Rv0100: An essential acyl carrier protein from M. tuberculosis important in dormancy.

We have identified an acyl-carrier protein, Rv0100, that is up-regulated in a dormancy model. This protein plays a critical role in the fatty acid biosynthesis pathway, which is important for energy storage and cell wall synthesis in Mycobacterium tuberculosis (MTB). Knocking out the Rv0100 gene resulted in a significant reduction of growth compared to wild-type MTB in the Wayne model of non-replicating persistence. We have also shown that Rv0100 is essential for the growth and survival of this pathogen during infection in mice and a macrophage model. Furthermore, knocking out Rv0100 disrupted the synthesis of phthiocerol dimycocerosates, the virulence-enhancing lipids produced by MTB and Mycobacterium bovis. We hypothesize that this essential gene contributes to MTB virulence in the state of latent infection. Therefore, inhibitors targeting this gene could prove to be potent antibacterial agents against this pathogen.

Installation of LYRM proteins in early eukaryotes to regulate the metabolic capacity of the emerging mitochondrion.

Core mitochondrial processes such as the electron transport chain, protein translation and the formation of Fe-S clusters (ISC) are of prokaryotic origin and were present in the bacterial ancestor of mitochondria. In animal and fungal models, a family of small Leu-Tyr-Arg motif-containing proteins (LYRMs) uniformly regulates the function of mitochondrial complexes involved in these processes. The action of LYRMs is contingent upon their binding to the acylated form of acyl carrier protein (ACP). This study demonstrates that LYRMs are structurally and evolutionarily related proteins characterized by a core triplet of α-helices. Their widespread distribution across eukaryotes suggests that 12 specialized LYRMs were likely present in the last eukaryotic common ancestor to regulate the assembly and folding of the subunits that are conserved in bacteria but that lack LYRM homologues. The secondary reduction of mitochondria to anoxic environments has rendered the function of LYRMs and their interaction with acylated ACP dispensable. Consequently, these findings strongly suggest that early eukaryotes installed LYRMs in aerobic mitochondria as orchestrated switches, essential for regulating core metabolism and ATP production.

Acyl carrier protein OsMTACP2 confers rice cold tolerance at the booting stage.

Low temperatures occurring at the booting stage in rice (Oryza sativa L.) often result in yield loss by impeding male reproductive development. However, the underlying mechanisms by which rice responds to cold at this stage remain largely unknown. Here, we identified MITOCHONDRIAL ACYL CARRIER PROTEIN 2 (OsMTACP2), the encoded protein of which mediates lipid metabolism involved in the cold response at the booting stage. Loss of OsMTACP2 function compromised cold tolerance, hindering anther cuticle and pollen wall development, resulting in abnormal anther morphology, lower pollen fertility, and seed setting. OsMTACP2 was highly expressed in tapetal cells and microspores during anther development, with the encoded protein localizing to both mitochondria and the cytoplasm. Comparative transcriptomic analysis revealed differential expression of genes related to lipid metabolism between the wild type and the Osmtacp2-1 mutant in response to cold. Through a lipidomic analysis, we demonstrated that wax esters, which are the primary lipid components of the anther cuticle and pollen walls, function as cold-responsive lipids. Their levels increased dramatically in the wild type but not in Osmtacp2-1 when exposed to cold. Additionally, mutants of two cold-induced genes of wax ester biosynthesis, ECERIFERUM1 and WAX CRYSTAL-SPARSE LEAF2, showed decreased cold tolerance. These results suggest that OsMTACP2-mediated wax ester biosynthesis is essential for cold tolerance in rice at the booting stage.

The acyl carrier proteins of lipid synthesis are busy having other affairs.

This is a review of the acyl carrier proteins (ACPs) of type II fatty acid synthesis in bacteria and mitochondria, their structures and protein interactions. Type II fatty acid synthesis in bacteria (Prog. Lipid Res. (2013) 52, 249-276; Biochim. Biophys. Acta (1996) 1302, 1-16; Annu. Rev. Biochem. (2005) 74, 791-831) and in the mitochondria of yeast and mammals (Biochim. Biophys. Acta Mol. Cell. Res. (2019) 1866, 118540; MedChemComm (2019) 10, 209-220; Elife (2016) 5, e17828; Mol. Cell (2018) 71, 567-580.e4) will be discussed only tangentially in this review. The above references are excellent recent reviews. Bacterial fatty acid synthesis has been a popular target for the development of new antimicrobials and an up-to-date review of the field has been published (Annu. Rev. Microbiol. (2022) 76, 281-304). The ACP-like proteins of secondary metabolites (e.g. polyketide synthesis will not be reviewed). Escherichia coli ACP is now called AcpP to distinguish it from the enzymes that attach (AcpS) and remove (AcpH) the 4'-phosphopantetheine (4'PP) prosthetic group. Note that the primary translation product of the acpP gene is called apo-AcpP. The addition of the 4'PP prosthetic group converts apo-AcpP to holo-AcpP (commonly referred to as AcpP). Acylation of the 4'PP prosthetic group gives acyl-AcpP species. The length of the acyl chain determines the properties of the acyl-AcpP as will be discussed below.

Growth of Enterococcus faecalis ∆plsX strains is restored by increased saturated fatty acid synthesis.

The Enterococcus faecalis acyl-acyl carrier protein (ACP) phosphate acyltransferase PlsX plays an important role in phospholipid synthesis and exogenous fatty acid incorporation. Loss of plsX almost completely blocks growth by decreasing de novo phospholipid synthesis, which leads to abnormally long-chain acyl chains in the cell membrane phospholipids. The ∆plsX strain failed to grow without supplementation with an appropriate exogenous fatty acid. Introduction of a ∆fabT mutation into the ∆plsX strain to increase fatty acid synthesis allowed very weak growth. The ∆plsX strain accumulated suppressor mutants. One of these encoded a truncated ß-ketoacyl-ACP synthase II (FabO) which restored normal growth and restored de novo phospholipid acyl chain synthesis by increasing saturated acyl-ACP synthesis. Saturated acyl-ACPs are cleaved by a thioesterase to provide free fatty acids for conversion to acyl-phosphates by the FakAB system. The acyl-phosphates are incorporated into position sn1 of the phospholipids by PlsY. We report the tesE gene encodes a thioesterase that can provide free fatty acids. However, we were unable to delete the chromosomal tesE gene to confirm that it is the responsible enzyme. TesE readily cleaves unsaturated acyl-ACPs, whereas saturated acyl-ACPs are cleaved much more slowly. Overexpression of an E. faecalis enoyl-ACP reductase either FabK or FabI which results in high levels of saturated fatty acid synthesis also restored the growth of the ∆plsX strain. The ∆plsX strain grew faster in the presence of palmitic acid than in the presence of oleic acid with improvement in phospholipid acyl chain synthesis. Positional analysis of the acyl chain distribution in the phospholipids showed that saturated acyl chains dominate the sn1-position indicating a preference for saturated fatty acids at this position. High-level production of saturated acyl-ACPs is required to offset the marked preference of the TesE thioesterase for unsaturated acyl-ACPs and allow the initiation of phospholipid synthesis.

Engineering acyl-ACP reductase with fusion tags enhances alka(e)ne synthesis in Escherichia coli.

Alka(e)nes are high-value chemicals with a potentially broad range of industrial applications because of their following advantages: (1) chemical and structural resemblance to petroleum hydrocarbons and (2) higher energy density and hydrophobicity than those of other biofuels. The low yield of bio-alka(e)nes, however, hinders their commercial application. The activity and solubility of acyl carrier protein (ACP) reductase (AAR) affect alka(e)ne biosynthesis in cyanobacteria. The enhancement of the activity and concentration of soluble AAR through genetic and process engineering can improve bio-alka(e)ne yield. Although fusion tags are used to enhance the expression or solubility of recombinant proteins, their effectiveness in improving the production of bio-alka(e)nes has not yet been reported. Fusion tags can be used to improve the amount or activity of soluble AAR in Escherichia coli and to increase the yield of alka(e)nes in E. coli cells co-expressing aldehyde deformylating oxygenase (ADO). Hence, in the present study, histidine (His6/His12), thioredoxin (Trx), maltose-binding protein (MBP), and N-utilization substance (NusA) were used as AAR fusion tags. The strain expressing SeAAR with His12 tag and NpADO showed a 7.2-fold higher yield of alka(e)nes than the strain expressing AAR without fusion tag and NpADO. The highest titer of alka(e)nes (194.78 mg/L) was achieved with the His12 tag.

Evaluation of strategies to narrow the product chain-length distribution of microbially synthesized free fatty acids.

The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by heterologous hosts is expression of a selective acyl-acyl carrier protein (ACP) thioesterase. However, few of these enzymes can generate a precise (greater than 90% of a desired chain-length) product distribution when expressed in a microbial or plant host. The presence of alternative chain-lengths can complicate purification in situations where blends of fatty acids are not desired. We report the assessment of several strategies for improving the dodecanoyl-ACP thioesterase from the California bay laurel to exhibit more selective production of medium-chain free fatty acids to near exclusivity. We demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was an effective library screening technique for identification of thioesterase variants with favorable shifts in chain-length specificity. This strategy proved to be a more effective screening technique than several rational approaches discussed herein. With this data, we isolated four thioesterase variants which exhibited a more selective FFA distribution over wildtype when expressed in the fatty acid accumulating E. coli strain, RL08. We then combined mutations from the MALDI isolates to generate BTE-MMD19, a thioesterase variant capable of producing free fatty acids consisting of 90% of C12 products. Of the four mutations which conferred a specificity shift, we noted that three affected the shape of the binding pocket, while one occurred on the positively charged acyl carrier protein landing pad. Finally, we fused the maltose binding protein (MBP) from E. coli to the N - terminus of BTE-MMD19 to improve enzyme solubility and achieve a titer of 1.9 g per L of twelve-carbon fatty acids in a shake flask.

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.

Amino acid (acyl carrier protein) ligase-associated biosynthetic gene clusters reveal unexplored biosynthetic potential.

This study aimed to evaluate the postulated cellular function of a novel family of amino acid (acyl carrier protein) ligases (AALs) in natural product biosynthesis. Here, we analyzed the manually curated, putative, aal-associated natural product biosynthetic gene clusters (NP BGCs) using two computational platforms for NP prediction, antiSMASH-BiG-SCAPE-CORASON and DeepBGC. The detected BGCs included a diversity of type I polyketide/nonribosomal peptide (PKS/NRPS) hybrid BGCs, exemplified by the guadinomine BGC, which suggested a dedicated function of AALs in the biosynthesis of rare (2S)-aminomalonyl-ACP extension units. Besides modular PKS/NRPSs and NRPSs, AAL-associated BGCs were predicted to assemble arylpolyenes, ladderane lipids, phosphonates, aminoglycosides, ß-lactones, and thioamides of both nonribosomal and ribosomal origins. Additionally, we revealed a frequent association of AALs with putative, seldom observed transglutaminase-like and BtrH-like transferases of the cysteine protease superfamily, which may form larger families of ACP-dependent amide bond catalysts used in NP synthesis. Our results disclosed an exceptional chemical novelty and biosynthetic potential of the AAL-associated BGCs in NP biosynthesis. The presented in silico evidence supports the initial hypothesis and provides an important foundation for future experimental studies aimed at discovering novel pharmaceutically relevant active compounds.

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.

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.

Genome-wide identification and analysis of ACP gene family in Sorghum bicolor (L.) Moench.

BACKGROUND: Acyl carrier proteins (ACP) constitute a very conserved carrier protein family. Previous studies have found that ACP not only takes part in the fatty acid synthesis process of almost all organisms, but also participates in the regulation of plant growth, development, and metabolism, and makes plants adaptable to stresses. However, this gene family has not been systematically studied in sorghum. RESULTS: Nine ACP family members were identified in the sorghum genome, which were located on chromosomes 1, 2, 5, 7, 8 and 9, respectively. Evolutionary analysis among different species divided the ACP family into four subfamilies, showing that the SbACPs were more closely related to maize. The prediction results of subcellular localization showed that SbACPs were mainly distributed in chloroplasts and mitochondria, while fluorescence localization showed that SbACPs were mainly localized in chloroplasts in tobacco leaf. The analysis of gene structure revealed a relatively simple genetic structure, that there were 1-3 introns in the sorghum ACP family, and the gene structure within the same subfamily had high similarity. The amplification method of SbACPs was mainly large fragment replication, and SbACPs were more closely related to ACPs in maize and rice. In addition, three-dimensional structure analysis showed that all ACP genes in sorghum contained four α helices, and the second helix structure was more conserved, implying a key role in function. Cis-acting element analysis indicated that the SbACPs might be involved in light response, plant growth and development regulation, biotic and abiotic stress response, plant hormone regulation, and other physiological processes. What's more, qRT-PCR analysis uncovered that some of SbACPs might be involved in the adaptive regulation of drought and salt stresses, indicating the close relationship between fatty acids and the resistance to abiotic stresses in sorghum. CONCLUSIONS: In summary, these results showed a comprehensive overview of the SbACPs and provided a theoretical basis for further studies on the biological functions of SbACPs in sorghum growth, development and abiotic stress responses.

Heterologous Expression, Purification, and Characterization of Type II Polyketide Synthase Acyl Carrier Proteins.

The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective and sustainable access to a range of pharmaceutically relevant molecules. Progress toward this goal hinges on gaining ample access to materials for in vitro characterizations and structural analysis of the components of these synthases. A central component of PKSs is the acyl carrier protein (ACP), which serves as a hub during the biosynthesis of type II polyketides. Herein, we share methods for accessing type II PKS ACPs via heterologous expression in E. coli . We also share how the installation of reactive and site-specific spectroscopic probes can be leveraged to study the conformational dynamics and interactions of type II PKS ACPs.

A phosphopantetheinyl transferase gene restricted to Porphyromonas.

The phosphopantetheinyl transferases (PPTases) catalyze the post-translational modification of carrier proteins (CPs) from fatty acid synthases (FASs) in primary metabolism and from polyketide synthases (PKSs) and non-ribosomal polypeptide synthases (NRPSs) in secondary metabolism. Based on the conserved sequence motifs and substrate specificities, two types (AcpS-type and Sfp-type) of PPTases have been identified in prokaryotes. We present here that Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, harbors merely one PPTase, namely PptP. Complementation and gene deletion experiments clearly show that PptP can replace the function of Escherichia coli AcpS and is essential for the growth of P. gingivalis. Purified PptP transfers the 4-phosphopantetheine moiety of CoA to inactive apo-acyl carrier protein (ACP) to form holo-ACP, which functions as an active carrier of the acyl intermediates of fatty acid synthesis. Moreover, PptP exhibits broad substrate specificity, modifying all ACP substrates tested and catalyzing the transfer of coenzyme A (CoA) derivatives. The lack of sequence alignment with known PPTases together with phylogenetic analyses revealed PptP as a new class of PPTases. Identification of the new PPTase gene pptP exclusive in Porphyromonas species reveals a potential target for treating P. gingivalis infections.

HetI-Like Phosphopantetheinyl Transferase Posttranslationally Modifies Acyl Carrier Proteins in Xanthomonas spp.

In Xanthomonas spp., the biosynthesis of the yellow pigment xanthomonadin and fatty acids originates in the type II polyketide synthase (PKS II) and fatty acid synthase (FAS) pathways, respectively. The acyl carrier protein (ACP) is the central component of PKS II and FAS and requires posttranslational phosphopantetheinylation to initiate these pathways. In this study, for the first time, we demonstrate that the posttranslational modification of ACPs in X. campestris pv. campestris is performed by an essential 4'-phosphopantetheinyl transferase (PPTase), XcHetI (encoded by Xc_4132). X. campestris pv. campestris strain XchetI could not be deleted from the X. campestris pv. campestris genome unless another PPTase-encoding gene such as Escherichia coli acpS or Pseudomonas aeruginosa pcpS was present. Compared with wild-type strain X. campestris pv. campestris 8004 and mutant XchetI::PapcpS, strain XchetI::EcacpS failed to generate xanthomonadin pigments and displayed reduced pathogenicity for the host plant, Brassica oleracea. Further experiments showed that the expression of XchetI restored the growth of E. coli acpS mutant HT253 and, when a plasmid bearing XchetI was introduced into P. aeruginosa, pcpS, which encodes the sole PPTase in P. aeruginosa, could be deleted. In in vitro enzymatic assays, XcHetI catalyzed the transformation of 4'-phosphopantetheine from coenzyme A to two X. campestris pv. campestris apo-acyl carrier proteins, XcAcpP and XcAcpC. All of these findings indicate that XcHetI is a surfactin PPTase-like PPTase with a broad substrate preference. Moreover, the HetI-like PPTase is ubiquitously conserved in Xanthomonas spp., making it a potential new drug target for the prevention of plant diseases caused by Xanthomonas.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Involvement of Arabidopsis Acyl Carrier Protein 1 in PAMP-Triggered Immunity.

Plant fatty acids (FAs) and lipids are essential in storing energy and act as structural components for cell membranes and signaling molecules for plant growth and stress responses. Acyl carrier proteins (ACPs) are small acidic proteins that covalently bind the fatty acyl intermediates during the elongation of FAs. The Arabidopsis thaliana ACP family has eight members. Through reverse genetic, molecular, and biochemical approaches, we have discovered that ACP1 localizes to the chloroplast and limits the magnitude of pattern-triggered immunity (PTI) against the bacterial pathogen Pseudomonas syringae pv. tomato. Mutant acp1 plants have reduced levels of linolenic acid (18:3), which is the primary precursor for biosynthesis of the phytohormone jasmonic acid (JA), and a corresponding decrease in the abundance of JA. Consistent with the known antagonistic relationship between JA and salicylic acid (SA), acp1 mutant plants also accumulate a higher level of SA and display corresponding shifts in JA- and SA-regulated transcriptional outputs. Moreover, methyl JA and linolenic acid treatments cause an apparently enhanced decrease of resistance against P. syringae pv. tomato in acp1 mutants than that in WT plants. The ability of ACP1 to prevent this hormone imbalance likely underlies its negative impact on PTI in plant defense. Thus, ACP1 links FA metabolism to stress hormone homeostasis to be negatively involved in PTI in Arabidopsis plant defense. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The C-terminal end of mycobacterial HadBC regulates AcpM interaction during the FAS-II pathway: a structural perspective.

Comprehending the molecular strategies employed by Mycobacterium tuberculosis (Mtb) in FAS-II regulation is of paramount significance for curbing tuberculosis progression. Mtb employs two sets of dehydratases, namely HadAB and HadBC (ß-hydroxyacyl acyl carrier protein dehydratase), for the regulation of the fatty acid synthase (FAS-II) pathway. We utilized a sequence similarity network to discern the basis for the presence of two copies of the dehydratase gene in Mtb. This analysis groups HadC and HadA in different clusters, which could be attributed to the variability in their physiological role with respect to the acyl chain uptake. Our study reveals structural details pertaining to the crystal structure of the last remaining enzyme of the FAS-II pathway. It also provides insights into the highly flexible hot-dog helix and substrate regulatory loop. Additionally, mutational studies assisted in establishing the role of the C-terminal end in HadC of HadBC in the regulation of acyl carrier protein from Mtb-mediated interactions. Complemented with surface plasmon resonance and molecular dynamics simulation studies, the present study provides the first evidence of the molecular mechanisms involved in the differential binding affinity of the acyl carrier protein from Mtb towards both mtbHadAB and mtbHadBC.

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase.

Plant plastidial acyl-acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Malonyl-acyl carrier protein decarboxylase activity promotes fatty acid and cell envelope biosynthesis in Proteobacteria.

Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the ß-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadAN) and a carboxy-terminal hot dog dimerization domain (MadAC) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadAC, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadAN did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.