[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

Serum ACSL4 levels in patients with ST-segment elevation myocardial infarction (STEMI) and its association with one-year major adverse cardiovascular events (MACE): A prospective cohort study.

In the present prospective cohort research, we aimed to explore the serum levels of Acyl-CoA synthetase long-chain family member 4 (ACSL4) in patients with ST-segment elevation myocardial infarction (STEMI) and its association with 1-year major adverse cardiovascular events (MACE). This prospective cohort study recruited 507 patients who underwent percutaneous coronary intervention for the treatment of STEMI at our hospital during August 2019 to July 2022. The serum ACSL4, tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, and C-reactive protein levels were measured by enzyme-linked immunosorbent assay. Demographic and clinical statistics were also collected. In addition, all patients were followed up for 1 year, and patients with MACE were defined as poor prognosis group. All data used SPSS 26.0 to statistical analyses. The poor prognosis group had significantly higher age and low-density leptin cholesterol (LDLC) levels compared to the favorable prognosis group (P < .05). STEMI patients exhibited significantly elevated serum levels of ACSL4, tumor necrosis factor-α, IL-6, IL-1ß, and C-reactive protein (P < .05). Serum ACSL4 and IL-1ß levels in the poor prognosis group were remarkably enhanced compared to the favorable prognosis group. Curvilinear regression analysis demonstrated that ACSL4 was associated with LDLC and IL-1ß. Moreover, ACSL4 (B = 0.138, 95% CI 1.108-1.189, P < .001), LDLC (B = 2.317, 95% CI 5.253-19.603, P < .001), and IL-1ß (B = 0.061, 95%CI 1.008-1.122, P = .025) levels were the risk factors for STEMI patients with 1-year MACE. This study showed that the serum ACSL4 levels was remarkably elevated in STEMI patients. This study might provide new targets and a comprehensive approach to cardiovascular protection in STEMI patients.

Structural Insights into Microbial One-Carbon Metabolic Enzymes Ni-Fe-S-Dependent Carbon Monoxide Dehydrogenases and Acetyl-CoA Synthases.

Ni-Fe-S-dependent carbon monoxide dehydrogenases (CODHs) are enzymes that interconvert CO and CO2 by using their catalytic Ni-Fe-S C-cluster and their Fe-S B- and D-clusters for electron transfer. CODHs are important in the microbiota of animals such as humans, ruminants, and termites because they can facilitate the use of CO and CO2 as carbon sources and serve to maintain redox homeostasis. The bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is responsible for acetate production via the Wood-Ljungdahl pathway, where acetyl-CoA is assembled from two CO2-derived one-carbon units. A Ni-Fe-S A-cluster is key to this chemistry. Whereas acetogens use the A- and C-clusters of CODH/ACS to produce acetate from CO2, methanogens use A- and C-clusters of an acetyl-CoA decarbonylase/synthase complex (ACDS) to break down acetate en route to CO2 and methane production. Here we review some of the recent advances in understanding the structure and mechanism of CODHs, CODH/ACSs, and ACDSs, their unusual metallocofactors, and their unique metabolic roles in the human gut and elsewhere.

The Peripherally Membrane-attached Protein MbFACL6 of Mycobacterium tuberculosis Activates a Broad Spectrum of Substrates.

The infectious disease tuberculosis is one of the fifteen most common causes of death worldwide (according to the WHO). About every fourth person is infected with the main causative agent Mycobacterium tuberculosis (Mb). A characteristic of the pathogen is its entrance into a dormant state in which a phenotypic antibiotic resistance is achieved. To target resistant strains, novel dormancy-specific targets are very promising. Such a possible target is the Mb "fatty acid-CoA ligase 6" (MbFACL6), which activates fatty acids and thereby modulates the accumulation of triacylglycerol-containing lipid droplets that are used by Mb as an energy source during dormancy. We investigated the membrane association of MbFACL6 in E. coli and its specific activity towards different substrates after establishing a novel MbFACL6 activity assay. Despite a high homology to the mammalian family of fatty acid transport proteins, which are typically transmembrane proteins, our results indicate that MbFACL6 is a peripheral membrane-attached protein. Furthermore, MbFACL6 tolerates a broad spectrum of substrates including saturated and unsaturated fatty acids (C12-C20), some cholic acid derivatives, and even synthetic fatty acids, such as 9(E)-nitrooleicacid. Therefore, the substrate selectivity of MbFACL6 appears to be much broader than previously assumed.

Characterization of a Cytosolic Acyl-Activating Enzyme Catalyzing the Formation of 4-Methylvaleryl-CoA for Pogostone Biosynthesis in Pogostemon Cablin.

Pogostone, a compound with various pharmaceutical activities, is a major constituent of the essential oil preparation called Pogostemonis Herba, which is obtained from the plant Pogostemon cablin. The biosynthesis of pogostone has not been elucidated, but 4-methylvaleryl-CoA (4MVCoA) is a likely precursor. We analyzed the distribution of pogostone in P. cablin using gas chromatography-mass spectrometry (GC-MS) and found that pogostone accumulates at high levels in the main stems and leaves of young plants. A search for the acyl-activating enzyme (AAE) that catalyzes the formation of 4MVCoA from 4-methylvaleric acid was launched, using an RNAseq-based approach to identify 31 unigenes encoding putative AAEs including the PcAAE2, the transcript profile of which shows a strong positive correlation with the distribution pattern of pogostone. The protein encoded by PcAAE2 was biochemically characterized in vitro and shown to catalyze the formation of 4MVCoA from 4-methylvaleric acid. Phylogenetic analysis showed that PcAAE2 is closely related to other AAE proteins in P. cablin and other species that are localized to the peroxisomes. However, PcAAE2 lacks a peroxisome targeting sequence 1 (PTS1) and is localized in the cytosol.

Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System.

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their formation de novo from minimal chemical and biological elements. Here, we describe a chemoenzymatic route to membrane-forming noncanonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding the fatty acyl-CoA ligase with suitable lipid precursors enabled one-pot de novo synthesis of membrane-bound vesicles. Noncanonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin was coexpressed alongside the acyl-CoA ligase, the in situ assembled membranes were spontaneously decorated with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

Functional insights into two Ocimum kilimandscharicum 4-coumarate-CoA ligases involved in phenylpropanoid biosynthesis.

Plant 4-coumarate-CoA ligase (4CL) catalyzes the ligation of CoA to cinnamic acid and its derivatives. Activated CoA esters are utilized for the biosynthesis of phenolic metabolites and lignin that play essential function in plants. Here, we characterize the diversity of Ocimum kilimandscharicum 4CLs (Ok4CLs). Phylogenetic analysis suggest that Ok4CLs could be grouped into three classes, class I - enzymes mostly involved in lignin biosynthesis, class II - non-structural phenylpropanoid biosynthesis and class III - yet to be characterized for specific role(s). We selected two Ok4CLs namely Ok4CL7 and Ok4CL15 for further characterization. Gene expression analysis suggested that Ok4CL7 is highly expressed in leaf trichomes, whereas Ok4CL15 is abundant in the roots. The recombinant Ok4CL7 and Ok4CL15 had optimal enzyme activities at 40 °C in pH 8 and 7, respectively. Ok4CL7 showed substrate preference towards p-coumaric acid, ferulic acid and caffeic acid. While, Ok4CL15 preferred p-coumaric acid, ferulic acid and sinapic acid. Feruloyl adenylate showed higher number of contacts and lowers binding energy with Ok4CL7 and 15 compared to cinnamoyl adenylate. Based on root-specific expression and preference for sinapic acid, Ok4CL15 might be involved in lignin biosynthesis. Further exploration is needed to unravel the role of diverse Ok4CLs in O. kilimandscharicum.

Mouse long-chain acyl-CoA synthetase 1 is active as a monomer.

Fatty acids are essential cellular building blocks and a major energy source. Regardless of their metabolic fate, fatty acids first need to be activated by forming a thioester with a coenzyme A group. This reaction is carried out by acyl-CoA synthetases (ACSs), of which ACSL1 (long-chain acyl-CoA synthetase 1) is an important member. Two bacterial homologues of ACSL1 crystal structures have been solved previously. One is a soluble dimeric protein, and the other is a monomeric peripheral membrane protein. The mammalian ACSL1 is a membrane protein with an N-terminal transmembrane helix. To characterize the mammalian ACSL1, we purified the full-length mouse ACSL1 and reconstituted it into lipid nanodiscs. Using enzymatic assays, mutational analysis, and cryo-electron microscopy, we show that mouse ACSL1 is active as a monomer.

Identification of the fatty acid coenzyme-A ligase FadD1 as an interacting partner of FptX in the Pseudomonas aeruginosa pyochelin pathway.

Pseudomonas aeruginosa is one of the most important nosocomial bacteria emerging as a highly multidrug-resistant pathogen. P. aeruginosa produces two siderophores including pyochelin (PCH) to fulfil its need for iron during infections. We know that both outer and inner membrane proteins FptA and FptX are involved in the ferri-PCH uptake, but this process requires increasing molecular and biochemical knowledge. Here, using bacterial two-hybrid and copurification assays we identified the fatty acid coenzyme-A ligase FadD1 as a novel interacting partner of the inner membrane transporter FptX and found that FadD1 may play a role in PCH production. We managed to purify the FadD1-FptX inner membrane complex and obtained low-resolution 3D models, opening the way for future high-resolution structures.

IPRO+/-: Computational Protein Design Tool Allowing for Insertions and Deletions.

Insertions and deletions (indels) in protein sequences alter the residue spacing along the polypeptide backbone and consequently open up possibilities for tuning protein function in a way that is inaccessible by amino acid substitution alone. We describe an optimization-based computational protein redesign approach centered around predicting beneficial combinations of indels along with substitutions and also obtain putative substrate-docked structures for these protein variants. This modified algorithmic capability would be of interest for enzyme engineering and broadly inform other protein design tasks. We highlight this capability by (1) identifying active variants of a bacterial thioesterase enzyme ('TesA) with experimental corroboration, (2) recapitulating existing active TEM-1 ß-Lactamase sequences of different sizes, and (3) identifying shorter 4-Coumarate:CoA ligases with enhanced in vitro activities toward non-native substrates. A separate PyRosetta-based open-source tool, Indel-Maker (http://www.maranasgroup.com/software.htm), has also been created to construct computational models of user-defined protein variants with specific indels and substitutions.

Thermomagneto-Responsive Smart Biocatalysts for Malonyl-Coenzyme A Synthesis.

Smart biocatalysts, in which enzymes are conjugated to stimuli-responsive polymers, have gained considerable attention because of their catalytic switchability and recyclability. Although many systems have been developed, they require separate laboratory techniques for their recovery, making them unsuitable for many practical applications. To address these issues, we designed a thermomagneto-responsive biocatalyst by immobilizing an enzyme on the terminal of thermo-responsive polymer brushes tethered on magnetic nanoparticle (NP) clusters. The concept is demonstrated by a system consisting of iron oxide NPs, poly(N-isopropyl-acrylamide), and a malonyl-Coenzyme A synthetase (MatB). By using free malonate and coenzyme A (CoA), the designed catalyst exhibits adequate activity for the production of malonyl-CoA. Thanks to the use of a magnetic NP cluster, whose magnetic moment is high, this system is fully recoverable under the magnetic field at above 32 °C because of the collapse of the thermo-responsive polymer shell in the clusters. In addition, the recycled catalyst maintains moderate activity even after three cycles, and it also shows excellent catalytic switchability, that is, negligible catalytic activity at 25 °C because of the blockage of the active sites of the enzyme by the extended hydrophilic polymer chains but great catalytic activity at a temperatures above the lower critical solution temperature at which the enzymes are exposed to the reaction medium because of the thermo-responsive contraction of polymer chains. Because the azide functionality in our system can be easily functionalized depending upon our need, such catalytically switchable, fully recoverable, and recyclable multiresponsive catalytic systems can be of high relevance for other cell-free biosynthetic approaches.

Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia colifadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2 In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenesIMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

Biochemical characterization of acyl activating enzymes for side chain moieties of Taxol and its analogs.

Taxol (paclitaxel) is a very widely used anticancer drug, but its commercial sources mainly consist of stripped bark or suspension cultures of members of the plant genus Taxus. Taxol accumulates as part of a complex mixture of chemical analogs, termed taxoids, which complicates its production in pure form, highlighting the need for metabolic engineering approaches for high-level Taxol production in cell cultures or microbial hosts. Here, we report on the characterization of acyl-activating enzymes (AAEs) that catalyze the formation of CoA esters of different organic acids relevant for the N-substitution of the 3-phenylisoserine side chain of taxoids. On the basis of similarities to AAE genes of known function from other organisms, we identified candidate genes in publicly available transcriptome data sets obtained with Taxus × media. We cloned 17 AAE genes, expressed them heterologously in Escherichia coli, purified the corresponding recombinant enzymes, and performed in vitro assays with 27 organic acids as potential substrates. We identified TmAAE1 and TmAAE5 as the most efficient enzymes for the activation of butyric acid (Taxol D side chain), TmAAE13 as the best candidate for generating a CoA ester of tiglic acid (Taxol B side chain), TmAAE3 and TmAAE13 as suitable for the activation of 4-methylbutyric acid (N-debenzoyl-N-(2-methylbutyryl)taxol side chain), TmAAE15 as a highly efficient candidate for hexanoic acid activation (Taxol C side chain), and TmAAE4 as suitable candidate for esterification of benzoic acid with CoA (Taxol side chain). This study lays important groundwork for metabolic engineering efforts aimed at improving Taxol production in cell cultures.

Enzymatic promiscuity and the evolution of bioluminescence.

Bioluminescence occurs when an enzyme, known as a luciferase, oxidizes a small-molecule substrate, known as a luciferin. Nature has evolved multiple distinct luciferases and luciferins independently, all of which accomplish the impressive feat of light emission. One of the best-known examples of bioluminescence is exhibited by fireflies, a class of beetles that use d-luciferin as their substrate. The evolution of bioluminescence in beetles is thought to have emerged from ancestral fatty acyl-CoA synthetase (ACS) enzymes present in all insects. This theory is supported by multiple lines of evidence: Beetle luciferases share high sequence identity with these enzymes, often retain ACS activity, and some ACS enzymes from nonluminous insects can catalyze bioluminescence from synthetic d-luciferin analogues. Recent sequencing of firefly genomes and transcriptomes further illuminates how the duplication of ACS enzymes and subsequent diversification drove the evolution of bioluminescence. These genetic analyses have also uncovered candidate enzymes that may participate in luciferin metabolism. With the publication of the genomes and transcriptomes of fireflies and related insects, we are now better positioned to dissect and learn from the evolution of bioluminescence in beetles.

4-Coumarate:coenzyme A ligase isoform 3 from Piper nigrum (Pn4CL3) catalyzes the CoA thioester formation of 3,4-methylenedioxycinnamic and piperic acids.

Black pepper, dried green fruit of Piper nigrum L., is a household spice most popular in the world. Piperine, the pungency compound of black pepper, is proposed to partially arise from phenylpropanoid pathway. In the biosynthesis of piperine, 4-coumarate:CoA ligase (4CLs) must play a pivotal role in activating intermediate acids to corresponding CoA thioesters to serve as substrates. Based on transcriptome data, we isolated three P. nigrum 4CL isoforms (Pn4CL1, -2, and -3) from unripe peppercorn. These Pn4CLs were expressed in E. coli for in vitro enzyme assay with putative substrates, namely cinnamic, coumaric, ferulic, piperonylic, 3,4-methylenedioxycinnamic (3,4-MDCA), and piperic acids. Phylogenetic analysis and substrate usage study indicated that Pn4CL1, active towards coumaric and ferulic acids, belongs to class I 4CL for lignin synthesis. Pn4CL2 was a typical cinnamate-specific coumarate:CoA ligase-like (CLL) protein. The Pn4CL3, as class II enzyme, exhibited general 4CL activity towards coumaric and ferulic acids. However, Pn4CL3 was also active towards piperonylic acid, 3,4-MDCA, and piperic acid. Pn4CL3 possessed ∼2.6 times higher catalytic efficiency (kcat/KM) towards 3,4-MDCA and piperic acid than towards coumaric and ferulic acids, suggesting its specific role in piperine biosynthesis. Different substrate preference among the Pn4CL isoforms can be explained by 3-dimensional protein structure modeling, which demonstrated natural variants in amino acid residues of binding pocket to accommodate different substrates. Quantitative PCR analysis of these isoforms indicated that Pn4CL1 transcript level was highest in the roots whereas Pn4CL2 in the fruits and Pn4CL3 in the leaves.

Structures of 2-Hydroxyisobutyric Acid-CoA Ligase Reveal Determinants of Substrate Specificity and Describe a Multi-Conformational Catalytic Cycle.

2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B12-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.

Synthesis, biological evaluation and computational study of novel isoniazid containing 4H-Pyrimido[2,1-b]benzothiazoles derivatives.

Synthesis of novel and potent hit molecules has an eternal demand. It is our continuous study to search novel bioactive hit molecules and as a part of this, a series of novel N'-isonicotinoyl-2-methyl-4-(pyridin-2-yl)-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carbohydrazide analogs (5a-5n) were synthesized with good yields by the conventional method. The various novel compounds have been characterized and identified by many analytical technique such as IR, 1H NMR, 13C NMR, mass spectral analysis, and elemental analysis. All the synthetic analogs (5a-5n) are evaluated for their in vitro antibacterial and anti-mycobacterial activities against different bacterial strains. Molecular docking and Molecular dynamics studies were helped in revealing the mode of action of these compounds through their interactions with the active site of the Mycobacterium tuberculosis enoyl reductase (InhA) enzyme. The calculated ADMET descriptors for the synthesized compounds validated good pharmacokinetic properties, confirming that these compounds could be used as templates for the development of new Anti-mycobacterial agents.

Structural and Functional Insights into Host Death Domains Inactivation by the Bacterial Arginine GlcNAcyltransferase Effector.

Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.

In Vitro Naringenin Biosynthesis from p-Coumaric Acid Using Recombinant Enzymes.

Naringenin is an important precursor for the production of a wide spectrum of flavonoids, and its production is of great interest in metabolic engineering. However, in cellular systems, identification of rate-limiting factors is often difficult because of complex regulatory networks. Cell-free catalytic systems emerge as a promising method to address this issue. Here, we explored the cell-free biosystem for naringenin production by combining different sources of 4-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). After systematic analysis of enzyme levels, substrate concentrations, and cofactors, 4CL and CHS were found to be crucial to the reaction. The best loading ratio of 4CL/CHS/CHI was 10:10:1, and malonyl-CoA was the limiting factor, as identified previously in fermentation. For the first time, we successfully constructed the system for naringenin production in vitro. Our study will deepen our understanding of the key factors in naringenin production and guide further engineering.

Adaptive evolution of the ACSL gene family in Carnivora.

Carnivores exhibit various fat contents and energy reserves to adapt to their environments. However, the molecular mechanisms underlying lipid metabolic differences among carnivores have not been well explored. Long-chain acyl-CoA synthetases (ACSLs) catalyze the initial step in lipid metabolism by activating fatty acids (FAs), and they drive acyl-CoAs toward anabolic lipid synthesis or catabolic ß-oxidation. We identified the sequences of the genes of the ACSL family (ACSL1, ACSL3, ACSL4, ACSL5 and ACSL6) in the sable (Martes zibellina) via transcriptome sequencing. The ACSL gene sequences of 13 other carnivores were obtained from NCBI. Phylogenetic results showed that unlike the widely accepted carnivore phylogeny, Canidae and Felidae tend to group together based on ACSL4 and ACSL6. The evolutionary analyses identified a series of positively selected amino acid residues in ACSL1, ACSL4 and ACSL5. Two radical amino acid substitutions detected in sable suggested potential insights into the molecular mechanism underlying the relatively low fat content in this animal. This is the first study to investigate the molecular mechanisms underlying the adaptive evolution of fat metabolism in carnivores. Overall, the ACSL genes were under different evolutionary forces in carnivores, and some genes have undergone adaptive evolution in lipid metabolism.