RESUMO
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.
Assuntos
Proteína de Transporte de Acila , Proteínas de Bactérias , Mycobacterium tuberculosis , Animais , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Camundongos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteína de Transporte de Acila/metabolismo , Proteína de Transporte de Acila/genética , Macrófagos/microbiologia , Macrófagos/metabolismo , Virulência , Regulação Bacteriana da Expressão Gênica , Tuberculose/microbiologia , Lipídeos/químicaRESUMO
The acyl carrier protein of Escherichia coli, termed AcpP, is a prototypical example of type II fatty acid synthase systems found in many bacteria. It serves as a central hub by accepting diverse acyl moieties (4-18 carbons) and shuttling them between its multiple enzymatic partners to generate fatty acids. Prior structures of acyl-AcpPs established that thioester-linked acyl cargos are sequestered within AcpP's hydrophobic lumen. In contrast, structures of enzyme-bound acyl-AcpPs showed translocation of AcpP-tethered acyl chains into the active sites of enzymes. The mechanistic underpinnings of this conformational interplay, termed chain-flipping, are unclear. Here, using heteronuclear NMR spectroscopy, we reveal that AcpP-tethered acyl chains (6-10 carbons) spontaneously adopt lowly populated solvent-exposed conformations. To this end, we devised a new strategy to replace AcpP's thioester linkages with 15N-labeled amide bonds, which facilitated direct "visualization" of these excited states using NMR chemical exchange saturation transfer and relaxation dispersion measurements. Global fitting of the corresponding data yielded kinetic rate constants of the underlying equilibrium and populations and lifetimes of solvent-exposed states. The latter were influenced by acyl chain composition and ranged from milliseconds to submilliseconds for chains containing six, eight, and ten carbons, owing to their variable interactions with AcpP's hydrophobic core. Although transient, the exposure of AcpP-tethered acyl chains to the solvent may allow relevant enzymes to gain access to its active thioester, and the enzyme-induced selection of this conformation will culminate in the production of fatty acids.
Assuntos
Proteína de Transporte de Acila , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/química , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Ácido Graxo Sintase Tipo IIRESUMO
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.
Assuntos
Mitocôndrias , Proteínas Mitocondriais , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Animais , Evolução Molecular , Eucariotos/metabolismo , Proteína de Transporte de Acila/metabolismo , Proteína de Transporte de Acila/genética , Filogenia , Modelos Moleculares , Humanos , Sequência de AminoácidosRESUMO
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.
Assuntos
Proteína de Transporte de Acila , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Oryza , Proteínas de Plantas , Pólen , Oryza/genética , Oryza/fisiologia , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , Pólen/metabolismo , Pólen/crescimento & desenvolvimento , Pólen/fisiologia , Proteína de Transporte de Acila/metabolismo , Proteína de Transporte de Acila/genética , Flores/genética , Flores/fisiologia , Flores/crescimento & desenvolvimento , Metabolismo dos Lipídeos/genética , Mutação/genética , Ceras/metabolismoRESUMO
The condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) by ß-ketoacyl-ACP synthase III (KAS III, FabH) and decarboxylation of malonyl-ACP by malonyl-ACP decarboxylase are the two pathways that initiate bacterial fatty acid synthesis (FAS) in Escherichia coli. In addition to these two routes, we report that Pseudomonas putida F1 ß-ketoacyl-ACP synthase I (FabB), in addition to playing a key role in fatty acid elongation, also initiates FAS in vivo. We report that although two P. putida F1 fabH genes (PpfabH1 and PpfabH2) both encode functional KAS III enzymes, neither is essential for growth. PpFabH1 is a canonical KAS III similar to E. coli FabH whereas PpFabH2 catalyzes condensation of malonyl-ACP with short- and medium-chain length acyl-CoAs. Since these two KAS III enzymes are not essential for FAS in P. putida F1, we sought the P. putida initiation enzyme and unexpectedly found that it was FabB, the elongation enzyme of the oxygen-independent unsaturated fatty acid pathway. P. putida FabB decarboxylates malonyl-ACP and condenses the acetyl-ACP product with malonyl-ACP for initiation of FAS. These data show that P. putida FabB, unlike the paradigm E. coli FabB, can catalyze the initiation reaction in FAS.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase , Pseudomonas putida , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Proteína de Transporte de Acila/metabolismo , Escherichia coli/metabolismo , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos , Glicogênio Sintase , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMO
Incomplete structural details of Mycobacterium tuberculosis (Mtb) fatty acid synthase-I (FAS-I) at near-atomic resolution have limited our understanding of the shuttling mechanism of its mobile acyl carrier protein (ACP). Here, we have performed atomistic molecular dynamics simulation of Mtb FAS-I with a homology-modeled structure of ACP stalled at dehydratase (DH) and identified key residues that mediate anchoring of the recognition helix of ACP near DH. The observed distance between catalytic residues of ACP and DH agrees with that reported for fungal FAS-I. Further, the conformation of the peripheral linker is found to be crucial in stabilizing ACP near DH. Correlated interdomain motion is observed between DH, enoyl reductase, and malonyl/palmitoyl transferase, consistent with prior experimental reports of fungal and Mtb FAS-I.
Assuntos
Proteína de Transporte de Acila , Mycobacterium tuberculosis , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Simulação de Dinâmica Molecular , CatáliseRESUMO
Megasynthases, such as typeâ I fatty acid and polyketide synthases (FASs and PKSs), are multienzyme complexes responsible for producing primary metabolites and complex natural products. Fatty acids (FAs) and polyketides (PKs) are built by assembling and modifying small acyl moieties in a stepwise manner. A central aspect of FA and PK biosynthesis involves the shuttling of substrates between the domains of the multienzyme complex. This essential process is mediated by small acyl carrier proteins (ACPs). The ACPs must navigate to the different catalytic domains within the multienzyme complex in a particular order to guarantee the fidelity of the biosynthesis pathway. However, the precise mechanisms underlying ACP-mediated substrate shuttling, particularly the factors contributing to the programming of the ACP movement, still need to be fully understood. This Review illustrates the current understanding of substrate shuttling, including concepts of conformational and specificity control, and proposes a confined ACP movement within typeâ I megasynthases.
Assuntos
Proteína de Transporte de Acila , Policetídeos , Proteína de Transporte de Acila/metabolismo , Ácidos Graxos , Complexos Multienzimáticos/química , Policetídeos/metabolismo , Policetídeo Sintases/metabolismoRESUMO
The emergence of multi-drug resistant Enterococcus faecalis raises a serious threat to global public health. E. faecalis is a gram-positive intestinal commensal bacterium found in humans. E. faecalis can endure extreme environments such as high temperature, pressure, and high salt, which facilitates them to cause infection in hospitals. E. faecalis has two acyl carrier proteins, AcpA (EfAcpA) in de novo fatty acid synthesis (FAS) and AcpB (EfAcpB) which utilizes exogenous fatty acids. Previously, we determined the tertiary structures of these two ACPs and investigated their structure-function relationships. Solution structures revealed that overall folding of these two ACPs is similar to those of other bacterial ACPs. However, circular dichroism (CD) experiments showed that the melting temperature of EfAcpA is 76.3°C and that of EfAcpB is 79.2°C, which are much higher than those of other bacterial ACPs. In this study, to understand the origin of their structural stabilities, we verified the important residues for stable folding of these two ACPs by monitoring thermal and chemical denaturation. Hydrogen/deuterium exchange and chemical denaturation experiments on wild-type and mutant proteins revealed that Ile10 of EfAcpA and Ile14 of EfAcpB mediate compact intramolecular packing and promote high thermostability and stable folding. E. faecalis may maximize efficiency of FAS and increase adaptability to the environmental stress by having two thermostable ACPs. This study may provide insight into bacterial adaptability and development of antibiotics against multi-drug-resistant E. faecalis.
Assuntos
Proteína de Transporte de Acila , Enterococcus faecalis , Humanos , Enterococcus faecalis/genética , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Antibacterianos/metabolismo , Ácidos Graxos/metabolismo , Dobramento de Proteína , Proteínas de Bactérias/metabolismoRESUMO
Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)-bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter-domain interactions after C-C bond formation in a chain-branching module of the rhizoxin PKS. Mechanism-based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C-C bond formation by cryo-electron microscopy (cryo-EM). The possible existence of two ACP binding sites, one of them a potential "parking position" for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C-C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines.
Assuntos
Proteína de Transporte de Acila , Policetídeo Sintases , Policetídeo Sintases/metabolismo , Microscopia Crioeletrônica , Sítios de Ligação , Domínio Catalítico , Proteína de Transporte de Acila/metabolismoRESUMO
Outside of their involvement in energy production, mitochondria play a critical role for the cell through their access to a discrete pathway for fatty acid biosynthesis. Despite decades of study in bacterial fatty acid synthases (the putative evolutionary mitochondrial precursor), our understanding of human mitochondrial fatty acid biosynthesis remains incomplete. In particular, the role of the key carrier protein, human mitochondrial acyl carrier protein (mACP), which shuttles the substrate intermediates through the pathway, has not been well-studied in part due to challenges in protein expression and purification. Herein, we report a reliable method for recombinant Escherichia coli expression and purification of mACP. Fundamental characteristics, including substrate sequestration and chain-flipping activity, are demonstrated in mACP using solvatochromic response. This study provides an efficient approach toward understanding the fundamental protein-protein interactions of mACP and its partner proteins, ultimately leading to a molecular understanding of human mitochondrial diseases such as mitochondrial fatty acid oxidation deficiencies.
Assuntos
Proteína de Transporte de Acila , Ácidos Graxos , Humanos , Proteína de Transporte de Acila/metabolismo , Escherichia coli/metabolismo , Ácido Graxo Sintases/química , Ácidos Graxos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismoRESUMO
Prokaryotes synthesize fatty acids using a type II synthesis pathway (FAS). In this process, the central player, i.e., the acyl carrier protein (ACP), sequesters the growing acyl chain in its internal hydrophobic cavity. As the acyl chain length increases, the cavity expands in size, which is reflected in the NMR chemical shift perturbations and crystal structures of the acyl-ACP intermediates. A few eukaryotic organelles, such as plastids and mitochondria, also harbor type II fatty acid synthesis machinery. Plastid FAS from spinach and Plasmodium falciparum has been characterized at the molecular level, but the mitochondrial pathway remains unexplored. Here, we report NMR studies of the mitochondrial acyl-acyl carrier protein intermediates of Leishmania major (acyl-LmACP). Our studies show that LmACP experiences remarkably small conformational changes upon acylation, with perturbations confined to helices II and III only. CastP determined that the cavity size of apo-LmACP (PDB entry 5ZWT) is less than that of Escherichia coli ACP (PDB 1T8K). Thus, the small chemical shift perturbations observed in the LmACP intermediates, coupled with CastP results, suggest an unusually small cavity when fully expanded. The faster rate of C8-LmACP chain hydrolysis compared to E. coli ACP (EcACP) also supports these convictions. Structure comparison of LmACP with other type II ACP disclosed unique differences in the helix I and loop I conformations, as well as several residues present there. Numerous hydrophobic residues in helix I and loop I (conserved in all mitochondrial ACPs) are substituted with hydrophilic residues in the bacterial/plastid type II ACP. For instance, Phe and leucine at positions 14 and 34 in LmACP are substituted with a hydrophilic residue and Ala in bacterial/plastid type II ACP. Mutation of Leu 34 to Ala (corresponding residue in EcACP) resulted in a complete loss of structure, underscoring its importance in maintaining the ACP fold. Thus, our NMR studies, combined with insights from the crystal structure, highlight several unique features of LmACP, distinct from the prokaryote and plastid type II ACP. Given the high sequence identity, the features might be conserved in all mitochondrial ACPs.
Assuntos
Proteína de Transporte de Acila , Leishmania major , Proteína de Transporte de Acila/metabolismo , Leishmania major/metabolismo , Escherichia coli/metabolismo , Modelos Moleculares , Conformação MolecularRESUMO
ACYL CARRIER PROTEIN4 (ACP4) is the most abundant ACP isoform in Arabidopsis (Arabidopsis thaliana) leaves and acts as a scaffold for de novo fatty acid biosynthesis and as a substrate for acyl-ACP-utilizing enzymes. Recently, ACP4 was found to interact with a protein-designated plastid RHOMBOID LIKE10 (RBL10) that affects chloroplast monogalactosyldiacylglycerol (MGDG) biosynthesis, but the cellular function of this interaction remains to be explored. Here, we generated and characterized acp4 rbl10 double mutants to explore whether ACP4 and RBL10 directly interact in influencing chloroplast lipid metabolism. Alterations in the content and molecular species of chloroplast lipids such as MGDG and phosphatidylglycerol were observed in the acp4 and rbl10 mutants, which are likely associated with the changes in the size and profiles of diacylglycerol (DAG), phosphatidic acid (PA), and acyl-ACP precursor pools. ACP4 contributed to the size and profile of the acyl-ACP pool and interacted with acyl-ACP-utilizing enzymes, as expected for its role in fatty acid biosynthesis and chloroplast lipid assembly. RBL10 appeared to be involved in the conversion of PA to DAG precursors for MGDG biosynthesis as evidenced by the increased 34:x PA and decreased 34:x DAG in the rbl10 mutant and the slow turnover of radiolabeled PA in isolated chloroplasts fed with [14C] acetate. Interestingly, the impaired PA turnover in rbl10 was partially reversed in the acp4 rbl10 double mutant. Collectively, this study shows that ACP4 and RBL10 affect chloroplast lipid biosynthesis by modulating substrate precursor pools and appear to act independently.
Assuntos
Proteína de Transporte de Acila , Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Ácidos Graxos/metabolismo , Ácidos Fosfatídicos/metabolismo , Plastídeos/metabolismo , Proteína de Transporte de Acila/metabolismoRESUMO
The N-acyl-L-homoserine lactone (AHL) quorum sensing regulates virulence in the opportunistic pathogen, Pseudomonas aeruginosa. The LasI and RhlI AHL synthases use acyl carrier protein substrates to synthesize, respectively, the 3-oxododecanoyl-L-homoserine lactone (3-oxoC12-HSL) and butyryl-L-homoserine lactone (C4-HSL) QS signals for this bacterium. Although P. aeruginosa genome contains three open reading frames to encode three acyl carrier proteins, namely the ACP1, ACP2 and ACP3, microarray and gene replacement studies show that only the ACP1 carrier protein is under quorum sensing regulation. In this study, we isotopically enriched one of the acyl carrier proteins, ACP1 from P. aeruginosa and describe the backbone resonance assignments for this protein to delineate the structural and molecular basis of ACP1 recognition in P. aeruginosa AHL quorum sensing signal synthesis.
Assuntos
Proteína de Transporte de Acila , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Proteína de Transporte de Acila/metabolismo , Ressonância Magnética Nuclear Biomolecular , Percepção de Quorum , Acil-Butirolactonas/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
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.
Assuntos
Enterococcus faecalis , Ácidos Graxos , Enterococcus faecalis/genética , Ácidos Graxos não Esterificados/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fosfolipídeos , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Fosfatos/metabolismoRESUMO
Acyl carrier proteins (ACPs) are universally conserved proteins amongst different species and are involved in fatty acid synthesis. Bacteria utilize ACPs as acyl carriers and donors for the synthesis of products such as endotoxins or acyl homoserine lactones (AHLs), which are used in quorum sensing mechanisms. In this study, wehave expressed isotopically labeled holo-ACP from Burkholderia mallei in Escherichia coli to assign 100% of non-proline backbone amide (HN) resonances, 95.5% of aliphatic carbon resonances and 98.6% of aliphatic hydrogen sidechain resonances.
Assuntos
Proteína de Transporte de Acila , Burkholderia mallei , Proteína de Transporte de Acila/metabolismo , Burkholderia mallei/metabolismo , Ressonância Magnética Nuclear Biomolecular , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
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.
Assuntos
Escherichia coli , Oxirredutases , Oxirredutases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Alcanos/metabolismo , Oxigenases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
Microbial fatty acids are synthesized by Type II fatty acid synthase and could be tailored by acyl-ACP thioesterase. With the prospects of medium-chain fatty-acid-derivative biofuels, the selectivity of thioesterase has been studied to control the fatty acid product chain length. Here, we report an alternative approach by manipulating the acyl carrier protein portion of acyl-ACP to switch the chain length propensity of the thioesterase. It was demonstrated that ChFatB2 from Cuphea hookeriana preferred C10-ACP to C8-ACP with ACP from E. coli, while converting preference to C8-ACP with ACP from Cuphea lanceolate. Circular dichroism (CD) results indicated that the C8-EcACP encountered a 34.4% α-helix increment compared to C10-EcACP, which resulted in an approximate binding affinity decrease in ChFatB2 compared to C10-EcACP. Similarly, the C10-ClACP2 suffered a 45% decrease in helix content compared to C8-ClACP2, and the conformational changes resulted in an 18% binding affinity decline with ChFatB2 compared with C10-ClACP2. In brief, the study demonstrates that the ACP portion of acyl-ACP contributes to the selectivity of acyl-ACP thioesterase, and the conformational changes of EcACP and ClACP2 switch the chain length preference of ChFatB2 between C8 and C10. The result provides fundamentals for the directed synthesis of medium-chain fatty acids based on regulating the conformational changes of ACPs.
Assuntos
Proteína de Transporte de Acila , Escherichia coli , Proteína de Transporte de Acila/metabolismo , Escherichia coli/metabolismo , Tioléster Hidrolases/metabolismo , Ácidos Graxos/metabolismoRESUMO
Natural products play critical roles as antibiotics, anticancer therapeutics, and biofuels. Polyketides are a distinct natural product class of structurally diverse secondary metabolites that are synthesized by polyketide synthases (PKSs). The biosynthetic gene clusters that encode PKSs have been found across nearly all realms of life, but those from eukaryotic organisms are relatively understudied. A type I PKS from the eukaryotic apicomplexan parasite Toxoplasma gondii,TgPKS2, was recently discovered through genome mining, and the functional acyltransferase (AT) domains were found to be selective for malonyl-CoA substrates. To further characterize TgPKS2, we resolved assembly gaps within the gene cluster, which confirmed that the encoded protein consists of three distinct modules. We subsequently isolated and biochemically characterized the four acyl carrier protein (ACP) domains within this megaenzyme. We observed self-acylationâor substrate acylation without an AT domainâfor three of the four TgPKS2 ACP domains with CoA substrates. Furthermore, CoA substrate specificity and kinetic parameters were determined for all four unique ACPs. TgACP2-4 were active with a wide scope of CoA substrates, while TgACP1 from the loading module was found to be inactive for self-acylation. Previously, self-acylation has only been observed in type II systems, which are enzymes that act in-trans with one another, and this represents the first report of this activity in a modular type I PKS whose domains function in-cis. Site-directed mutagenesis of specific TgPKS2 ACP3 acidic residues near the phosphopantetheinyl arm demonstrated that they influence self-acylation activity and substrate specificity, possibly by influencing substrate coordination or phosphopantetheinyl arm activation. Further, the lack of TgPKS2 ACP self-acylation with acetoacetyl-CoA, which is utilized by previously characterized type II PKS systems, suggests that the substrate carboxyl group may be critical for TgPKS2 ACP self-acylation. The unexpected properties observed from T. gondii PKS ACP domains highlight their distinction from well-characterized microbial and fungal systems. This work expands our understanding of ACP self-acylation beyond type II systems and helps pave the way for future studies on biosynthetic enzymes from eukaryotes.
Assuntos
Proteína de Transporte de Acila , Policetídeo Sintases , Toxoplasma , Proteína de Transporte de Acila/metabolismo , Acilação , Aciltransferases/química , Malonil Coenzima A/metabolismo , Policetídeo Sintases/metabolismo , Toxoplasma/metabolismoRESUMO
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.