acetyl-CoA Synthase Research Articles

Optimizing Huangjiu Fermentation for Enhanced Aroma: Insights into Saccharomyces cerevisiae jiangnan1# Strain

Higher alcohols (HAs) and acetate esters (AEs) are crucial volatile flavor compounds in Chinese huangjiu. The imbalance between HAs and AEs leads to poor flavor quality, lack of drinking comfort and headaches after drinking. By comparing the metabolic profiles and gene expression of Saccharomyces cerevisiae jiangnan1# with control strain (85#), 12.38% reduction in HA levels during unilateral fermentation was identified, especially a significant 47.32% drop in 2-phenethyl alcohol, without affecting AEs levels (P > 0.05). Bilateral fermentation strategies further optimized HA reduction by 8.33% and increased AEs significantly. Transcriptome data highlighted the down-regulation of key enzymes ARO9 and ARO10 in the Ehrlich pathway, linked to reduced HA synthesis, and the up-regulation of ALDH and acetyl-CoA synthase, promoting AE accumulation. The results showed that jiangnan1# strain-specific fermentation characteristics, particularly the regulation of key metabolic pathways, are the primary drivers of differences in HAs and AEs in huangjiu fermentation. These findings are instrumental in developing targeted metabolic strategies for enhancing the aroma profile of huangjiu, emphasizing the role of microbial modulation in food fermentation processes.

The transcription factor FgSfp1 orchestrates mycotoxin deoxynivalenol biosynthesis in Fusarium graminearum

Fusarium graminearum (F. graminearum) and its derivative mycotoxin deoxynivalenol (DON) are highly concerned with food safety and sustainability worldwide. Although several transcription factors (TFs) had been elucidated, molecular mechanism participates in DON biosynthesis regulation remains largely unrevealed. Here, we first characterized a zinc finger-contained TF in F. graminearum, FgSfp1, which is indispensable for DON production since its depletion resulting in a 95.4% DON yielding reduction. Interestingly, contrast to previous knowledge, all TRI-cluster genes were abnormally upregulated in ΔFgSfp1 while Tri proteins abundance rationally decreased simultaneously. Further evidence show FgSfp1 could coordinate genetic translation pace by manipulating ribosomal biogenesis process. Specifically, FgSfp1-depletion leads to ribosome biogenesis assembly factor (RiBi) expression attenuation along with DON precursor acetyl-CoA synthase reduction since FgSfp1 actively interacts with RNA 2’-O-methylation enzyme FgNop1 revealed by Bi-FC. It subsequently influences mRNA translation pace. In conclusion, we elucidated that the FgSfp1 orchestrates DON biosynthesis via participating RNA posttranscriptional modification for ribosomal RNA maturation, offering insights into the DON biosynthesis regulation. Ultimately, this TF might be a key regulator for DON contamination control in the whole food chain.

Capturing a methanogenic carbon monoxide dehydrogenase/acetyl-CoA synthase complex via cryogenic electron microscopy

Approximately two-thirds of the estimated one-billion metric tons of methane produced annually by methanogens is derived from the cleavage of acetate. Acetate is broken down by a Ni-Fe-S-containing A-cluster within the enzyme acetyl-CoA synthase (ACS) to carbon monoxide (CO) and a methyl group (CH3+). The methyl group ultimately forms the greenhouse gas methane, whereas CO is converted to the greenhouse gas carbon dioxide (CO2) by a Ni-Fe-S-containing C-cluster within the enzyme carbon monoxide dehydrogenase (CODH). Although structures have been solved of CODH/ACS from acetogens, which use these enzymes to make acetate from CO2, no structure of a CODH/ACS from a methanogen has been reported. In this work, we use cryo-electron microscopy to reveal the structure of a methanogenic CODH and CODH/ACS from Methanosarcina thermophila (MetCODH/ACS). We find that the N-terminal domain of acetogenic ACS, which is missing in all methanogens, is replaced by a domain of CODH. This CODH domain provides a channel for CO to travel between the two catalytic Ni-Fe-S clusters. It generates the binding surface for ACS and creates a remarkably similar CO alcove above the A-cluster using residues from CODH rather than ACS. Comparison of our MetCODH/ACS structure with our MetCODH structure reveals a molecular mechanism to restrict gas flow from the CO channel when ACS departs, preventing CO escape into the cell. Overall, these long-awaited structures of a methanogenic CODH/ACS reveal striking functional similarities to their acetogenic counterparts despite a substantial difference in domain organization.

Enhancing effect of conductive materials and rumen microorganisms on the anaerobic digestion performance of a real traditional Chinese medicine wastewater

This work evaluated the effect of different concentrations of conductive materials and rumen microorganisms on the anaerobic digestion (AD) performance of real traditional Chinese medicine (TCM) wastewater. The experiments first determined the optimal organic concentration of 0.25 L/L for the AD system and removed suspended solids from real TCM wastewater to reduce the inhibition effect on methanogenesis. Analysis of the modified Gompertz model showed that the addition of activated carbon, biochar, cow manure, and rumen fluid at doses of 12 g/L, 12 g/L, 12 mL/L, and 125 mL/L, respectively, had the greatest effect on CH4 production, increasing the cumulative CH4 yield by 13.5 %, 10.4 %, 26.8 %, and 32.9 %. Through microbial community and metabolism pathway analyses, the conductive materials promoted direct interspecies electron transfer (DIET) between Clostridium and Methanothrix, and the acetoclastic methanogenic pathway dominated by acetyl-CoA synthase. Rumen microorganisms enhanced AD performance by promoting the growth of hydrogenotrophic methanogens and the abundance of genes dominated by formylmethanofuran dehydrogenase in the hydrogenotrophic methanogenic pathway, illustrating the relationship between microbial community and metabolism pathway. Rumen microorganisms increased CH4 production more than conductive materials in real TCM wastewater. This study helps to better understand the internal mechanisms by which different materials enhance AD performance.

An alcove at the acetyl-CoA synthase nickel active site is required for productive substrate CO binding and anaerobic carbon fixation

One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. Invitro spectroscopy, kinetics, binding, and invivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.

Nuclear position and local acetyl-CoA production regulate chromatin state

Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid β-oxidation, which is also increased in the rim. Inhibition of fatty acid β-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.

Acetate derived from the intestinal tract has a critical role in maintaining skeletal muscle mass and strength in mice.

Acetate is a short-chain fatty acid (SCFA) that is produced by microbiota in the intestinal tract. It is an important nutrient for the intestinal epithelium, but also has a high plasma concentration and is used in the various tissues. Acetate is involved in endurance exercise, but its role in resistance exercise remains unclear. To investigate this, mice were administered either multiple antibiotics with and without oral acetate supplementation or fed a low-fiber diet. Antibiotic treatment for 2 weeks significantly reduced grip strength and the cross-sectional area (CSA) of muscle fiber compared with the control group. Intestinal concentrations of SCFAs were reduced in the antibiotic-treated group. Oral administration of acetate with antibiotics prevented antibiotic-induced weakness of skeletal muscle and reduced CSA of muscle fiber. Similarly, a low-fiber diet for 1 year significantly reduced the CSA of muscle fiber and fecal and plasma acetate concentrations. To investigate the role of acetate as an energy source, acetyl-CoA synthase 2 knockout mice were used. These mice had a shorter lifespan, reduced skeletal muscle mass and smaller CSA of muscle fiber than their wild type littermates. In conclusion, acetate derived from the intestinal microbiome can contribute to maintaining skeletal muscle performance.

Role of Sphingosine Kinase 1 in Glucolipotoxicity-Induced Early Activation of Autophagy in INS-1 Pancreatic β Cells.

Insulin-producing pancreatic β cells play a crucial role in the regulation of glucose homeostasis, and their failure is a key event for diabetes development. Prolonged exposure to palmitate in the presence of elevated glucose levels, termed gluco-lipotoxicity, is known to induce β cell apoptosis. Autophagy has been proposed to be regulated by gluco-lipotoxicity in order to favor β cell survival. However, the role of palmitate metabolism in gluco-lipotoxcity-induced autophagy is presently unknown. We therefore treated INS-1 cells for 6 and 24 h with palmitate in the presence of low and high glucose concentrations and then monitored autophagy. Gluco-lipotoxicity induces accumulation of LC3-II levels in INS-1 at 6 h which returns to basal levels at 24 h. Using the RFP-GFP-LC3 probe, gluco-lipotoxicity increased both autophagosomes and autolysosmes structures, reflecting early stimulation of an autophagy flux. Triacsin C, a potent inhibitor of the long fatty acid acetyl-coA synthase, completely prevents LC3-II formation and recruitment to autophagosomes, suggesting that autophagic response requires palmitate metabolism. In contrast, etomoxir and bromo-palmitate, inhibitors of fatty acid mitochondrial β-oxidation, are unable to prevent gluco-lipotoxicity-induced LC3-II accumulation and recruitment to autophagosomes. Moreover, bromo-palmitate and etomoxir potentiate palmitate autophagic response. Even if gluco-lipotoxicity raised ceramide levels in INS-1 cells, ceramide synthase 4 overexpression does not potentiate LC3-II accumulation. Gluco-lipotoxicity also still stimulates an autophagic flux in the presence of an ER stress repressor. Finally, selective inhibition of sphingosine kinase 1 (SphK1) activity precludes gluco-lipotoxicity to induce LC3-II accumulation. Moreover, SphK1 overexpression potentiates autophagic flux induced by gluco-lipotxicity. Altogether, our results indicate that early activation of autophagy by gluco-lipotoxicity is mediated by SphK1, which plays a protective role in β cells.

Site specific redox properties in ligand differentiated di-nickel complexes inspired by the acetyl CoA synthase active site.

Bimetallic transition metal complexes with site-specific redox properties offer a versatile platform for understanding electron polarization, intramolecular electron transfer processes, and customizing electronic and magnetic properties that might impact reactivity and catalyst design. Inspired by the dissymmetric nickel sites in the Acetyl CoA Synthase (ACS) Active Site, three new bimetallic Ni(N2S2)-Ni(S2C2R2) complexes based on Ni(N2S2) metalloligand donor synthons, Nid, in mimicry of the nickel site distal to the redox-active iron sulfur cluster of ACS, and nickel dithiolene receiver units, designated as Nip, the nickel proximal to the 4Fe4S cluster, were combined to explore the influence of ligand environment on electronic structure and redox properties of each unit. The combination of synthons gave a matrix of three S-bridged dinickel complexes, characterized by X-ray crystallography, and appropriate spectroscopies. Computational modeling is connected to the electronic characteristics of the nickel donor and receiver units. This study demonstrated the intricacies of identifying sites of electrochemical redox processes, within multi-metallic systems containing non-innocent ligands.

Genomic potential and physiological characteristics of C1 metabolism in novel acetogenic bacteria.

Acetogenic bacteria can utilize C1 compounds, such as carbon monoxide (CO), formate, and methanol, via the Wood-Ljungdahl pathway (WLP) to produce biofuels and biochemicals. Two novel acetogenic bacteria of the family Eubacteriaceae ES2 and ES3 were isolated from Eulsukdo, a delta island in South Korea. We conducted whole genome sequencing of the ES strains and comparative genome analysis on the core clusters of WLP with Acetobacterium woodii DSM1030T and Eubacterium limosum ATCC8486T. The methyl-branch cluster included a formate transporter and duplicates or triplicates copies of the fhs gene, which encodes formyl-tetrahydrofolate synthetase. The formate dehydrogenase cluster did not include the hydrogenase gene, which might be replaced by a functional complex with a separate electron bifurcating hydrogenase (HytABCDE). Additionally, duplicated copies of the acsB gene, encoding acetyl-CoA synthase, are located within or close to the carbonyl-branch cluster. The serum bottle culture showed that ES strains can utilize a diverse range of C1 compounds, including CO, formate, and methanol, as well as CO2. Notably, ES2 exhibited remarkable resistance to high concentrations of C1 substrates, such as 100% CO (200 kPa), 700 mM formate, and 500 mM methanol. Moreover, ES2 demonstrated remarkable growth rates under 50% CO (0.45 h-1) and 200 mM formate (0.34 h-1). These growth rates are comparable to or surpassing those previously reported in other acetogenic bacteria. Our study introduces novel acetogenic ES strains and describes their genetic and physiological characteristics, which can be utilized in C1-based biomanufacturing.

THU558 Combination Therapy Of Acetyl-CoA Synthetase Inhibitor And Fulvestrant Reverse Epigenetic Alterations Of Metastatic Breast Cancer Cells And Reduce Cancer Cell Proliferation

Abstract Disclosure: A.N. Mogol: None. Z. Madak Erdogan: None. Endocrine therapies exploit the hormone dependencies of the cancer cells in various ways, including by targeting estrogen receptor alpha (ERα) that is expressed by approximately 70% of human breast cancers. After treatment, recurrence and metastasis are still observed in 30-40% of ER+ patients and the 5-year relative overall survival rate for these patients is just 24%. Acetyl-CoA production significantly regulates histone acetylation and gene regulation, which support tumor progression. In our previous study, we showed the synergistic effect of the ERα agonist Fulvestrant (Fulv) and Acetyl-CoA Synthase Inhibitor (ACSI) in reducing cell viability in ER+ metastatic breast cancer (MBC) cells in vitro. To study the synergistic contribution of ACSS2 to endocrine resistance, we used ChIP analysis, which showed that ACSS2 was recruited to ERα binding sites with Fulv treatment in cell line xenograft tumors and PDX samples. We used the CUT&RUN method to investigate specific protein (H3k27ac, ERα and ACSS2) and DNA interactions in ER+ MBC cells globally. Enriched chromatin samples were then used for qPCR and sequencing. Combination therapy of the ACSI and Fulv significantly altered the epigenetic landscape of the breast cancer cells in a manner to decrease cell viability. High histone acetylation at lysine 27 residue was observed in Veh treatment group and combination therapy of ACSI+Fulv further decreased the enrichment of this histone modification in classical ER binding sites. Interestingly, ACSS2 binding was increased with Fulv treatment. Targeting epigenetic alterations caused by cancer cell adaptations is a novel approach that could potentially lead to new therapies to reduce metastasis mortality. Presentation: Thursday, June 15, 2023

Metabolic Profiling of a Fast Neutron Soybean Mutant Reveals an Increased Abundance of Isoflavones.

A total of 718 metabolites were identified in leaves and seeds of the soybean (Glycine max (L.) Merr., Fabaceae) fast neutron (FN) mutant 2012CM7F040p05ar154bMN15, which was previously shown to have 21 genes deleted and higher protein content in seeds as compared to wild-type. Among the identified metabolites, 164 were found only in seeds, 89 only in leaves, and 465 in both leaves and seeds. Metabolites that exhibited higher abundance in the mutant leaf than in the wild type include the flavonoids afromosin, biochanin A, dihydrodaidzein, and apigenin. Mutant leaves also exhibited a higher accumulation of glycitein-glucoside, dihydrokaempferol, and pipecolate. The seed-only metabolites that were found in higher abundance in the mutant compared to the wild type included 3-hydroxybenzoate, 3-aminoisobutyrate, coenzyme A, N-acetyl-β-alanine, and 1-methylhistidine. Among several amino acids, the cysteine content increased in the mutant leaf and seed when compared to the wild type. We anticipate that the deletion of acetyl-CoA synthase created a negative feedback effect on carbon dynamics, resulting in increased amounts of cysteine and isoflavone-associated metabolites. Metabolic profiling provided new insight into the cascading effect of gene deletions that helps breeders to produce value-added nutritional seed traits.

A De Novo Designed Trimeric Metalloprotein as a Nip Model of the Acetyl-CoA Synthase.

We present a Nip site model of acetyl coenzyme-A synthase (ACS) within a de novo-designed trimer peptide that self-assembles to produce a homoleptic Ni(Cys)3 binding motif. Spectroscopic and kinetic studies of ligand binding demonstrate that Ni binding stabilizes the peptide assembly and produces a terminal NiI-CO complex. When the CO-bound state is reacted with a methyl donor, a new species is quickly produced with new spectral features. While the metal-bound CO is albeit unactivated, the presence of the methyl donor produces an activated metal-CO complex. Selective outer sphere steric modifications demonstrate that the physical properties of the ligand-bound states are altered differently depending on the location of the steric modification above or below the Ni site.

#3566 ACSS2 AGGRAVATES RENAL TUBULAR INFLAMMATION BY INDUCING THE DYSREGULATION OF FATTY ACID METABOLISM AND MITOCHONDRIAL OXIDATIVE STRESS IN DIABETIC MICE

Abstract Background and Aims Treatment options for diabetic nephropathy (DN) are currently limited. Chronic renal inflammation has been widely recognized as a major promoter of DN. The tubular epithelial cells are a major source of IL-1β in diabetic db/db mice. Inhibition of Acetyl CoA Synthase 2 (ACSS2) has been shown to promote high-fat diet-induced liver injuries, but the underlying mechanism of action of ACSS2 in diabetic renal injuries has not been elucidated. Method Streptozotocin (STZ) was intraperitoneally injected to establish the diabetic mouse model. We investigated the effects of ACSS2 on diabetes-induced renal tubular injury in transgenic mice with global acss2 knockout (acss2−/−). PAS staining was carried out to evaluate pathological injury of kidneys in mice. The renal tissue immunostaining was conducted to detect IL-1β expression and macrophage distribution in kidney tissues of mice. HK-2 cell culture model was achieved by high glucose stimulation in vitro. Real-time PCR analysis was applied to evaluate gene expression of inflammatory factors IL-1β, TNF-α, and MCP-1. Western Blot and immunofluorescence staining were conducted to the activation of the NLRP3 pathway and mitochondrial reactive oxygen species (ROS). Results ACSS2 was upregulated in STZ-induced diabetic renal tubular cells, which is significantly co-expressed in IL-1β-positive renal tubular cells of STZ-induced diabetic mice. The mice with acss2−/- exhibited decreased renal tubular inflammation and inhibited NOD-like receptor protein 3 (NLRP3) inflammasome activation as evidenced by elevated protein levels of NLRP3 and cleaved caspase-1 when compared with diabetic wild-type mice. The selective inhibition of ACSS2 protected HK-2 cells in vitro when exposed to a high glucose environment by downregulating NLRP3 inflammasome and ameliorating mitochondrial oxidative stress. Mechanistically, we showed that mitochondrial dysfunction preceded inflammatory response in vitro. The elimination of mitochondrial ROS using mitochondria-targeted antioxidants (mito-TEMPO) reversed HG-mediated NLRP3 inflammation activation in HK-2 cells, which recapitalized the phenotype observed in acss2-/- mice. We also found that cytoplasm ACSS2, which was increased after HG stimulation, induced fatty acid synthase (FASN) mediated overproduction of free fatty acid. Downregulating FASN using FASN-small interfering RNA restored mitochondrial function, and suppressed NLRP3 inflammasome activation. Conclusion In conclusion, diabetes-induced ACSS2 upregulation led to subsequent FASN-mediated dysregulation of fatty acid metabolism and mitochondrial ROS overproduction, eventually causing NLRP3 inflammasome activation and renal inflammation. Therefore, our findings established the role of ACSS2 in diabetes-induced inflammation of renal tubular epithelium and unveiled the underlying mechanism of its action. ACSS2 appears as a promising target for treating diabetic renal tubular epithelial inflammation.

Characterization of Methyl- and Acetyl-Ni Intermediates in Acetyl CoA Synthase Formed during Anaerobic CO2 and CO Fixation.

The Wood-Ljungdahl Pathway is a unique biological mechanism of carbon dioxide and carbon monoxide fixation proposed to operate through nickel-based organometallic intermediates. The most unusual steps in this metabolic cycle involve a complex of two distinct nickel-iron-sulfur proteins: CO dehydrogenase and acetyl-CoA synthase (CODH/ACS). Here, we describe the nickel-methyl and nickel-acetyl intermediates in ACS completing the characterization of all its proposed organometallic intermediates. A single nickel site (Nip) within the A cluster of ACS undergoes major geometric and redox changes as it transits the planar Nip, tetrahedral Nip-CO and planar Nip-Me and Nip-Ac intermediates. We propose that the Nip intermediates equilibrate among different redox states, driven by an electrochemical-chemical (EC) coupling process, and that geometric changes in the A-cluster linked to large protein conformational changes control entry of CO and the methyl group.

Continuous fed-batch strategy decreases acetic acid production and increases volatile ester formation in wines under high-gravity fermentation

High sugar fermentation elevates acetic acid levels in wines, which can be avoided by applying the continuous fed-batch strategy. In this study, yeast gene expressions and wine volatile compounds were evaluated by quantitative real-time PCR (RT-qPCR) and gas chromatograph mass spectrometry (GC-MS) in high-gravity (HG, 320 g/L sugars) fermentations with different batch strategies. The acetic acid concentration in continuous fed-batch fermentation wine was reduced by 51.69 %, compared with that in whole-batch fermentation wine. The acetyl-CoA synthase gene (ACS2) expression was up-regulated, whereas the glycerol-3-phosphate dehydrogenase gene (GPD1) expression was down-regulated on day 3 and day 7 during the continuous fed-batch fermentation. The volatile ester concentration in continuous fed-batch fermentation wine was 36.74 % higher than that in whole-batch fermentation wine. Overall, the continuous fed-batch strategy can modulate the expression of yeast genes involved in acetic acid metabolism and can increase volatile esters in wine under high sugar fermentation. Our findings provide a reference for the application of a continuous fed-batch strategy in high-sugar fermentation so as to improve the quality of the wine.

Conversion of acetate and glyoxylate to fumarate by a cell-free synthetic enzymatic biosystem

Fumarate is a value-added chemical that is widely used in food, medicine, material, and agriculture industries. With the rising attention to the demand for fumarate and sustainable development, many novel alternative ways that can replace the traditional petrochemical routes emerged. The in vitro cell-free multi-enzyme catalysis is an effective method to produce high value chemicals. In this study, a multi-enzyme catalytic pathway comprising three enzymes for fumarate production from low-cost substrates acetate and glyoxylate was designed. The acetyl-CoA synthase, malate synthase, and fumarase from Escherichia coli were selected and the coenzyme A achieved recyclable. The enzymatic properties and optimization of reaction system were investigated, reaching a fumarate yield of 0.34 mM with a conversion rate of 34% after 20 h of reaction. We proposed and realized the conversion of acetate and glyoxylate to fumarate in vitro using a cell-free multi-enzyme catalytic system, thus providing an alternative approach for the production of fumarate.

Design of a minimal di-nickel hydrogenase peptide.

Ancestral metabolic processes involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13-amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe] hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase, two ancient, extant proteins central to metabolism. These experimental results demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth.

Production of S-methyl-methionine using engineered Saccharomyces cerevisiae sake K6.

Introducing a plant-derived MMT gene encoding methionine S-methyltransferase into engineered Saccharomyces cerevisiae sake K6 allowed microbial production of S-methyl-methionine (SMM).

The two-electron reduced A cluster in acetyl-CoA synthase: Preparation, characteristics and mechanistic implications

Acetyl-CoA synthase (ACS) is a central enzyme in the carbon and energy metabolism of certain anaerobic species of bacteria and archaea that catalyzes the direct synthesis and cleavage of the acetyl CC bond of acetyl-CoA by an unusual enzymatic mechanism of special interest for its use of organonickel intermediates. An Fe4S4 cluster associated with a proximal, reactive Nip and distal spectator Nid comprise the active site metal complex, known as the A cluster. Experimental and theoretical methods have uncovered much about the ACS mechanism, but have also opened new unanswered questions about the structure and reactivity of the A cluster in various intermediate forms. Here we report a method for large scale isolation of ACS with its A cluster in the acetylated state. Isolated acetyl-ACS and the two-electron reduced ACS, produced by acetyl-ACS reaction with CoA, were characterized by UV–visible and EPR spectroscopy. Reactivity with electron acceptors provided an assessment of the apparent Em for two-electron reduction of the A cluster. The results help to distinguish between alternative electronic states of the reduced cluster, provide evidence for a role of the Fe/S cluster in catalysis, and offer an explanation of why one-electron reductive activation is observed for a reaction cycle involving 2-electron chemistry.