrio have the potential to catabolize DMSP via the demethylation pathway, and that only a small portion of Psychrobacter strains may catabolize DMSP via the cleavage pathway. This study provides novel insights into DMSP catabolism in marine bacteria. IMPORTANCE Dimethylsulfoniopropionate (DMSP) is abundant in the oceans. The catabolism of DMSP is an important step of the global sulfur cycle. Although Gammaproteobacteria are widespread in the oceans, the contribution of Gammaproteobacteria in global DMSP catabolism is not fully understood. Here, we found that bacteria of four genera belonging to Gammaproteobacteria ( Psychrobacter , Pseudoalteromonas , Alteromonas and Vibrio ), which were isolated from Arctic samples, were able to grow on DMSP. The DMSP catabolic pathways of representative strains were proposed. Bioinformatic analysis indicates that most bacteria of Psychrobacter and Vibrio have the potential to catabolize DMSP via the demethylation pathway, and that only a small portion of Psychrobacter strains may catabolize DMSP via the cleavage pathway. Our results suggest that novel DMSP dethiomethylases/demethylases may exist in Pseudoalteromonas , Alteromonas and Vibrio , and that Gammaproteobacteria may be important participants in marine, especially in polar DMSP cycling.

Hepatic global DNA Hypomethylation Phenotype in Rainbow Trout Fed Diets Varying in Carbohydrate to Protein Ratio

Background A high carbohydrate-low protein diet can induce hepatic global DNA hypomethylation in trout. The mechanisms remain unclear. Objective We aimed to investigate whether increase in dietary carbohydrates (dHC) or decrease in dietary proteins (dLP) can cause hepatic global DNA hypomethylation, and to explore the underlying mechanisms in trout. Methods Two feeding trials were conducted on juvenile males, both of which involved a 4-day fasting and 4-day refeeding protocol. In Trial 1, trout were fed either a high protein-no carbohydrate (HP-NC, protein 60% dry matter (DM), carbohydrates 0% DM) or a moderate protein-high carbohydrate (MP-HC, protein 40% DM, carbohydrates 30% DM) diet. In Trial 2, fish were fed either a moderate protein-no carbohydrate (MP-NC, protein 40% DM, carbohydrates 0% DM), a MP-HC (protein 40% DM, carbohydrates 30% DM), or a low protein-no carbohydrate (LP-NC, protein 20% DM, carbohydrates 0% DM) diet to separate the effects of dHC and dLP on the hepatic methylome. Global CmCGG methylation, DNA demethylation derivative levels, and mRNA expression of DNA (de)methylation-related genes were measured. Differences were tested by one-way ANOVA when data were normally distributed or by Kruskal-Wallis non-parametric test if not. Results In both trails, global CmCGG methylation levels remained unaffected, but the hepatic 5-mdC content decreased after refeeding (1–3%). The MP-HC group had 3.4-fold higher hepatic 5-hmdC and a similar 5-mdC level compared to the HP-NC group in Trial 1. Both MP-HC and LP-NC diets lowered the hepatic 5-mdC content (1–2%), but only the LP-NC group had a significantly lower 5-hmdC level (P < 0.01) compared with MP-NC group in Trail 2. Conclusions dHC and dLP independently induced hepatic global DNA demethylation in trout. The alterations in other methylation derivatives levels indicated the demethylation process was achieved through an active demethylation pathway and probably occurred at non-CmCGG sites.

Demethylation of Non-CpG Sites in DNA Is Initiated by TET2 5-Methylcytosine Dioxygenase

In the mammalian genome, cytosine methylation predominantly occurs at CpG sites. In addition, a number of recent studies have uncovered extensive C5 cytosine methylation (5mC) at non-CpG (5mCpH, where H = A/C/T) sites. Little is known about the enzyme responsible for active demethylation of 5mCpH sites. Using a very sensitive and quantitative LC–MS/MS method, we demonstrate that the human TET2, an iron (II)- and 2OG-dependent dioxygenase, which is a frequently mutated gene in several myeloid malignancies, as well as in a number of other types of cancers, can oxidize 5mCpH sites in double-stranded DNA in vitro. Similar to oxidation of 5mCpG, oxidation of 5mC at CpH sites produces 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) bases in DNA. After 5mCpG, which is the most preferred substrate, TET2 prefers 5mCpC as a substrate, followed by 5mCpA and then 5mCpT. Since the TDG/BER pathway and deformylation or decarboxylation of 5fC or 5caC, respectively, can convert 5fCpH and 5caCpH to an unmodified cytosine base in DNA, our results suggest a novel demethylation pathway of 5mCpH sites initiated by TET2 dioxygenase.

An Expanded Genetic Code Enables Trimethylamine Metabolism in Human Gut Bacteria

ABSTRACT Cardiovascular disease (CVD) has been linked to animal-based diets, which are a major source of trimethylamine (TMA), a precursor of the proatherogenic compound trimethylamine-N-oxide (TMAO). Human gut bacteria in the genus Bilophila have genomic signatures for genetic code expansion that could enable them to metabolize both TMA and its precursors without production of TMAO. We uncovered evidence that the Bilophila demethylation pathway is actively transcribed in gut microbiomes and that animal-based diets cause Bilophila to rapidly increase in abundance. CVD occurrence and Bilophila abundance in humans were significantly negatively correlated. These data lead us to propose that Bilophila, which is commonly regarded as a pathobiont, may play a role in mitigating cardiovascular disease. Human gut microbiomes have been shown to affect the development of a myriad of disease states, but mechanistic connections between diet, health, and microbiota have been challenging to establish. The hypothesis that Bilophila reduces cardiovascular disease by circumventing TMAO production offers a clearly defined mechanism with a potential human health impact, but investigations of Bilophila cell biology and ecology will be needed to fully evaluate these ideas. IMPORTANCE Links between trimethylamine-N-oxide (TMAO) and cardiovascular disease (CVD) have focused attention on mechanisms by which animal-based diets have negative health consequences. In a meta-analysis of data from foundational gut microbiome studies, we found evidence that specialized bacteria have and express a metabolic pathway that circumvents TMAO production and is often misannotated because it relies on genetic code expansion. This naturally occurring mechanism for TMAO attenuation is negatively correlated with CVD. Ultimately, these findings point to new avenues of research that could increase microbiome-informed understanding of human health and hint at potential biomedical applications in which specialized bacteria are used to curtail CVD development.

Distinct RNAN-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates

The AlkB family of nonheme Fe(II)/2-oxoglutarate–dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases ofN6-methyladenosine (m6A) but furnish different major products,N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidativeN-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a direct m6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for nonredundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.

Evolutionary history of dimethylsulfoniopropionate (DMSP) demethylation enzyme DmdA in marine bacteria

Dimethylsulfoniopropionate (DMSP), an osmolyte produced by oceanic phytoplankton and bacteria, is primarily degraded by bacteria belonging to the Roseobacter lineage and other marine Alphaproteobacteria via DMSP-dependent demethylase A protein (DmdA). To date, the evolutionary history of DmdA gene family is unclear. Some studies indicate a common ancestry between DmdA and GcvT gene families and a co-evolution between Roseobacter and the DMSP-producing-phytoplankton around 250 million years ago (Mya). In this work, we analyzed the evolution of DmdA under three possible evolutionary scenarios: (1) a recent common ancestor of DmdA and GcvT, (2) a coevolution between Roseobacter and the DMSP-producing-phytoplankton, and (3) an enzymatic adaptation for utilizing DMSP in marine bacteria prior to Roseobacter origin. Our analyses indicate that DmdA is a new gene family originated from GcvT genes by duplication and functional divergence driven by positive selection before a coevolution between Roseobacter and phytoplankton. Our data suggest that Roseobacter acquired dmdA by horizontal gene transfer prior to an environment with higher DMSP. Here, we propose that the ancestor that carried the DMSP demethylation pathway genes evolved in the Archean, and was exposed to a higher concentration of DMSP in a sulfur-rich atmosphere and anoxic ocean, compared to recent Roseobacter eco-orthologs (orthologs performing the same function under different conditions), which should be adapted to lower concentrations of DMSP.

Taking the “Me” out of meat: A new demethylation pathway dismantles a toxin's precursor

Carnitine, a molecule found in red meat, is metabolized to trimethylamine (TMA) by the gut microbiota. TMA is then converted in the liver to trimethylamine oxide, a causative agent for atherosclerosis. Kountz et al. have discovered an alternative pathway for carnitine metabolism in the gut bacterium Eubacterium limosum. Instead of forming TMA, carnitine is demethylated by the newly discovered methyltransferase MtcB, sending one-carbon units into production of short-chain fatty acids. These results suggest that bacterial metabolic activities could promote cardiovascular health by preventing the buildup of toxin precursors.

Distinct RNA N-Demethylation Pathways Catalyzed by Non-Heme Iron ALKBH5 and FTO Enzymes Enable Regulation of Formaldehyde Release Rates

<p>Abstract</p><p><br></p><p>The AlkB family of non-heme-Fe(II)/2-oxoglutarate(2OG)-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6 methyladenosine (m6A) but furnish different major products, N6 hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a direct</p><p>m6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for non-redundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.</p><p><br></p><p><br></p><p>Significance Statement</p><p><br></p><p>Non-heme iron enzymes FTO and ALKBH5 play central roles in epigenetic RNA regulation by catalyzing the oxidation of N6-methyladenosine (m6A) to produce N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here, we provide a mechanistic rationale for these distinct biochemical outcomes by identifying that ALKBH5 performs m6A demethylation via an unprecedented covalent-based mechanism with concomitant and rapid release of A and formaldehyde (FA), whereas FTO liberates hm6A to release A and FA over longer timescales. This work reveals foundational biochemical differences between these closely related but non-redundant epigenetic enzymes and identifies ALKBH5 as an endogenous source of rapid formaldehyde generation in cells.</p>