Acyl Carrier Protein Research Articles

Divergent Tandem Acyl Carrier Proteins Necessitate In‐Series Polyketide Processing in the Leinamycin Family

AbstractThe leinamycin family of polyketides are promising antitumor antibiotics, yet several aspects of their biosynthesis remain elusive. All leinamycin family members bear a sulfur‐containing moiety which is essential for the anticancer activity exhibited by leinamycin. The key building blocks required for the incorporation of these functionalities are introduced in the final module of the polyketide synthase (PKS), which elegantly combines β‐branching and thiocysteine incorporation to generate a diverse library of sulfur‐based molecular scaffolds. Two acyl carrier proteins (ACPs) form a key didomain component of this module, but their amino acid sequence divergence has brought into question the common notion of functional equivalence. Here, we provide unprecedented functional evidence that these tandem ACPs play distinct roles in the final module of polyketide assembly. Using the weishanmycin biosynthetic pathway as a template, the in vitro reconstitution of key polyketide chain extension and β‐branching steps in this module has revealed strict functional selectivity for a single ACP. Furthermore, we propose a cryptic transacylation step must occur prior to polyketide off‐loading and cyclization. Altogether, these mechanistic investigations suggest that an atypical in‐series mechanism underpins sulfur incorporation in the leinamycin family, and provides significant progress towards delineating their late‐stage assembly.

Mitochondrial acyl carrier protein, Acp1, required for iron-sulfur cluster assembly in mitochondria and cytoplasm in Saccharomyces cerevisiae

Mitochondria perform vital biosynthetic processes, including fatty acid synthesis and iron-sulfur (FeS) cluster biogenesis. In Saccharomyces cerevisiae mitochondria, the acyl carrier protein Acp1 participates in type II fatty acid synthesis, requiring a 4-phosphopantetheine (PP) prosthetic group. Acp1 also interacts with the mitochondrial FeS cluster assembly complex that contains the cysteine desulfurase Nfs1. Here we investigated the role of Acp1 in FeS cluster biogenesis in mitochondria and cytoplasm. In the Acp1-depleted (Acp1↓) cells, biogenesis of mitochondrial FeS proteins was impaired, likely due to greatly reduced Nfs1 protein and/or its persulfide-forming activity. Formation of cytoplasmic FeS proteins was also deficient, suggesting a disruption in generating the (Fe-S)int intermediate, that is exported from mitochondria and is utilized for cytoplasmic FeS cluster assembly. Iron homeostasis was perturbed, with enhanced iron uptake into the cells and accumulation of iron in mitochondria. The Δppt2 strain, lacking the mitochondrial ability to add PP to Acp1, phenocopied the Acp1↓ cells. These data suggest that the holo form of Acp1 with the PP-conjugated acyl chain is required for stability of the Nfs1 protein and/or stimulation of its persulfide-forming activity. Thus, mitochondria lacking Acp1 (or Ppt2) cannot support FeS cluster biogenesis in mitochondria or cytoplasm, leading to disrupted iron homeostasis.

A Study of the Bioactive Compounds, Antioxidant Capabilities, Antibacterial Effectiveness, and Cytotoxic Effects on Breast Cancer Cell Lines Using an Ethanolic Extract from the Aerial Parts of the Indigenous Plant Anabasis aretioïdes Coss. & Moq.

Anabasis aretioïdes contain numerous bioactive compounds that provide several advantages, including antioxidant, antibacterial, anticancer, neuroprotective, anti-inflammatory, and antidiabetic characteristics. This study aimed to make a hydroethanolic extract from the aerial part of the plant, analyze its biochemical compounds, and test its biological activities. From HPLC-DAD analysis, cinnamic acid, sinapic acid, and vanillin bioactives were found to be the main compounds in the extract. The spectrometric tests revealed that the extract was rich in flavonoids (8.52 ± 0.32 mg RE/100 g DW), polyphenols (159.32 ± 0.63 mg GAE/100 g DW), and condensed tannins (8.73 ± 0.23 mg CE/100 g DW). The extract showed significant antioxidant activity. There were strong correlations between the amount of flavonoid or polyphenol and the antioxidant assays, including ABTS, DPPH, β-carotene, and TAC. The extract also showed highly effective results against Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis as well as against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and showed promising cytotoxicity against breast cancer cell lines MCF-7 and MDA-MB-231. The in silico modeling of the bioactive compounds contained in the extract illustrated their interaction mode with the active sites of particular target proteins, and it showed that rutin had the strongest effect on stopping NADPH oxidase enzyme, with a glide score of −6.889 Kcal/mol. Sinapic acid inhibited E. coli beta-ketoacyl-[acyl carrier protein] synthase (−7.517 kcal/mol), and apigenin showed high binding affinity to S. aureus nucleoside di-phosphate kinase, with −8.656 kcal/mol. Succinic acid has the strongest anticancer effect for caspase-3, with a glide score of −8.102 kcal/mol. These bioactive components may be beneficial as antioxidant and antibacterial applications in medicine, foods, natural cosmetics, and breast cancer prevention in the future. As a result, the use of this indigenous plant must be considered to maximize its value and preservation.

Cryo-EM structure of the Mycobacterium smegmatis MmpL5-AcpM complex.

The emergence and spread of multidrug-resistant tuberculosis (TB) present enormous challenges to the global public health. The causative agent, Mycobacterium tuberculosis, has now infected more than one-third of the world's population. Here, we report the first structure of the mycobacterial membrane protein large 5 (MmpL5), an essential transporter for iron acquisition, bound with the meromycolate extension acyl carrier protein M (AcpM), indicating a plausible pathway for mycobactin translocation. Our studies will ultimately inform an era in structure-guided drug design to combat TB infection.

Evaluating delivery of peptide nucleic acids to Gram-negative bacteria using differently linked membrane-active peptides and their stapled analogs

Antisense oligonucleotides have been developed as therapeutic compounds, with peptide nucleic acid (PNA) emerging as a promising nucleic acid mimic for antimicrobial applications. To be effective, PNAs must be internalized into bacterial cells, as they are not naturally absorbed. A strategy to improve PNA membrane penetration and cellular uptake involves covalently conjugating them to cell-penetrating peptides. However, these membrane-active peptides can exhibit cytotoxicity, and their efficiency as PNA carriers needs to be enhanced. Therefore, we explored new peptide–PNA conjugates and their linkers to understand how they affect PNA uptake into bacteria. We conjugated PNA to two peptides, anoplin and (KFF)3K, along with their structurally stabilized hydrocarbon-stapled derivatives, and evaluated their transport into various bacterial strains. The PNA sequence targeted bacterial mRNA encoding the essential acyl carrier protein. As linkages, we used either a non-cleavable 8-amino-2,6-dioxaoctanoyl (ethylene glycol, eg1) linker or a reducible disulfide bridge. We found that the hydrocarbon-stapled peptides did not enhance PNA delivery, despite the strong inner- and outer-membrane-penetrating capabilities of the standalone peptides. Additionally, the disulfide bridge linkage, which is cleavable in the bacterial cytoplasm, decreased the antimicrobial activity of the peptide–PNA conjugates. Notably, we identified anoplin as a new potent PNA carrier peptide, with the anoplin–eg1–PNA conjugate demonstrating antibacterial activity against E. coli and S. Typhimurium strains in the 2–4 µM range.

Reshaping the substrate-binding pocket of acyl-ACP reductase to enhance the production of sustainable aviation fuel in Escherichia coli.

To reduce carbon emissions and address environmental concerns, the aviation industry is exploring the use of sustainable aviation fuel (SAF) as an alternative to traditional fossil fuels. In this context, bio-alkane is considered a potentially high-value solution. The present study focuses on the enzymes acyl-acyl carrier protein [ACP] reductase (AAR) and aldehyde-deformylating oxygenase (ADO), which are crucial enzymes for alka(e)ne biosynthesis. By using protein engineering techniques, including semi-rational design and site-directed mutagenesis, we aimed to enhance the substrate specificity of AAR and improve alkane production efficiency. The co-expression of a modified AAR (Y26G/Q40M mutant) with wild-type ADO in Escherichia coli significantly increased alka(e)ne production from 28.92 mg/L to 167.30 mg/L, thus notably demonstrating a 36-fold increase in alkane yield. This research highlights the potential of protein engineering in optimizing SAF production, thereby contributing to the development of more sustainable and efficient SAF production methods and promoting greener air travel.

The Enteric Bacterium Enterococcus faecalis Elongates and Incorporates Exogenous Short and Medium Chain Fatty Acids Into Membrane Lipids.

Enterococcus faecalis incorporates and elongates exogeneous short- and medium-chain fatty acids to chains sufficiently long to enter membrane phospholipid synthesis. The acids are activated by the E. faecalis fatty acid kinase (FakAB) system and converted to acyl-ACP species that can enter the fatty acid synthesis cycle to become elongated. Following elongation the acyl chains are incorporated into phospholipid by the PlsY and PlsC acyltranferases. This process has little effect on de novo fatty acid synthesis in the case of short-chain acids, but a greater effect with medium-chain acids. Incorporation of exogenous short-chain fatty acids in E. faecalis was greatly increased by overexpression of either AcpA, the acyl carrier protein of fatty acid synthesis, or the phosphate acyl transferase PlsX. The PlsX of Lactococcus lactis was markedly superior to the E. faecalis PlsX in incorporation of short-chain but not long-chain acids. These manipulations also allowed unsaturated fatty acids of lengths too short for direct transfer to the phospholipid synthesis pathway to be elongated and support growth of E. faecalis unsaturated fatty acid auxotrophic strains. Short- and medium-chain fatty acids can be abundant in the human gastrointestinal tract and their elongation by E. faecalis would conserve energy and carbon by relieving the requirement for total de novo synthesis of phospholipid acyl chains.

The double-edged role of FASII regulator FabT in Streptococcus pyogenes infection.

In Streptococcus pyogenes, the type II fatty acid (FA) synthesis pathway FASII is feedback-controlled by the FabT repressor bound to an acyl-Acyl carrier protein. Although FabT defects confer reduced virulence in animal models, spontaneous fabT mutants arise in vivo. We resolved this paradox by characterizing the conditions and mechanisms requiring FabT activity, and those promoting fabT mutant emergence. The fabT defect leads to energy dissipation, limiting mutant growth on human tissue products, which explains the FabT requirement during infection. Conversely, emerging fabT mutants show superior growth in biotopes rich in saturated FAs, where continued FASII activity limits their incorporation. We propose that membrane alterations and continued FASII synthesis are the primary causes for increased fabT mutant mortality in nutrient-limited biotopes, by failing to stop metabolic consumption. Our findings elucidate the rationale for emerging fabT mutants that improve bacterial survival in lipid-rich biotopes, but lead to a genetic impasse for infection.

Molecular insights into the structure and function of the Staphylococcus aureus fatty acid kinase

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. This complex consists of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, and is known to impact virulence and disease outcomes. Despite some recent studies, there remains many outstanding questions as to the enzymatic mechanism and structure of FAK . To better address this gap in knowledge, we used a combination of modeling, biochemical, and cell-based approaches to build on prior proposed models and identify critical details of FAK activity. Using bio-layer interferometry, we demonstrated nanomolar affinity between FakA and FakB that also indicates that FakA is dimer when binding FakB. Additionally, targeted mutagenesis of the FakA Middle domain demonstrates it possesses a metal binding pocket that is critical for FakA dimer stability and FAK function in vitro and in vivo. Lastly, we solved structures of the apo and ligand-bound FakA kinase domain to capture the molecular changes in the protein following ATP binding and hydrolysis. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

Divergent Tandem Acyl Carrier Proteins Necessitate In-Series Polyketide Processing in the Leinamycin Family.

The leinamycin family of polyketides are promising antitumour antibiotics, yet several aspects of their biosynthesis remain elusive. All leinamycin family members bear a sulfur-containing moiety which is essential for the anticancer activity exhibited by leinamycin. The key building blocks required for the incorporation of these functionalities are introduced in the final module of the polyketide synthase (PKS), which elegantly combines β-branching and thiocysteine incorporation to generate a diverse library of sulfur-based molecular scaffolds. Two acyl carrier proteins (ACPs) form a key didomain component of this module, but their amino acid sequence divergence has brought into question the common notion of functional equivalence. Here, we provide unprecedented functional evidence that these tandem ACPs play distinct roles in the final module of polyketide assembly. Using the weishanmycin biosynthetic pathway as a template, the in vitro reconstitution of key polyketide chain extension and β-branching steps in this module has revealed strict functional selectivity for a single ACP. Furthermore, we propose a cryptic transacylation step must occur prior to polyketide off-loading and cyclization. Altogether, these mechanistic investigations suggest that an atypical in-series mechanism underpins sulfur incorporation in the leinamycin family, and provides significant progress towards delineating their late-stage assembly.

Structural Plasticity within 3-Hydroxy-3-Methylglutaryl Synthases Catalyzing the First Step of β-Branching in Polyketide Biosynthesis Underpins a Dynamic Mechanism of Substrate Accommodation.

Understanding how enzymes have been repurposed by evolution to carry out new functions is a key goal of mechanistic enzymology. In this study we aimed to identify the adaptations required to allow the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) enzymes of primary isoprenoid assembly to function in specialized polyketide biosynthetic pathways, where they initiate β-branching. This role notably necessitates that the HMG synthases (HMGSs) act on substrates tethered to noncatalytic acyl carrier protein (ACP) domains instead of coenzyme A, and accommodation of substantially larger chains within the active sites. Here, we show using a combination of X-ray crystallography and small-angle X-ray scattering, that a model HMGS from the virginiamycin system exhibits markedly increased flexibility relative to its characterized HMGCS counterparts. This mobility encompasses multiple secondary structural elements that define the dimensions and chemical nature of the active site, as well the catalytic residues themselves. This result was unexpected given the well-ordered character of the HMGS within the context of an HMGS/ACP complex, but analysis by synchrotron radiation circular dichroism demonstrates that this interaction leads to increased HMGS folding. This flexible to more rigid transition is notably not accounted for by AlphaFold2, which yielded a structural model incompatible with binding of the native substrates. Taken together, these results illustrate the continued necessity of an integrative structural biology approach combining crystallographic and solution-phase data for elucidating the mechanisms underlying enzyme remodeling, information which can inform strategies to replicate such evolution effectively in the laboratory.

Development of Mycobacterium tuberculosis Enoyl Acyl Reductase (InhA) Inhibitors: A Mini-Review

This review article delves into the critical role of Enoyl acyl carrier protein Reductase (InhA; ENR), a vital enzyme in the NADH-dependent acyl carrier protein reductase family, emphasizing its significance in fatty acid synthesis and, more specifically, the biosynthesis of mycolic acid. The primary objective of this literature review is to elucidate diverse scaffolds and their developmental progression targeting InhA inhibition, thereby disrupting mycolic acid biosynthesis. Various scaffolds, including thiourea, piperazine, thiadiazole, triazole, quinazoline, benzamide, rhodanine, benzoxazole, and pyridine, have been systematically explored for their potential as InhA inhibitors. Noteworthy findings highlight thiadiazole and triazole derivatives, demonstrating promising IC50 values within the nanomolar concentration range. The review offers comprehensive insights into InhA's structure, structure-activity relationships, and a detailed overview of distinct scaffolds as effective inhibitors of InhA.

Exploring the role of FAT genes in Solanaceae species through genome-wide analysis and genome editing.

Plants produce numerous fatty acid derivatives, and some of these compounds have significant regulatory functions, such as governing effector-induced resistance, systemic resistance, and other defense pathways. This study systematically identified and characterized eight FAT genes (Acyl-acyl carrier protein thioesterases), four in the Solanum lycopersicum and four in the Solanum tuberosum genome. Phylogenetic analysis classified these genes into four distinct groups, exhibiting conserved domain structures across different plant species. Promoter analysis revealed various cis-acting elements, most of which are associated with stress responsiveness and growth and development. Micro-RNA (miRNA) analysis identified specific miRNAs, notably miRNA166, targeting different FAT genes in both species. Utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated knockout, mutant lines for SlFATB1 and SlFATB3 were successfully generated and exhibited diverse mutation types. Biochemical evaluation of selected mutant lines revealed significant changes in fatty acid composition, with linoleic and linolenic acid content variations. The study also explored the impact of FAT gene knockout on tomato leaf architecture through scanning electron microscopy, providing insights into potential morphological alterations. Knocking out of FAT genes resulted in a significant reduction in both trichome and stoma density. These findings contribute to a comprehensive understanding of FAT genes in Solanaceous species, encompassing genetic, functional, and phenotypic aspects.

Multi-omics approach for understanding the response of Bacteroides fragilis to carbapenems

BackgroundThe prevalence of Bacteroides fragilis isolates resistant to first-line beta-lactam drugs is increasing, resulting in reduced treatment efficacy. Investigating the bacterial transcriptome and proteome can uncover links between bacterial genes and resistance mechanisms. In this study, we experimentally assessed in vitro the transcriptional and proteomic profiles of B. fragilis exposed to SICs of meropenem, an effective antimicrobial agent, collected from patients with intra-abdominal diseases at Astana City Hospital, Kazakhstan. MethodsB. fragilis was cultured in brain heart infusion broth and sub-cultured every 48 h for 8 days in media with and without meropenem. Total RNA was extracted from the liquid cultures using a commercial RNeasy mini kit, and strand-specific RNA sequencing (RNA-seq) was performed on the DNBSEQ platform. Raw RNA-seq data were retrieved from BioProject No. PRJNA531645 and uploaded to the NCBI Sequence Read Archive (accession no. SRX22081155). Proteins of B. fragilis were extracted and separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis, followed by analysis of the eluted peptides using liquid chromatography–tandem mass spectrometry. Cluster analysis utilised the Database for Annotation, Visualisation, and Integrated Discovery. ResultsThe subinhibitory concentration (SIC) of meropenem was determined to be 0.5 μg/L (minimum inhibitory concentration: 1). Mapping of reads to the reference genome identified 2477 expressed genes in all B. fragilis BFR KZ01 samples. Ten differentially expressed genes (DEGs) were common across comparison groups during and post-antibiotic exposure (wMEM vs. MEM2 and MEM2 vs. rMEM8); however, no substantially enriched Gene Ontology terms were identified. The cluster analysis highlighted a significant enrichment cluster (W-0560 oxidoreductase) of DEGs following antibiotic withdrawal. In total, 859 B. fragilis proteins were identified, with the expressions of three proteins, 3-oxoacyl-[acyl carrier protein] reductase, acetyl-CoA carboxylase biotin carboxylase subunit, and beta-ketoacyl-ACP synthase III, being upregulated in the enriched protein folding category. Notably, chaperone proteins such as FKBP-type peptidyl-prolyl cis-trans isomerases (involved in the cis-trans isomerisation of prolyl peptide bonds) and GroES (a co-chaperone functioning with GroEL) were also identified. ConclusionsUnder the influence of low doses of antibiotics defense mechanisms are activated which contribute to the emergence of resistance. These results provide insight into the response of B. fragilis to meropenem exposure, mainly at the SIC, contributing to the understanding bacterial survival strategies under stress conditions.

Lysophospholipid remodeling mediated by the LplT and Aas protein complex in the bacterial envelope

Lysophospholipid transporter LplT and acyltransferase Aas consist of a lysophospholipid-remodeling system ubiquitously found in gram-negative microorganisms. LplT flips lysophospholipids across the inner membrane, which are subsequently acylated by Aas on the cytoplasmic membrane surface. Our previous study showed that the proper functioning of this system is important to protecting E. coli from phospholipase-mediated host attack by maintaining the integrity of the bacterial cell envelope. However, the working mechanism of this system is still unclear. Herein, we report that LplT and Aas form a membrane protein complex in E. coli which allows these two enzymes to cooperate efficiently to move lysophospholipids across the bacterial membrane and catalyze their acylation. The direct interaction of LplT and Aas was demonstrated both in vivo and in vitro with a binding affinity of 2.3 μM. We found that a cytoplasmic loop of LplT adjacent to the exit of the substrate translocation pathway plays an important role in maintaining its interaction with Aas. Aas contains an acyl-acyl carrier protein synthase domain and an acyl-transferase domain. Its interaction with LplT is mediated exclusively by its transferase domain. Mutations within the three loops near the putative catalytic site of the transferase domain, respectively, disrupt its interaction with LplT and lysophospholipid acylation activity. These results support a hypothesis of the functional coupling mechanism, in which LplT directly interacts with the transferase domain of Aas for specific substrate membrane migration, providing synchronization of substrate translocation and biosynthetic events.

Molecular insights into the oleic acid accumulation in safflower

AbstractMost of the Indian safflower (Carthamus tinctorius L.) varieties produce oil rich in linoleic acid (LA, ~75%) and low in oleic acid (OA, ~15%). In the fatty acid biosynthetic pathway, the fatty acid desaturase 2 (FAD2) enzyme converts OA to LA. Safflower is reported to have 12–20 FAD2 genes. Gene expression analysis of four FAD2 genes during seed development in a high LA variety, PBNS‐12, revealed high expression of FAD2‐1 at 21 days after flowering (DAF), correlating with high LA accumulation. Fatty acid profiling of 448 Indian safflower germplasm accessions revealed four lines to have high (58%–77%) OA content, with NASF‐39 having the highest OA content. Interestingly, all four high OA lines showed the same mutation in the FAD2‐1 gene. The DNA sequence of FAD2‐1 from the four high OA lines showed a deletion of C at the +606 position, resulting in a premature stop codon at the +733 position and a truncated protein of 244 amino acids. Hence, despite the high expression levels of FAD2‐1 in NASF‐39 at 18–21 DAF, it exhibited high OA (77%). The dysfunctional nature of the truncated FAD2‐1 in NASF‐39 was evident in molecular docking studies with 1‐stearoyl‐2‐oleoyl phosphatidylcholine. We also sequenced FATB, a thioesterase responsible for releasing stearic acid from acyl carrier protein for further desaturation to oleic acid, where an A773G substitution was observed. This resulted in E258G substitution in NASF‐39 FATB compared to that of PBNS‐12. This probably made the acyl‐binding pocket of NASF‐39 FATB unstable, contributing to high OA accumulation. Thus, the outcomes of this study can help develop super and ultra‐high oleic safflower varieties through various genetics and genomics approaches.

Synthesis and biological profile of substituted hexahydrofuro[3,4-b]furans, a novel class of bicyclic acyl-acyl carrier protein (ACP) thioesterase inhibitors.

Weed control is a significant challenge for farmers around the globe. Of the various methods available for combatting weeds, small molecules remain the most effective and versatile technology to date. In the search for novel chemical entities with new modes of action toward herbicide-resistant weeds, we have investigated hexahydrofuro[3,4-b]furan-based acyl-acyl carrier protein (ACP) thioesterase inhibitors inspired by X-ray co-crystal structure-based modeling studies. By exploiting scaffold hopping concepts and molecular modeling studies we were able to identify new hexahydrofuro[3,4-b]furan-based lead structures showing promising activity in vivo against commercially important grass weeds in line with strong target affinity. The present work covers a series of novel herbicidal lead structures that possess a hexahydrofuro[3,4-b]furan scaffold as a structural key feature, carrying ortho-substituted aryloxy side chains. Based on an optimized synthetic approach a broad structure-activity relationship (SAR) study was carried out. The new compounds emerging from our modeling-inspired structural variations show good acyl-ACP thioesterase inhibition in line with promising initial herbicidal activity. Glasshouse trials showed that the hexahydrofuro[3,4-b]furans outlined herein display good control of cold and warm season grass-weed species in pre-emergence application. Remarkably, some of the novel acyl-ACP thioesterase-inhibitors also showed promising efficacy against warm season weeds that are difficult to control. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Computational Studies to Explore Plant-based Inhibitors for Enoyl- (Acyl Carrier Protein)- Reductase (InhA) of Mycobacterium tuberculosis

Aims: A computational approach has been adopted to find therapeutically potent herbal compounds with anti-TB properties Background: The second largest cause of death globally is Mycobacterium tuberculosis. Considering that the BCG vaccine is only marginally effective. This study has focused on enoyl- (acyl carrier protein)- reductase (InhA), one of the important enzymes in M. tuberculosis's type II fatty acid (mycolic acid synthesis) biosynthesis pathway. Bioinformatics-based tools have been used to explore therapeutically sound phytocompounds against InhA. Objective: To conduct an insightful study using bioinformatics-based tools to explore phytocompounds originating from different medicinal plants which would act as potent inhibitors of enoyl - (acyl carrier protein)- reductase (InhA) to obstruct the growth of M. tuberculosis. Methods: Molecular docking (using EasyDockVina) has been used for screening the 150 phytocompounds against Enoyl - (acyl carrier protein)- reductase (InhA). AMDET analysis was performed using DruLito and protox II to test the drug-likeness properties of phytocompounds. Results: From the results of molecular docking and two-dimensional interaction, it is concluded that Licoflavone B, Tembaterine, Colubrine and Shinpterocarpin are the potent inhibitors of InhA. Conclusion: According to this study, Licoflavone B, Tembaterine, Columbin, and Shinpterocarpin have positively passed AMDET screening and have high docking scores. These phytocompounds can be considered safe drug candidates against InhA of Mycobacterium tuberculosis.

Mutagenesis Supports AlphaFold Prediction of How Modular Polyketide Synthase Acyl Carrier Proteins Dock With Downstream Ketosynthases.

The docking of an acyl carrier protein (ACP) domain with a downstream ketosynthase (KS) domain in each module of a polyketide synthase (PKS) helps ensure accurate biosynthesis. If the polyketide chain bound to the ACP has been properly modified by upstream processing enzymes and is compatible with gatekeeping residues in the KS tunnel, a transacylation reaction can transfer it from the 18.1-Å phosphopantetheinyl arm of the ACP to the reactive cysteine of the KS. AlphaFold-Multimer predicts a general interface for these transacylation checkpoints. Half of the solutions obtained for 50 ACP/KS pairs show the KS motif TxLGDP forming the first turn of an α-helix, as in reported structures, while half show it forming a type I β-turn not previously observed. Solutions with the latter conformation may represent how these domains are relatively positioned during the transacylation reaction, as the entrance to the KS active site is relatively open and the phosphopantetheinylated ACP serine and the reactive KS cysteine are relatively closer-17.2 versus 20.9 Å, on average. To probe the predicted interface, 20 mutations were made to KS surface residues within the model triketide lactone synthase P1-P6-P7. The activities of these mutants are consistent with the proposed interface.

Biosynthesis of the Unusual Epoxy Isonitrile-Containing Antibiotics Aerocyanidin and Amycomicin.

Aerocyanidin and amycomicin are two antibiotics derived from long-chain acids with a rare epoxy isonitrile moiety, the complexity of which renders the total synthesis of these two natural products rather challenging. How this functionality is biosynthesized has also remained obscure. While the biosynthetic gene clusters for these compounds have been identified, both appear to be deficient in genes encoding enzymes seemingly necessary for the oxidative modifications observed in these antibiotics. Herein, the biosynthetic pathways of aerocyanidin and amycomicin are fully elucidated. They share a conserved pathway to isonitrile intermediates that involves a bifunctional thioesterase and a nonheme iron α-ketoglutarate-dependent enzyme. In both cases, the isonitrile intermediates are then loaded onto an acyl carrier protein (ACP) catalyzed by a ligase. The isonitrile-tethered ACP is subsequently processed by polyketide synthase(s) to undergo chain extension, thereby assembling a long-chain γ-hydroxy isonitrile acid skeleton. The epoxide is installed by the cupin domain-containing protein AecF to conclude the biosynthesis of aerocyanidin. In contrast, three P450 enzymes AmcB, AmcC, and AmcQ are involved in epoxidation and keto formation to finalize the biosynthesis of amycomicin. These results thus explain the sequence of oxidation events that result in the final structures of aerocyanidin and amycomicin as well as the biosynthesis of the key γ-hydroxy epoxy isonitrile functional group.