N-acyl-4-arylaminopiperidines: Design and synthesis of a potential antimicrobial scaffold.

We report a concise synthesis of N-acylated piperidines through a Knoevenagel-Doebner condensation/amide construction/ amination sequence. The design of the piperidines considered the pharmacophoric features found in previously reported inhibitors of FabI, an enzyme implicated in bacterial fatty acid biosynthesis. After the microbiological evaluation at 50 µM, the analogs displayed moderate activity against some pathogens from the ESKAPE group, reaching up to 42 % of growth inhibition for MRSA, 54 % for K. pneumoniae, and 37 % for P. aeruginosa (multiresistant strains). Docking studies demonstrate that almost all of them docked satisfactorily into the catalytic domain of S. aureus FabI, maintaining a similar pose as other reported inhibitors. The results shown herein propose the N-acyl-4-arylaminopiperidines as the basis for the development of more active candidates.

An inhibitory mechanism of AasS, an exogenous fatty acid scavenger: Implications for re-sensitization of FAS II antimicrobials.

Antimicrobial resistance is an ongoing "one health" challenge of global concern. The acyl-ACP synthetase (termed AasS) of the zoonotic pathogen Vibrio harveyi recycles exogenous fatty acid (eFA), bypassing the requirement of type II fatty acid synthesis (FAS II), a druggable pathway. A growing body of bacterial AasS-type isoenzymes compromises the clinical efficacy of FAS II-directed antimicrobials, like cerulenin. Very recently, an acyl adenylate mimic, C10-AMS, was proposed as a lead compound against AasS activity. However, the underlying mechanism remains poorly understood. Here we present two high-resolution cryo-EM structures of AasS liganded with C10-AMS inhibitor (2.33 Å) and C10-AMP intermediate (2.19 Å) in addition to its apo form (2.53 Å). Apart from our measurements for C10-AMS' Ki value of around 0.6 µM, structural and functional analyses explained how this inhibitor interacts with AasS enzyme. Unlike an open state of AasS, ready for C10-AMP formation, a closed conformation is trapped by the C10-AMS inhibitor. Tight binding of C10-AMS blocks fatty acyl substrate entry, and therefore inhibits AasS action. Additionally, this intermediate analog C10-AMS appears to be a mixed-type AasS inhibitor. In summary, our results provide the proof of principle that inhibiting salvage of eFA by AasS reverses the FAS II bypass. This facilitates the development of next-generation anti-bacterial therapeutics, esp. the dual therapy consisting of C10-AMS scaffold derivatives combined with certain FAS II inhibitors.

Cryptic Cerulenin Rearrangement in Ketosynthase Covalent Inhibition.

The antibiotic cerulenin is a fungal natural product identified as a covalent inhibitor of ketosynthases within fatty acid and polyketide biosynthesis. Due to its selective and potent inhibitory activity, cerulenin has found significant utility in multidisciplinary biochemical, biomedical, and clinical studies. Although its covalent inhibition profile has been confirmed, cerulenin's mechanism has not been fully determined at a molecular level, frustrating the drug development of related analogues. Herein, we describe the use of stable isotopic tracking with NMR and MS methods to unravel the covalent mechanism of cerulenin against type II fatty acid ketosynthases. We detail the discovery of a unique C2-C3 retro-aldol bond cleavage and a structural rearrangement upon covalent inhibition of cerulenin at the active cysteine residue in E. coli type II fatty acid ketosynthases FabB and FabF.

Divergent unsaturated fatty acid synthesis in two highly related model pseudomonads.

The genomes of the best-studied pseudomonads, Pseudomonas aeruginosa and Pseudomonas putida, which share 85% of the predicted coding regions, contain a fabA fabB operon (demonstrated in P. aeruginosa, putative in P. putida). The enzymes encoded by the fabA and fabB genes catalyze the introduction of a double bond into a 10-carbon precursor which is elongated to the 16:1Δ9 and 18:1Δ11 unsaturated fatty acyl chains required for functional membrane phospholipids. A detailed analysis of transcription of the P. putida fabA fabB gene cluster showed that fabA and fabB constitute an operon and disclosed an unexpected and essential fabB promoter located within the fabA coding sequence. Inactivation of the fabA fabB operon fails to halt the growth of P. aeruginosa PAO1 but blocks growth of P. putida F1 unless an exogenous unsaturated fatty acid is provided. We report that the asymmetry between these two species is due to the P. aeruginosa PAO1 desA gene which encodes a fatty acid desaturase that introduces double bonds into the 16-carbon acyl chains of membrane phospholipids. Although P. putida F1 encodes a putative DesA homolog that is 84% identical to the P. aeruginosa PAO1, the protein fails to provide sufficient unsaturated fatty acid synthesis for growth when the FabA FabB pathway is inactivated. We report that the P. putida F1 DesA homolog can functionally replace the P. aeruginosa DesA. Hence, the defect in P. putida F1 desaturation is not due to a defective P. putida F1 DesA protein but probably to a weakly active component of the electron transfer process.

Control of Unsaturation in De Novo Fatty Acid Biosynthesis by FabA.

Carrier protein-dependent biosynthesis provides a thiotemplated format for the production of natural products. Within these pathways, many reactions display exquisite substrate selectivity, a regulatory framework proposed to be controlled by protein-protein interactions (PPIs). In Escherichia coli, unsaturated fatty acids are generated within the de novo fatty acid synthase by a chain length-specific interaction between the acyl carrier protein AcpP and the isomerizing dehydratase FabA. To evaluate PPI-based control of reactivity, interactions of FabA with AcpP bearing multiple sequestered substrates were analyzed through NMR titration and guided high-resolution docking. Through a combination of quantitative binding constants, residue-specific perturbation analysis, and high-resolution docking, a model for substrate control via PPIs has been developed. The in silico results illuminate the mechanism of FabA substrate selectivity and provide a structural rationale with atomic detail. Helix III positioning in AcpP communicates sequestered chain length identity recognized by FabA, demonstrating a powerful strategy to regulate activity by allosteric control. These studies broadly illuminate carrier protein-dependent pathways and offer an important consideration for future inhibitor design and pathway engineering.

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.

Engineered Chimeras Unveil Swappable Modular Features of Fatty Acid and Polyketide Synthase Acyl Carrier Proteins.

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein, we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the Escherichia coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based cross-linking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step toward the strategic engineering of "un-natural" natural products.

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.

Cadaverine regulates biotin synthesis to modulate primary root growth in Arabidopsis.

Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root-growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3-BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8-diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3-BIO1 activity. Furthermore, cadaverine-treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl-coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants.

Genetic Suppression of Lethal Mutations in Fatty Acid Biosynthesis Mediated by a Secondary Lipid Synthase.

The biosynthesis and incorporation of polyunsaturated fatty acids into phospholipid membranes are unique features of certain marine Gammaproteobacteria inhabiting high-pressure and/or low-temperature environments. In these bacteria, monounsaturated and saturated fatty acids are produced via the classical dissociated type II fatty acid synthase mechanism, while omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) are produced by a hybrid polyketide/fatty acid synthase-encoded by the pfa genes-also referred to as the secondary lipid synthase mechanism. In this work, phenotypes associated with partial or complete loss of monounsaturated biosynthesis are shown to be compensated for by severalfold increased production of polyunsaturated fatty acids in the model marine bacterium Photobacterium profundum SS9. One route to suppression of these phenotypes could be achieved by transposition of insertion sequences within or upstream of the fabD coding sequence, which encodes malonyl coenzyme A (malonyl-CoA) acyl carrier protein transacylase. Genetic experiments in this strain indicated that fabD is not an essential gene, yet mutations in fabD and pfaA are synthetically lethal. Based on these results, we speculated that the malonyl-CoA transacylase domain within PfaA compensates for loss of FabD activity. Heterologous expression of either pfaABCD from P. profundum SS9 or pfaABCDE from Shewanella pealeana in Escherichia coli complemented the loss of the chromosomal copy of fabD in vivo. The co-occurrence of independent, yet compensatory, fatty acid biosynthetic pathways in selected marine bacteria may provide genetic redundancy to optimize fitness under extreme conditions. IMPORTANCE A defining trait among many cultured piezophilic and/or psychrophilic marine Gammaproteobacteria is the incorporation of both monounsaturated and polyunsaturated fatty acids into membrane phospholipids. The biosynthesis of these different classes of fatty acid molecules is linked to two genetically distinct co-occurring pathways that utilize the same pool of intracellular precursors. Using a genetic approach, new insights into the interactions between these two biosynthetic pathways have been gained. Specifically, core fatty acid biosynthesis genes previously thought to be essential were found to be nonessential in strains harboring both pathways due to functional overlap between the two pathways. These results provide new routes to genetically optimize long-chain omega-3 polyunsaturated fatty acid biosynthesis in bacteria and reveal a possible ecological role for maintaining multiple pathways for lipid synthesis in a single bacterium.

Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis.

Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.

Effects of lysine 2-hydroxyisobutyrylation on bacterial FabI activity and resistance to triclosan.

Lysine 2-hydroxyisobutyrylation (Khib) is a novel protein posttranslational modification conserved in eukaryotes and prokaryotes. However, the biological significance of Khib remains largely unknown. Here, through screening the proteome-wide Khib modification sites in bacteria using a bioinformatic method, we identified a potential Khib site (K201hib) targeted by de-2-hyroxyisobutyrylase CobB at the substrate-binding site of FabI, an enoyl-acyl carry protein reductase (EnvM or FabI) in fatty acid biosynthesis pathway. First, we confirmed that the previously identified de-2-hyroxyisobutyrylase CobB can remove Khib of FabI in an in vitro experiment. To investigate the biological effects of the Khib on FabI's activity, amino acid substitutes were introduced to the modification sites of the protein of E. coli origin to mimic modified/unmodified status. We found that the mutant mimicking K201hib reduced FabI activity with decreased Michaelis constant (Km) and catalytic turnover number (kcat), while the mutant mimicking the unmodified form and the recombinant wild-type protein treated with CobB exhibited increased activity. However, the dissociation constant (KD) between FabI and NADH was not affected by the mutation mimicking the modification, suggesting that K201hib didn't alter the binding between NADH and FabI. We also found that K201hib tended to increase the resistance of E. coli to triclosan (TCL), a widely-used antibiotics targeting FabI. Taken together, this study identified the regulatory role of Khib on FabI activity and pointed to a novel mechanism related to antibiotic resistance.

The Escherichia coli FadR transcription factor: Too much of a good thing?

Escherichia coli FadR is a transcription factor regulated by acyl-CoA thioester binding that optimizes fatty acid (FA) metabolism in response to environmental FAs. FadR represses the fad genes of FA degradation (ß-oxidation) and activates the fab genes of FA synthesis thereby allowing E. coli to have its cake (acyl chains for phospholipid synthesis) and eat it (degrade acyl chains to acetyl-CoA). Acyl-CoA binding of FadR derepresses the transcription of the fad genes and cancels fab gene transcriptional activation. Activation of fab genes was thought restricted to the fabA and fabB genes of unsaturated FA synthesis, but FadR overproduction markedly increases yields of all FA acyl chains. Subsequently, almost all of the remaining fab genes were shown to be transcriptionally activated by FadR binding, but binding was very weak. Why are the low-affinity sites retained? What effects on cell physiology would result from their conversion to high-affinity sites (thereby mimicking FadR overproduction)? Investigations of E. coli cell size determinants showed that FA synthesis primarily determines E. coli cell size. Upon modest induction of FadR, cell size increases, but at the cost of growth rate and accumulation of intracellular membranes. Greater induction resulted in further growth rate decreases and abnormal cells. Hence, too much FadR is bad. FadR is extraordinarily conserved in γ-proteobacteria but has migrated. Mycobacterium tuberculosis encodes FadR orthologs one of which is functional in E. coli. Strikingly, the FadR theme of acyl-CoA-dependent transcriptional regulation is found in a different transcription factor family where two Bacillus species plus bacterial and archaeal thermophiles contain related proteins of similar function.

Design of Arylsulfonylhydrazones as Potential FabH Inhibitors: Synthesis, Antimicrobial Evaluation and Molecular Docking.

BACKGROUND: Antimicrobial resistance is a persistent problem regarding infection treatment and calls for developing new antimicrobial agents. Inhibition of bacterial ß-ketoacyl acyl carrier protein synthase III (FabH), which catalyzes the condensation reaction between a CoAattached acetyl group and an ACP-attached malonyl group in bacteria is an interesting strategy to find new antibacterial agents. OBJECTIVE: The aim of this work was to design and synthesize arylsulfonylhydrazones potentially FabH inhibitors and evaluate their antimicrobial activity. METHODS: MIC50 values of sulfonylhydrazones against E. coli and S. aureus were determined. Antioxidant activity was evaluated by DPPH (1-1'-diphenyl-2-picrylhydrazyl) assay and cytotoxicity against LL24 lung fibroblast cells was verified by MTT method. Principal component analysis (PCA) was performed in order to suggest a structure-activity relationship. Molecular docking allowed to propose sulfonylhydrazones interactions with FabH. RESULTS: The most active compound showed activity against S. aureus and E. coli, with MIC50 = 0.21 and 0.44 µM, respectively. PCA studies correlated better activity to lipophilicity and molecular docking indicated that sulfonylhydrazone moiety is important to hydrogen-bond with FabH while methylcatechol ring performs π-π stacking interaction. The DPPH assay revealed that some sulfonylhydrazones derived from the methylcatechol series had antioxidant activity. None of the evaluated compounds was cytotoxic to human lung fibroblast cells, suggesting that the compounds might be considered safe at the tested concentration. CONCLUSION: Arylsufonylhydrazones is a promising scaffold to be explored for the design of new antimicrobial agents.

Structure and Mechanism of the Ketosynthase-Chain Length Factor Didomain from a Prototypical Polyunsaturated Fatty Acid Synthase.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFASs) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes that are homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KSc) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon-carbon bond formation. We report the X-ray crystal structure of the KS-CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, provides a foundation for understanding the mechanism of substrate recognition and chain length control by the KS-CLF didomain as well as its interaction with a cognate acyl carrier protein partner.

The food-grade antimicrobial xanthorrhizol targets the enoyl-ACP reductase (FabI) in Escherichia coli.

Xanthorrhizol, isolated from the Indonesian Java turmeric Curcuma xanthorrhiza, displays broad-spectrum antibacterial activity. We report herein the evidence that mechanism of action of xanthorrhizol may involve FabI, an enoyl-(ACP) reductase, inhibition. The predicted Y156V substitution in the FabI enzyme promoted xanthorrhizol resistance, while the G93V mutation originally known for triclosan resistance was not effective against xanthorrhizol. Two other mutations, F203L and F203V, conferred FabI enzyme resistance to both xanthorrhizol and triclosan. These results showed that xanthorrhizol is a food-grade antimicrobial compound targeting FabI but with a different mode of binding from triclosan.

A kinetic rationale for functional redundancy in fatty acid biosynthesis.

Cells build fatty acids with biocatalytic assembly lines in which a subset of enzymes often exhibit overlapping activities (e.g., two enzymes catalyze one or more identical reactions). Although the discrete enzymes that make up fatty acid pathways are well characterized, the importance of catalytic overlap between them is poorly understood. We developed a detailed kinetic model of the fatty acid synthase (FAS) of Escherichia coli and paired that model with a fully reconstituted in vitro system to examine the capabilities afforded by functional redundancy in fatty acid synthesis. The model captures-and helps explain-the effects of experimental perturbations to FAS systems and provides a powerful tool for guiding experimental investigations of fatty acid assembly. Compositional analyses carried out in silico and in vitro indicate that FASs with multiple partially redundant enzymes enable tighter (i.e., more independent and/or broader range) control of distinct biochemical objectives-the total production, unsaturated fraction, and average length of fatty acids-than FASs with only a single multifunctional version of each enzyme (i.e., one enzyme with the catalytic capabilities of two partially redundant enzymes). Maximal production of unsaturated fatty acids, for example, requires a second dehydratase that is not essential for their synthesis. This work provides a kinetic, control-theoretic rationale for the inclusion of partially redundant enzymes in fatty acid pathways and supplies a valuable framework for carrying out detailed studies of FAS kinetics.

Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD.

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.

Activity Mapping the Acyl Carrier Protein: Elongating Ketosynthase Interaction in Fatty Acid Biosynthesis.

Elongating ketosynthases (KSs) catalyze carbon-carbon bond-forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, the ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways. Recent structures of the Escherichia coli FAS ACP, AcpP, in covalent complexes with its two cognate elongating KSs, FabF and FabB, provide high-resolution details of these interfaces, but a systematic analysis of specific interfacial interactions responsible for stabilizing these complexes has not yet been undertaken. Here, we use site-directed mutagenesis with both in vitro and in vivo activity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interacting regions and demonstrate the effects of point mutants, double mutants, and region deletion variants. Results from these analyses reveal a robust and modular FabF interface capable of tolerating seemingly critical interface mutations with only the deletion of an entire region significantly compromising activity. Structure and sequence analyses of FabF orthologs from related type II FAS pathways indicate significant conservation of type II FAS KS interface residues and, overall, support its delineation into interaction regions. These findings strengthen our mechanistic understanding of molecular recognition events between ACPs and FAS enzymes and provide a blueprint for engineering ACP-dependent biosynthetic pathways.

Fatty Acid Biosynthesis in Chromerids.

Fatty acids are essential components of biological membranes, important for the maintenance of cellular structures, especially in organisms with complex life cycles like protozoan parasites. Apicomplexans are obligate parasites responsible for various deadly diseases of humans and livestock. We analyzed the fatty acids produced by the closest phototrophic relatives of parasitic apicomplexans, the chromerids Chromera velia and Vitrella brassicaformis, and investigated the genes coding for enzymes involved in fatty acids biosynthesis in chromerids, in comparison to their parasitic relatives. Based on evidence from genomic and metabolomic data, we propose a model of fatty acid synthesis in chromerids: the plastid-localized FAS-II pathway is responsible for the de novo synthesis of fatty acids reaching the maximum length of 18 carbon units. Short saturated fatty acids (C14:0-C18:0) originate from the plastid are then elongated and desaturated in the cytosol and the endoplasmic reticulum. We identified giant FAS I-like multi-modular enzymes in both chromerids, which seem to be involved in polyketide synthesis and fatty acid elongation. This full-scale description of the biosynthesis of fatty acids and their derivatives provides important insights into the reductive evolutionary transition of a phototropic algal ancestor to obligate parasites.