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Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA 1 . The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine 2 , and the acetylation of histones and proteins 3 , 4 . In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA 5 , 6 . In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue 1 and in cholinergic neurons 2 , 7 . The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer 8 – 11 and hyperlipidaemia 12 , 13 . Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth. Crystal structures of ATP citrate lyase from bacteria, archaea and humans unravel how the enzyme directs the formation of the central metabolite acetyl-CoA, and shed light onto the evolutionary origins of the Krebs cycle.

ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA 1 – 5 . The acetyl-CoA product is crucial for the metabolism of fatty acids 6 , 7 , the biosynthesis of cholesterol 8 , and the acetylation and prenylation of proteins 9 , 10 . There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation 2 , 5 , 11 . ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials 4 , 5 . Many inhibitors of ACLY have been reported, but most of them have weak activity 5 . Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have also determined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure–activity relationships of these compounds. This allosteric site greatly enhances the ‘druggability’ of ACLY and represents an attractive target for the development of new ACLY inhibitors. The structure of human ATP-citrate lyase, in complex with a newly developed small-molecule inhibitor, shows extensive conformational changes that reveal an allosteric site for the inhibitor to bind and indirectly compete with the citrate substrate.

ObjectivesBifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate.ResultsA new BP-2 strain that produces alginate lyase was screened and identified from rotted Sargassum. A new alginate lyase, Alg17B, belonging to the polysaccharide lyase family 17, was isolated and purified from BP-2 fermentation broth by freeze-drying, dialysis, and ion exchange chromatography. The enzymatic properties of the purified lyase were investigated. The molecular weight of Alg17B was approximately 77 kDa, its optimum reaction temperature was 40–45 °C, and its optimum reaction pH was 7.5–8.0. The enzyme was relatively stable at pH 7.0–8.0, with a temperature range of 25–35 °C, and the specific activity of the purified enzyme reached 4036 U/mg. A low Na+ concentration stimulated Alg17B enzyme activity, but Ca2+, Zn2+, and other metal ions inhibited it. Substrate specificity analysis, thin-layer chromatography, and mass spectrometry showed that Alg17B is an alginate lyase that catalyses the hydrolysis of sodium alginate, polymannuronic acid (polyM) and polyguluronic acid to produce monosaccharides and low molecular weight oligosaccharides. Alg17B is also bifunctional, exhibiting both endolytic and exolytic activities toward alginate, and has a wide substrate utilization range with a preference for polyM.ConclusionsAlg17B can be used to saccharify the main carbohydrate, alginate, in the ethanolic production of brown algae fuel as well as in preparing and researching oligosaccharides.

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood 1 . Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs. Glucocorticoids reprogram the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and, as a consequence, inhibition of the inflammatory response.

Rhodotorula toruloides is an oleaginous yeast recognized for its robustness and the production of high content of neutral lipids. Early biochemical studies have linked ATP citrate lyase (ACL), phosphoketolase (PK), and cytosolic malic enzyme (cMAE) with de novo lipid synthesis. In this study, we discovered that upon a CRISPR/Cas9-mediated knockout of the ACL gene, lipid content in R. toruloides IFO0880 decreased from 50 to 9% of its dry cell weight (DCW) in glucose medium and caused severe growth defects (reduced specific growth rate, changes in cell morphology). In xylose medium, the lipid content decreased from 43 to 38% of DCW. However, when grown on acetate as the sole carbon source, the lipid content decreased from 45 to 20% of DCW. Significant growth defects as a result of ACL knockout were observed on all substrates. In contrast, PK knockout resulted in no change in growth or lipid synthesis. Knocking out cMAE gene resulted in lipid increase of 2.9% of DCW and 23% increase in specific growth rate on glucose. In xylose or acetate medium, no change in lipid production as a result of cMAE gene knockout was observed. These results demonstrated that ACL plays a crucial role in lipid synthesis in R. toruloides IFO0880, as opposed to PK pathway or cMAE, whose presence in some conditions even disfavors lipid production. These results provided valuable information for future metabolic engineering of R. toruloides . Graphical Abstract Key points • ACL is crucial for the fatty acid synthesis and growth in R. toruloides IFO0880. • Lipid production and cell growth is are unchanged as a result of PK knockout. • Cytosolic malic enzyme does not play a significant role in lipogenesis.

Nicotinamide adenine dinucleotide (NAD + ) is a co-substrate for several enzymes, including the sirtuin family of NAD + -dependent protein deacylases. Beneficial effects of increased NAD + levels and sirtuin activation on mitochondrial homeostasis, organismal metabolism and lifespan have been established across species. Here we show that α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), the enzyme that limits spontaneous cyclization of α-amino-β-carboxymuconate-ε-semialdehyde in the de novo NAD + synthesis pathway, controls cellular NAD + levels via an evolutionarily conserved mechanism in Caenorhabditis elegans and mouse. Genetic and pharmacological inhibition of ACMSD boosts de novo NAD + synthesis and sirtuin 1 activity, ultimately enhancing mitochondrial function. We also characterize two potent and selective inhibitors of ACMSD. Because expression of ACMSD is largely restricted to kidney and liver, these inhibitors may have therapeutic potential for protection of these tissues from injury. In summary, we identify ACMSD as a key modulator of cellular NAD + levels, sirtuin activity and mitochondrial homeostasis in kidney and liver. Genetic or pharmacological inhibition of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase increases NAD + and improves mitochondrial function in nematodes and mice, and may have therapeutic potential in kidney and liver disease.

Although many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga Chlorella variabilis NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or -alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid–binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons.

Alkaline pectate lyases (Pels) have potential application in bioscouring of the textile industry. In this study, a thermo-alkaline Pel (BacPelA) gene from an alkaliphilic Bacillus clausii strain was cloned and overexpressed in Escherichia coli . The mature BacPelA exhibited maximum activity at pH 10.5 and 70 °C and showed high cleavage capability on methylated pectins. BacPelA showed the highest specific activity of 936.2 U mg −1 on ≥85% methylated pectin and 675.5 U mg −1 on standard substrate polygalacturonic acid (PGA) upon evaluation of the absorbance at 235 nm (A 235 ). The K m and k cat values for PGA were 0.54 g l −1 and 346.5 s −1 , respectively. Moreover, the 3,5-dinitrosalicylic acid (DNS) assay, which detects the released reducing oligogalacturonic acids, was confirmed to be inaccurate and unsuitable for endo-acting pectinase activity assay because of the difference in the reducibility by DNS reagent between the standard galacturonic acid and the catalytic oligomer products. Significant ramie fiber weight loss was observed following treatment with BacPelA (24.8%) and combined enzyme-chemical method (30.9%), which indicated that the degumming efficiency of BacPelA was the highest of all alkaline and thermostable Pels reported to date. The total activity of the recombinant mature BacPelA reached 8378.2 U ml −1 (A 235 ) by high-cell-density cultivation in fed-batch fermentation with productivity of 239.4 U ml −1  h −1 using E. coli as host, which represents the highest Pel yield reported to date. Therefore, BacPelA, with promising properties for bioscouring, shows potential applications for ramie degumming in the textile industry.

Macroalgae are considered to be promising biomass for fuels and chemicals production. To utilize brown macroalgae as biomass, the degradation of alginate, which is the main carbohydrate of brown macroalgae, into monomeric units is a critical prerequisite step. Saccharophagus degradans 2-40 is capable of degrading more than ten different polysaccharides including alginate, and its genome sequence demonstrated that this bacterium contains several putative alginate lyase genes including alg17C. The gene for Alg17C, which is classified into the PL-17 family, was cloned and overexpressed in Escherichia coli. The recombinant Alg17C was found to preferentially act on oligoalginates with degrees of polymerization higher than 2 to produce the alginate monomer, 4-deoxy-l-erythro-5-hexoseulose uronic acid. The optimal pH and temperature for Alg17C were found to be 6 and 40 °C, respectively. The K ^sub M^ and V ^sub max^ of Alg17C were 35.2 mg/ml and 41.7 U/mg, respectively. Based on the results of this study, Alg17C could be used as the key enzyme to produce alginate monomers in the process of utilizing alginate for biofuels and chemicals production.[PUBLICATION ABSTRACT]

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonasarctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.