Acyl carrier protein tag can enhance tobacco etch virus protease stability and promote its covalent immobilisation.

Fusion expression is widely employed to enhance the solubility of recombinant proteins. However, removal of the fusion tag is often required due to its potential impact on the structure and activity of passenger proteins. Tobacco etch virus (TEV) protease is widely used for this purpose due to its stringent sequence recognition. In the present work, fusion to the acyl carrier protein from E. coli fatty acid synthase (ACP) significantly increased the yield of recombinant soluble TEV, and the ACP tag also greatly improved TEV stability. The cleavage activity of TEV was not affected by the ACP fusion tag, and ACP-TEV retained high activity, even at unfavourable pH values. Moreover, ACP-TEV could be efficiently modified by co-expressed E. coli holo-ACP synthase (AcpS), leading to covalent attachment of 4'-phosphopantetheine (4'-PP) group to ACP. The sulfhydryl group of the long, flexible 4'-PP chain displayed high specific reactivity with iodoacetyl groups on the solid support. Thus, TEV could be immobilised effectively and conveniently via the active holo-ACP, and immobilised TEV retained high cleavage activity after a long storage period and several cycles of reuse. As a low-cost and recyclable biocatalyst, TEV immobilised by this method holds promise for biotechnological research and development. KEY POINTS: • The ACP tag greatly increased the soluble expression and stability of TEV protease. • The ACP tag did not affect the cleavage activity of TEV. • The holo-ACP Tag effectively mediated the covalent immobilisation of TEV.

Anammox Bacterial S-Adenosyl-l-Methionine Dependent Methyltransferase Crystal Structure and Its Interaction with Acyl Carrier Proteins.

Ladderane lipids (found in the membranes of anaerobic ammonium-oxidizing [anammox] bacteria) have unique ladder-like hydrophobic groups, and their highly strained exotic structure has attracted the attention of scientists. Although enzymes encoded in type II fatty acid biosynthesis (FASII) gene clusters in anammox bacteria, such as S-adenosyl-l-methionine (SAM)-dependent enzymes, have been proposed to construct a ladder-like structure using a substrate connected to acyl carrier protein from anammox bacteria (AmxACP), no experimental evidence to support this hypothesis was reported to date. Here, we report the crystal structure of a SAM-dependent methyltransferase from anammox bacteria (AmxMT1) that has a substrate and active site pocket between a class I SAM methyltransferase-like core domain and an additional α-helix inserted into the core domain. Structural comparisons with homologous SAM-dependent C-methyltransferases in polyketide synthase, AmxACP pull-down assays, AmxACP/AmxMT1 complex structure predictions by AlphaFold, and a substrate docking simulation suggested that a small compound connected to AmxACP could be inserted into the pocket of AmxMT1, and then the enzyme transfers a methyl group from SAM to the substrate to produce branched lipids. Although the enzymes responsible for constructing the ladder-like structure remain unknown, our study, for the first time, supports the hypothesis that biosynthetic intermediates connected to AmxACP are processed by SAM-dependent enzymes, which are not typically involved in the FASII system, to produce the ladder-like structure of ladderane lipids in anammox bacteria.

Identification and Functional Analysis of Acyl-Acyl Carrier Protein Δ9 Desaturase from Nannochloropsis oceanica.

The oleaginous marine microalga Nannochloropsis oceanica strain IMET1 has attracted increasing attention as a promising photosynthetic cell factory due to its unique excellent capacity to accumulate large amounts of triacylglycerols and eicosapentaenoic acid. To complete the genomic annotation for genes in the fatty acid biosynthesis pathway of N. oceanica, we conducted the present study to identify a novel candidate gene encoding the archetypical chloroplast stromal acyl-acyl carrier protein Δ9 desaturase. The full-length cDNA was generated using rapid-amplification of cDNA ends, and the structure of the coding region interrupted by four introns was determined. The RT-qPCR results demonstrated the upregulated transcriptional abundance of this gene under nitrogen starvation condition. Fluorescence localization studies using EGFP-fused protein revealed that the translated protein was localized in chloroplast stroma. The catalytic activity of the translated protein was characterized by inducible expression in Escherichia coli and a mutant yeast strain BY4389, indicating its potential desaturated capacity for palmitoyl-ACP (C16:0-ACP) and stearoyl-ACP (C18:0-ACP). Further functional complementation assay using BY4839 on plate demonstrated that the expressed enzyme restored the biosynthesis of oleic acid. These results support the desaturated activity of the expressed protein in chloroplast stroma to fulfill the biosynthesis and accumulation of monounsaturated fatty acids in N. oceanica strain IMET1.

Activity of Fatty Acid Biosynthesis Initiating Ketosynthase FabH with Acetyl/Malonyl-oxa/aza(dethia)CoAs.

Fatty acid and polyketide biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. A prime example is FabH, which initiates fatty acid biosynthesis in many bacteria and plants. FabH performs an acyltransferase reaction with acetyl-CoA to generate an acetyl-S-FabH acyl-enzyme intermediate and subsequent decarboxylative Claisen-condensation with a malonyl-thioester carried by an acyl carrier protein (ACP). We envision that crystal structures of FabH with substrate analogues can provide insight into the conformational changes and enzyme/substrate interactions underpinning the distinct reactions. Here, we synthesize acetyl/malonyl-CoA analogues with esters or amides in place of the thioester and characterize their stability and behavior as Escherichia coli FabH substrates or inhibitors to inform structural studies. We also characterize the analogues with mutant FabH C112Q that mimics the acyl-enzyme intermediate allowing dissection of the decarboxylation reaction. The acetyl- and malonyl-oxa(dethia)CoA analogues undergo extremely slow hydrolysis in the presence of FabH or the C112Q mutant. Decarboxylation of malonyl-oxa(dethia)CoA by FabH or C112Q mutant was not detected. The amide analogues were completely stable to enzyme activity. In enzyme assays with acetyl-CoA and malonyl-CoA (rather than malonyl-ACP) as substrates, acetyl-oxa(dethia)CoA is surprisingly slightly activating, while acetyl-aza(dethia)CoA is a moderate inhibitor. The malonyl-oxa/aza(dethia)CoAs are inhibitors with Ki's near the Km of malonyl-CoA. For comparison, we determine the FabH catalyzed decomposition rates for acetyl/malonyl-CoA, revealing some fundamental catalytic traits of FabH, including hysteresis for malonyl-CoA decarboxylation. The stability and inhibitory properties of the substrate analogues make them promising for structure-function studies to reveal fatty acid and polyketide enzyme/substrate interactions.

Structural Basis of Transient Interactions of Acyltransferase VinK with the Loading Acyl Carrier Protein of the Vicenistatin Modular Polyketide Synthase.

Acyltransferase (AT) recognizes its cognate acyl carrier protein (ACP) for functional transfer of an acyl unit in polyketide biosynthesis. However, structural characterization of AT-ACP complexes is limited because of the weak and transient interactions between them. In the biosynthesis of macrolactam polyketide vicenistatin, the trans-acting loading AT VinK transfers a dipeptidyl unit from the stand-alone ACP VinL to the ACP domain (VinP1ACPL) of the loading module of modular polyketide synthase VinP1. Although the previously determined structure of the VinK-VinL complex clearly illustrates the VinL recognition mechanism of VinK, how VinK recognizes VinP1ACPL remains unclear. Here, the crystal structure of a covalent VinK-VinP1ACPL complex formed with a pantetheine-type cross-linking probe is reported at 3.0 Å resolution. The structure of the VinK-VinP1ACPL complex provides detailed insights into the transient interactions between VinK and VinP1ACPL. The importance of residues in the binding interface was confirmed by site-directed mutational analyses. The binding interface between VinK and VinP1ACPL is similar to that between VinK and VinL, although some of the interface residues are different. However, the ACP orientation and interaction mode observed in the VinK-VinP1ACPL complex are different from those observed in other AT-ACP complexes such as the disorazole trans-AT-ACP complex and cis-AT-ACP complexes of modular polyketide synthases. Thus, AT-ACP binding interface interactions are different in each type of AT-ACP pair.

Heterologous expression, purification, and partial characterisation of the apicoplast protein 3-oxoacyl-[acyl-carrier-protein] reductase from Toxoplasma gondii.

Recombinant expression and purification of proteins have become a staple of modern drug discovery as it enables more precise in vitro analyses of drug targets, which may help obtain biochemical and biophysical parameters of a known enzyme and even uncover unknown characteristics indicative of novel enzymatic functions. Such information is often necessary to prepare adequate screening assays and drug-discovery experiments in general. Toxoplasma gondii is an obligate protozoan parasite that is a member of the phylum Apicomplexa, can develop several neuro-degenerative symptoms and, in specific cases, certain death for human hosts. Its relict non-photosynthetic plastid, the apicoplast, harbours a unique de novo long-chain fatty acid synthesis pathway of a prokaryotic character, FASII. The FASII pathway shows plasticity and, is essential for many intracellular and membranal components, along with fatty acid uptake via salvaging from the host, therefore, its disruption causes parasite death. TgFabG, a FASII enzyme responsible for a single reduction step in the pathway, was recombinantly expressed, purified and biochemically and biophysically characterised in this study. The bioengineering hurdle of expressing the recombinant gene of a eukaryotic, signal peptide-containing protein in a prokaryotic system was overcome for the apicomplexan enzyme TgFabG, by truncating the N-terminal signal peptide. TgFabG was ultimately recombinantly produced in a plasmid expression vector from its 1131 base pair gene, purified as 260 and 272 amino acid proteins using a hexahistidine (6 × Histag) affinity chromatography and its biochemical (enzyme activity and kinetics) and biophysical characteristics were analysed in vitro.

Multiple Functions of the Type II Thioesterase Associated with the Phoslactomycin Polyketide Synthase.

Polyketide synthases (PKSs) are molecular assembly lines that condense basic chemical building blocks for the production of structurally diverse polyketides. Many PKS biosynthetic gene clusters contain a gene encoding for a type II thioesterase (TEII). It is believed that TEIIs exert a proofreading function and restore or increase the productivity of PKSs by removing aberrant modifications on the acyl-carrier proteins (ACPs) of the PKS assembly line. Yet biochemical evidence is still sparse. Here, we investigated the function of PnG, the TEII of the phoslactomycin PKS (Pn PKS), in the context of its cognate assembly line in vitro. Biochemical analysis revealed that PnG preferentially hydrolyzes alkyl-ACPs over (alkyl)malonyl-ACPs by up to three orders of magnitude, supporting a proofreading role of the enzyme. We further demonstrate that PnG increases the in vitro production of different native and non-native tetra-, penta-, and hexaketide derivatives of phoslactomycin by more than one order of magnitude and show that these effects are caused by the initial clearing of the Pn PKS, as well as proofreading of the active assembly line. Finally, we demonstrate that PnG is able to release intermediate but notably also terminal polyketides from the Pn PKS. This allows PnG to functionally replace and overcome the terminal TEI activity of chimeric in vitro Pn PKS systems, as showcased with a phoslactomycin hexaketide system. Altogether, our experiments provide detailed insights into the molecular mechanisms and the multiple functions of PnG in its native context, as well as their potential use in future applications.

Structural study of acyl carrier protein of Enterococcus faecalis and its interaction with enzymes in de novo fatty acid synthesis.

Enterococcus faecalis has recently shown signs of high antibiotic resistance. These bacteria can endure extremes of temperature and this may be due to the high thermostability of its proteins. E. faecalis has two acyl carrier proteins (ACPs), AcpA (EfAcpA), which is essential for de novo fatty acid synthesis (FAS), and EfAcpB, which plays an auxiliary role in the incorporation of exogenous fatty acids. Structural studies on EfAcpA and its interaction with FAS enzymes have not yet been reported. Here, we investigated the structures of EfAcpA using NMR spectroscopy, showing that EfAcpA consists of three α-helices with a long α2α3 loop, while the other ACPs have four α-helices. CD experiments showed that the melting temperature of EfAcpA is 76.3 °C and the Ala mutation for Ile10 reduced it dramatically by 29.5 °C. Highly conserved Ile10 of EfAcpA mediates compact intramolecular packing and promotes high thermostability. A docking simulation of EfAcpA and ß-ketoacyl-ACP synthase III (EfKAS III) showed that the α2α3 loop of EfAcpA contributes to specific protein-protein interactions (PPI) with EfKAS III. Unconserved charged residues, Lys52 and Glu54, in the α2α3 loop of EfAcpA formed specific electrostatic interactions with Asp 226 and Arg217 of EfKAS III, respectively. Binding interactions between EfAcpA and EfKASIII may provide insights for designing PPI inhibitors targeting FAS in E. faecalis to overcome its antibacterial resistance.

Mycobacterium tuberculosis KasA as a drug target: Structure-based inhibitor design.

Recent studies have reported the ß-ketoacyl-acyl carrier protein KasA as a druggable target for Mycobacterium tuberculosis. This review summarizes the current status of major classes of KasA inhibitors with an emphasis on significant contributions from structure-based design methods leveraging X-ray crystal structures of KasA alone and in complex with inhibitors. The issues addressed within each inhibitor class are discussed while detailing the characterized interactions with KasA and structure-activity relationships. A critical analysis of these findings should lay the foundation for new KasA inhibitors to study the basic biology of M. tuberculosis and to form the basis of new antitubercular molecules of clinical significance with activity against drug-sensitive and drug-resistant infections.

Mycobacterial acyl carrier protein suppresses TFEB activation and upregulates miR-155 to inhibit host defense.

Mycobacterial acyl carrier protein (AcpM; Rv2244), a key protein involved in Mycobacterium tuberculosis (Mtb) mycolic acid production, has been shown to suppress host cell death during mycobacterial infection. This study reports that mycobacterial AcpM works as an effector to subvert host defense and promote bacterial growth by increasing microRNA (miRNA)-155-5p expression. In murine bone marrow-derived macrophages (BMDMs), AcpM protein prevented transcription factor EB (TFEB) from translocating to the nucleus in BMDMs, which likely inhibited transcriptional activation of several autophagy and lysosomal genes. Although AcpM did not suppress autophagic flux in BMDMs, AcpM reduced Mtb and LAMP1 co-localization indicating that AcpM inhibits phagolysosomal fusion during Mtb infection. Mechanistically, AcpM boosted the Akt-mTOR pathway in BMDMs by upregulating miRNA-155-5p, a SHIP1-targeting miRNA. When miRNA-155-5p expression was inhibited in BMDMs, AcpM-induced increased intracellular survival of Mtb was suppressed. In addition, AcpM overexpression significantly reduced mycobacterial clearance in C3HeB/FeJ mice infected with recombinant M. smegmatis strains. Collectively, our findings point to AcpM as a novel mycobacterial effector to regulate antimicrobial host defense and a potential new therapeutic target for Mtb infection.

Structural Insight of KSIII (ß-Ketoacyl-ACP Synthase)-like Acyltransferase ChlB3 in the Biosynthesis of Chlorothricin.

Chlorothricin (CHL) belongs to a spirotetronate antibiotic family produced by Streptomyces antibioticus that inhibits pyruvate carboxylase and malate dehydrogenase. For the biosynthesis of CHL, ChlB3 plays a crucial role by introducing the 6-methylsalicylic acid (6MSA) moiety to ChlB2, an acyl carrier protein (ACP). However, the structural insight and catalytic mechanism of ChlB3 was unclear. In the current study, the crystal structure of ChlB3 was solved at 3.1 Å-resolution and a catalytic mechanism was proposed on the basis of conserved residues of structurally related enzymes. ChlB3 is a dimer having the same active sites as CerJ (a structural homologous enzyme) and uses a KSIII-like fold to work as an acyltransferase. The relaxed substrate specificity of ChlB3 was defined by its catalytic efficiencies (kcat/Km) for non-ACP tethered synthetic substrates such as 6MSA-SNAC, acetyl-SNAC, and cyclohexonyl-SNAC. ChlB3 successfully detached the 6MSA moiety from 6MSA-SNAC substrate and this hydrolytic activity demonstrated that ChlB3 has the potential to catalyze non-ACP tethered substrates. Structural comparison indicated that ChlB3 belongs to FabH family and showed 0.6-2.5 Å root mean square deviation (RMSD) with structural homologous enzymes. Molecular docking and dynamics simulations were implemented to understand substrate active site and structural behavior such as the open and closed conformation of the ChlB3 protein. The resultant catalytic and substrate recognition mechanism suggested that ChlB3 has the potential to use non-native substrates and minimize the labor of expressing ACP protein. This versatile acyltransferase activity may pave the way for manufacturing CHL variants and may help to hydrolyze several thioester-based compounds.

Physiological and Genetic Regulation for High Lipid Accumulation by Chlorella sorokiniana Strains from Different Environments of an Arctic Glacier, Desert, and Temperate Lake under Nitrogen Deprivation Conditions.

Microalgae can adapt to extreme environments with specialized metabolic mechanisms. Here, we report comparative physiological and genetic regulation analyses of Chlorella sorokiniana from different environmental regions of an arctic glacier, desert, and temperate native lake in response to N deprivation, for screening the optimal strain with high lipid accumulation. Strains from the three regions showed different growth and biochemical compositions under N deprivation. The arctic glacier and desert strains produced higher soluble sugar content than strains from the native lake. The arctic glacier strains produced the highest levels of lipid content and neutral lipids under N deprivation compared with strains from desert and native lake. At a molecular level, the arctic strain produced more differentially expressed genes related to fatty acid biosynthesis, glycolysis gluconeogenesis, and glycerolipid metabolism. The important functional genes acetyl coenzyme A (acetyl-CoA) carboxylase (ACCase), fatty acid synthase complex, pyruvate dehydrogenase component, and fatty acyl-acyl carrier protein (acyl-ACP) thioesterase were highly expressed in arctic strains. More acetyl-CoA was produced from glycolysis gluconeogenesis and glycerolipid metabolism, which then produced more fatty acid with the catalytic function of ACCase and acyl-ACP thioesterase in fatty acid biosynthesis. Our results indicated that the C. sorokiniana strains from the arctic region had the fullest potential for biodiesel production due to special genetic regulation related to fatty acid synthesis, glycolysis gluconeogenesis, and glycerolipid metabolism. IMPORTANCE It is important to reveal the physiological and genetic regulation mechanisms of microalgae for screening potential strains with high lipid production. Our results showed that Chlorella sorokiniana strains from arctic glacier, desert, and temperate native lake had different growth, biochemical composition, and genetic expression under N deprivation. The strains from an arctic glacier produced the highest lipid content (including neutral lipid), which was related to the genetic regulation of fatty acid biosynthesis, glycolysis gluconeogenesis, and glycerolipid metabolism. The functional genes for acetyl-CoA carboxylase, fatty acid synthase complex, pyruvate dehydrogenase component, and fatty acyl-ACP thioesterase in the three pathways were highly expressed in arctic strains. The revelation of physiological and genetic regulation of strains from different environmental regions will contribute to the microalgae selection for high lipid accumulation.

Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin.

Mupirocin is a clinically important antibiotic produced by Pseudomonas fluorescens NCIMB 10586 that is assembled by a complex trans-AT polyketide synthase. The polyketide fragment, monic acid, is esterified by a 9-hydroxynonanoic acid (9HN) side chain which is essential for biological activity. The ester side chain assembly is initialised from a 3-hydroxypropionate (3HP) starter unit attached to the acyl carrier protein (ACP) MacpD, but the fate of this species is unknown. Herein we report the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis. These investigations reveal a complex interplay between a novel iterative or "stuttering" KS-AT didomain (MmpF), the multidomain module MmpB and multiple ACPs. This work has important implications for understanding the late-stage biosynthetic steps of mupirocin and will be important for future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol).

High Expression of ACOT2 Predicts Worse Overall Survival and Abnormal Lipid Metabolism: A Potential Target for Acute Myeloid Leukemia.

Acyl-CoA thioesterase (ACOT) plays a considerable role in lipid metabolism, which is closely related to the occurrence and development of cancer, nevertheless, its role has not been fully elucidated in acute myeloid leukemia (AML). To explore the role of ACOT2 in AML and to provide a potential therapeutic target for AML, the expression pattern of ACOT was investigated based on the TNMplot, Gene Expression Profiling Interactive Analysis (GEPIA), and Cancer Cell Line Encyclopedia (CCLE) database, and diagnostic value, prognostic value, and clinical phenotype of ACOT were explored based on data from The Cancer Genome Atlas (TCGA). Functional annotation and enrichment analysis of the common targets between ACOT2 coexpressed and AML-related genes were further performed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) analyses. The protein-protein interaction (PPI) network of ACOT2 coexpressed genes and functional ACOT2-related metabolites association network were constructed based on GeneMANIA and Human Metabolome Database. Among ACOTs, ACOT2 was highly expressed in AML compared to normal control subjects according to TNMplot, GEPIA, and CCLE database, which was significantly associated with poor overall survival (OS) in AML (P=0.003). Moreover, ACOT2 exhibited excellent diagnostic efficiency for AML (AUC: 1.000) and related to French-American-British (FAB) classification and cytogenetics. GO, KEGG, and GSEA analyses of 71 common targets between ACOT2 coexpressed and AML-related genes revealed that ACOT2 is closely related to ACOT1, ACOT4, enoyl-acyl carrier protein reductase, mitochondrial (MECR), puromycin-sensitive aminopeptidase (NPEPPS), SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), and long-chain fatty acid-CoA ligase 1 (ACSL1) in PPI network, and plays a significant role in lipid metabolism, that is, involved in fatty acid elongation and biosynthesis of unsaturated fatty acids. Collectively, the increase of ACOT2 may be an important characteristic of worse OS and abnormal lipid metabolism, suggesting that ACOT2 may become a potential therapeutic target for AML.

Uncovering Lasonolide A Biosynthesis Using Genome-Resolved Metagenomics.

Invertebrates, particularly sponges, have been a dominant source of new marine natural products. For example, lasonolide A (LSA) is a potential anticancer molecule isolated from the marine sponge Forcepia sp., with nanomolar growth inhibitory activity and a unique cytotoxicity profile against the National Cancer Institute 60-cell-line screen. Here, we identified the putative biosynthetic pathway for LSA. Genomic binning of the Forcepia sponge metagenome revealed a Gram-negative bacterium belonging to the phylum Verrucomicrobia as the candidate producer of LSA. Phylogenetic analysis showed that this bacterium, here named "Candidatus Thermopylae lasonolidus," only has 88.78% 16S rRNA identity with the closest relative, Pedosphaera parvula Ellin514, indicating that it represents a new genus. The lasonolide A (las) biosynthetic gene cluster (BGC) was identified as a trans-acyltransferase (AT) polyketide synthase (PKS) pathway. Compared with its host genome, the las BGC exhibits a significantly different GC content and pentanucleotide frequency, suggesting a potential horizontal acquisition of the gene cluster. Furthermore, three copies of the putative las pathway were identified in the candidate producer genome. Differences between the three las repeats were observed, including the presence of three insertions, two single-nucleotide polymorphisms, and the absence of a stand-alone acyl carrier protein in one of the repeats. Even though the verrucomicrobial producer shows signs of genome reduction, its genome size is still fairly large (about 5 Mbp), and, compared to its closest free-living relative, it contains most of the primary metabolic pathways, suggesting that it is in the early stages of reduction. IMPORTANCE While sponges are valuable sources of bioactive natural products, a majority of these compounds are produced in small quantities by uncultured symbionts, hampering the study and clinical development of these unique compounds. Lasonolide A (LSA), isolated from marine sponge Forcepia sp., is a cytotoxic molecule active at nanomolar concentrations, which causes premature chromosome condensation, blebbing, cell contraction, and loss of cell adhesion, indicating a novel mechanism of action and making it a potential anticancer drug lead. However, its limited supply hampers progression to clinical trials. We investigated the microbiome of Forcepia sp. using culture-independent DNA sequencing, identified genes likely responsible for LSA synthesis in an uncultured bacterium, and assembled the symbiont's genome. These insights provide future opportunities for heterologous expression and cultivation efforts that may minimize LSA's supply problem.

Tracing of Acyl Carrier Protein-channeled Mitomycin Intermediates in Streptomyces caespitosus Facilitates Characterization of the Biosynthetic Steps for AHBA-GlcN Formation and Processing.

Mitomycins are a family of naturally occurring, potent alkylating agents in which the C member has been clinically used for cancer chemotherapy for over 5 decades. In Streptomyces caespitosus, mitomycins are derived from an N-glycoside composed of a 3-amino-5-hydroxybenzoic acid (AHBA) unit and a d-glucosamine (GlcN) unit; however, how this N-glycoside is formed and rearranged to a mitosane, for example, the compact polycyclic ring system of mitomycin C, remains elusive. Benefiting from the development of a method used to trace the mitomycin intermediates that accumulate on an acyl carrier protein (ACP), we here dissect the enzymatic steps for AHBA-GlcN formation and processing to underlie the mitosane structure. Following the N-glycosylation of AHBA with activated N-acetyl-GlcN, deacetylation occurs on ACP to provide AHBA-GlcN. Then, the sugar portion of this N-glycoside is transformed into a linear aminodiol that terminates with an epoxyethane, yielding an ACP-channeled intermediate that is ready for mitosane formation through crosslinking between the AHBA and linearized sugar units. This transformation is unusual and relies on the functional association of a dihydronicotinamide adenine dinucleotide (phosphate)-dependent protein with a radical S-adenosyl-l-methionine protein. Characterization of these ACP-based enzymatic steps for AHBA-GlcN formation and processing sheds light on the poorly understood biosynthetic pathway of mitomycins.

The Two Acyl Carrier Proteins of Enterococcus faecalis Have Nonredundant Functions.

Enterococcus faecalis encodes two proteins, AcpA and AcpB, having the characteristics of acyl carrier proteins (ACPs). We report that the acpA gene located in the fatty acid synthesis operon is essential for fatty acid synthesis and the ΔacpA strain requires unsaturated fatty acids for growth. The ΔacpA strain could be complemented by a plasmid carrying a wild-type acpA gene, but not by a plasmid carrying a wild-type acpB gene. Substitution of four AcpA residues for those of AcpB resulted in a protein that modestly complemented the ΔacpA strain and restored fatty acid synthesis, although the acyl chains synthesized were unusually short. IMPORTANCE Enterococcus faecalis, as well as related species, has two genes-acpA and acpB-encoding putative acyl carrier proteins (ACPs). It has been assumed that AcpA is essential for fatty acid synthesis whereas AcpB is involved utilization of environmental fatty acids. We report here the first experimental test of the essentiality of acpA and show that it is indeed an essential gene that cannot be replaced by acpB.

Whole genome analysis and elucidation of docosahexaenoic acid (DHA) biosynthetic pathway in Aurantiochytrium sp. SW1.

Aurantiochytrium sp., a fungoid marine protist that belongs to Stramenophila has proven its potential in the production of polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acids (DHA). In this study, genomic characterisation of a potential producer for commercial production of DHA, Aurantiochytrium sp. SW1 has been carried out via whole genome sequencing analysis. The genome size of this strain is 60.89 Mb, with a total of 11,588 protein-coding genes. Among these, 9,127 genes could be functionally annotated into a total of 7,248 (62.5 %) from UniProt, 6,554 (56.6 %) from KEGG and 8,643 (74.6 %) genes from eggNOG protein database. The highest proportion of genes belongs to the protein family of metabolism were further assigned into 11 metabolic categories. The highest number of genes belonging to lipid metabolism (321 genes) followed by carbohydrate metabolism (290 genes), metabolism of cofactors and vitamins (197 genes) and amino acid metabolism (188 genes). Further analysis into the biosynthetic pathway for DHA showed evidence of all genes involved in PKS (polyketide synthase)-like PUFA synthase pathway and incomplete fatty acid synthase-elongase/desaturase pathway. Analysis of PUFA synthase showed the presence of up to ten tandem acyl carrier protein (ACP) domains which might have contributed to high DHA production in this organism. In addition, a hybrid system incorporating elements of FAS, Type I PKS and Type II PKS systems were found to be involved in the biosynthetic pathways of fatty acids in Aurantiochytrium sp. SW1. This study delivers an important reference for future research to enhance the lipid, especially DHA production in Aurantiochytrium sp, SW1 and establishment of this strain as an oleaginous thraustochytrid model.

Desaturases: Structural and mechanistic insights into the biosynthesis of unsaturated fatty acids.

This review highlights the key role of fatty acid desaturases in the synthesis of naturally occurring, more common and not unsaturated fatty acids. The three major classes of fatty acid desaturases, such as acyl-lipid, acyl-acyl carrier protein and acyl-coenzyme A, are described in detail, with particular attention to the cellular localisation, the structure, the substrate and product specificity and the expression and regulation of desaturase genes. The review also gives an insight into the biocatalytic reaction of fatty acid desaturation by covering the general and more class-specific mechanistic studies around the synthesis of unsaturated fatty acids Finally, we conclude the review by looking at the numerous novel applications for desaturases in order to meet the very high demand for polyunsaturated fatty acids, taking into account the opportunity for the development of new, more efficient, easily reproducible, sustainable bioengineering advances in the field.

Understanding and Circumventing the Requirement for Native Thioester Substrates for α-Oxoamine Synthase Reactions.

Many enzyme classes require thioester electrophiles such as acyl-carrier proteins and acyl-coenzyme A substrates. For in vitro applications, these substrates can render these chemical transformations impractical. To address this challenge, we have investigated the mechanism of coenzyme A in gating catalysis of one α-oxoamine synthase, SxtA AOS. Through investigating the reactivity of SxtA AOS and corresponding enzyme variants against a panel of substrates and coenzyme A mimics, we determined that activity is gated through the binding of the pantetheine arm and a phosphate group that hydrogen bonds to residue Lys154 that is predicted by an AlphaFold2 model to be located in a tunnel leading to the active site. To provide an economical solution for preparative-scale reactions, in situ transthioesterification was used with pantetheine and simple thioester substrate precursors, resulting in productive reactions. These findings outline a strategy for employing ACP- and CoA-dependent enzymes that are inaccessible through other means without the need for cost-prohibitive coenzyme A or carrier protein-activated substrates.