Structure of Type-I Mycobacterium tuberculosis fatty acid synthase at 3.3 Å resolution.

Tuberculosis (TB) is a devastating and rapidly spreading disease caused by Mycobacterium tuberculosis (Mtb). Therapy requires prolonged treatment with a combination of multiple agents and interruptions in the treatment regimen result in emergence and spread of multi-drug resistant (MDR) Mtb strains. MDR Mtb poses a significant global health problem, calling for urgent development of novel drugs to combat TB. Here, we report the 3.3 Å resolution structure of the ~2 MDa type-I fatty acid synthase (FAS-I) from Mtb, determined by single particle cryo-EM. Mtb FAS-I is an essential enzymatic complex that contributes to the virulence of Mtb, and thus a prime target for anti-TB drugs. The structural information for Mtb FAS-I we have obtained enables computer-based drug discovery approaches, and the resolution achieved by cryo-EM is sufficient for elucidating inhibition mechanisms by putative small molecular weight inhibitors.

Expression of a recombinant, 4'-Phosphopantetheinylated, active M. tuberculosis fatty acid synthase I in E. coli.

BACKGROUND: Fatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS). METHODS: In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. RESULTS: Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. CONCLUSION: Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

Cryo-EM structure of fatty acid synthase (FAS) from Rhodosporidium toruloides provides insights into the evolutionary development of fungal FAS.

Fungal fatty acid synthases Type I (FAS I) are up to 2.7 MDa large molecular machines composed of large multifunctional polypeptides. Half of the amino acids in fungal FAS I are involved in structural elements that are responsible for scaffolding the elaborate barrel-shaped architecture and turning fungal FAS I into highly efficient de novo producers of fatty acids. Rhodosporidium toruloides is an oleaginous fungal species and renowned for its robust conversion of carbohydrates into lipids to over 70% of its dry cell weight. Here, we use cryo-EM to determine a 7.8-Å reconstruction of its FAS I that reveals unexpected features; its novel form of splitting the multifunctional polypeptide chain into the two subunits α and ß, and its duplicated ACP domains. We show that the specific distribution into α and ß occurs by splitting at one of many possible sites that can be accepted by fungal FAS I. While, therefore, the specific distribution in α and ß chains in R. toruloides FAS I is not correlated to increased protein activities, we also show that the duplication of ACP is an evolutionary late event and argue that duplication is beneficial for the lipid overproduction phenotype.

Overexpression and characterization of a novel (S)-specific extended short-chain dehydrogenase/reductase from Candida parapsilosis.

The gene encoding a putative protein from Candida parapsilosis CDC317 (CPE) was cloned and overexpressed in Escherichia coli. The protein was amenable to overexpression in E. coli and constituted 35% of the total cell protein content. The optimal activity was determined at pH 5.5 and 40°C with the substrate 4-chloro-3-oxobutanoate ethyl ester (COBE). The optical purity of the product was over 99% enantiomeric excess for the (S)-isomer, and the molar conversion yield of the product was 91.1%. The apparent k(m) value for COBE was 0.19±0.01mM, which is an order of magnitude lower than that of other enzymes in the literature.

The fatty acid synthase of the basidiomycete Omphalotus olearius is a single polypeptide.

Fatty acids are essential components of almost all biological membranes. Additionally, they are important in energy storage, as second messengers during signal transduction, and in post-translational protein modification. De novo synthesis of fatty acids is essential for almost all organisms, and entails the iterative elongation of the growing fatty acid chain through a set of reactions conserved in all kingdoms. During our work on the biosynthesis of secondary metabolites, a 450-kDa protein was detected by SDS-PAGE of enriched fractions from mycelial lysates from the basidiomycete Omphalotus olearius. Protein sequencing of this protein band revealed the presence of peptides with homology to both alpha and beta subunits of the ascomycete fatty acid synthase (FAS) family. The FAS encoding gene of O. olearius was sequenced. The positions of its predicted 21 introns were verified. The gene encodes a 3931 amino acids single protein, with an equivalent of the ascomycetous beta subunit at the N-terminus and the a subunit at the C-terminus. This is the first report on an FAS protein from a homobasidiomycete and also the first fungal FAS which is comprised of a single polypeptide.

An orthogonal purification strategy for isolating crosslinked domains of modular synthases.

Chemo-enzymatic methods for covalently crosslinking carrier proteins with partner enzymes within modular synthases hold promise for elucidating and engineering metabolic pathways. Our efforts to crystallize the ACP-KS complexes of fatty acid synthases have been complicated by difficulties in the purification of the crosslinked complex from the other proteins in the reaction. Here we present a solution that employs an orthogonal purification strategy to achieve the quantity and level of purity necessary for further studies of this complex.

Inhibition of isolated Mycobacterium tuberculosis fatty acid synthase I by pyrazinamide analogs.

An analog of pyrazinamide (PZA), 5-chloropyrazinamide (5-Cl-PZA), has previously been shown to inhibit mycobacterial fatty acid synthase I (FASI). FASI has been purified from a recombinant strain of M. smegmatis (M. smegmatis Deltafas1 attB::M. tuberculosis fas1). Following purification, FASI activity and inhibition were assessed spectrophotometrically by monitoring NADPH oxidation. The observed inhibition was both concentration and structure dependent, being affected by both substitution at the 5 position of the pyrazine nucleus and the nature of the ester or N-alkyl group. Under the conditions studied, both 5-Cl-PZA and PZA exhibited concentration and substrate dependence consistent with competitive inhibition of FASI with K(i)s of 55 to 59 microM and 2,567 to 2,627 microM, respectively. The results were validated utilizing a radiolabeled fatty acid synthesis assay. This assay showed that FASI was inhibited by PZA and pyrazinoic acid as well as by a series of PZA analogs.

Mass spectrometry-based systems approach for identification of inhibitors of Plasmodium falciparum fatty acid synthase.

The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (beta-ketoacyl-ACP synthase), FabG (beta-ketoacyl-ACP reductase), FabZ (beta-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (-)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite.

Tandem affinity purification tagging of fatty acid biosynthetic enzymes in Synechocystis sp. PCC6803 and Arabidopsis thaliana.

De novo fatty acid synthesis in plants occurs primarily in the plastids and is catalysed by a type-II fatty acid synthase (FAS) in which separate enzymes catalyse sequential reactions. Genes encoding all of the plant FAS components have been identified, following enzyme purification or by homology to Escherichia coli genes, and the structure of a number of the individual proteins determined. There are several lines of biochemical evidence indicating that FAS enzymes form a multi-protein complex and both in vitro and in vivo strategies can be used to investigate the association and interactions between them. To investigate protein interactions in vivo, tandem affinity purification-tagged FAS components are being used to purify complexes from both Arabidopsis thaliana and Synechocystis PCC6803. Here, the development of the tandem affinity purification method, its modification, and its use in plants is described and the experimental results achieved so far are reported.

Architecture of mammalian fatty acid synthase at 4.5 A resolution.

The homodimeric mammalian fatty acid synthase is one of the most complex cellular multienzymes, in that each 270-kilodalton polypeptide chain carries all seven functional domains required for fatty acid synthesis. We have calculated a 4.5 angstrom-resolution x-ray crystallographic map of porcine fatty acid synthase, highly homologous to the human multienzyme, and placed homologous template structures of all individual catalytic domains responsible for the cyclic elongation of fatty acid chains into the electron density. The positioning of domains reveals the complex architecture of the multienzyme forming an intertwined dimer with two lateral semicircular reaction chambers, each containing a full set of catalytic domains required for fatty acid elongation. Large distances between active sites and conformational differences between the reaction chambers demonstrate that mobility of the acyl carrier protein and general flexibility of the multienzyme must accompany handover of the reaction intermediates during the reaction cycle.

Architecture of a fungal fatty acid synthase at 5 A resolution.

All steps of fatty acid synthesis in fungi are catalyzed by the fatty acid synthase, which forms a 2.6-megadalton alpha6beta6 complex. We have determined the molecular architecture of this multienzyme by fitting the structures of homologous enzymes that catalyze the individual steps of the reaction pathway into a 5 angstrom x-ray crystallographic electron density map. The huge assembly contains two separated reaction chambers, each equipped with three sets of active sites separated by distances up to approximately 130 angstroms, across which acyl carrier protein shuttles substrates during the reaction cycle. Regions of the electron density arising from well-defined structural features outside the catalytic domains separate the two reaction chambers and serve as a matrix in which domains carrying the various active sites are embedded. The structure rationalizes the compartmentalization of fatty acid synthesis, and the spatial arrangement of the active sites has specific implications for our understanding of the reaction cycle mechanism and of the architecture of multienzymes in general.

Purification and characterization of the Mycobacterium tuberculosis FabD2, a novel malonyl-CoA:AcpM transacylase of fatty acid synthase.

Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in fatty acid and mycolic acid biosynthesis of Mycobacterium tuberculosis. fabd2 is a novel gene coding MCAT in M. tuberculosis besides another known fabd. In our study, fabd2 was inserted into a bacterial expression vector pET28a resulting in a 6x Histidine-tag fabd2 fusion gene construction. The protein was purified by nickel affinity chromatography and the characterizations of FabD2 have been investigated. The molecular weight of FabD2 was estimated to be 26 kDa by MALDI-TOF. Consistent with the biosynthesis specialty of reported MCATs, FabD2 resulted in a typical activity of bacterial MCATs, which catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP. Some physical and chemical differences between FabD2 and FabD also have been found. FabD2 shows dissimilarity with FabD in secondary structure in different pH buffer and MCAT genes RT-PCR results reveal different transcript condition with each other. Furthermore, FabD2 shows low similarity in protein sequence when alignment with other MCATs.

Two functional FAS-I type fatty acid synthases in Corynebacterium glutamicum.

The lipid-rich Corynebacterianeae, to which Corynebacterium glutamicum and Mycobacterium species belong, produce both fatty acids and mycolic acids. Compared with most other bacteria, C. glutamicum possesses two fatty acid synthases, encoded by fasA (8907 kb; FAS-IA) and fasB (8988 kb; FAS-IB). Here, it was shown by mutational analyses that fasA is essential but fasB is not. However, in a fasA background, the fasB mutation results in a slightly reduced growth yield, l-glutamate production is increased, and comparative lipid analysis suggests that in vivo FAS-IB is active primarily to supply palmitate. Transcript quantifications revealed that the fasB transcript contributes 32 % to both fas transcripts during growth on glucose, affirmative for fasB expression, and that fasB is subordinate to fasA. The fasA transcript is downregulated by 8.3-fold during growth on acetate as compared with glucose. The lipid analyses also demonstrate that cells grown on propionate produce a number of uneven fatty acids (e.g. 15 : 0, 17 : 0, 17 : 1), which are not present in cells grown on glucose or acetate, suggesting that fatty acid synthase in vivo may also use propionyl-CoA as the priming unit in fatty acid synthesis. The fatty acid auxotrophic fasAB double mutant was used to determine the suggested incorporation of fatty acids into mycolic acids. Supplementation of this mutant with uniformly labelled [(13)C]oleate and analysis of isolated mycolic acids confirmed that mature mycolic acids in the mutant consist exclusively of two fused [(13)C]oleate molecules. In addition to an altered phospholipid profile, the fasB mutant also exhibits differences in its mycolic acid profile. Taken together, the results show that although FAS-IA is the most relevant fatty acid synthase of C. glutamicum and FAS-IB is supplementary, both synthases are necessary to produce the characteristic lipid environment of this organism.

Head-to-head coiled arrangement of the subunits of the animal fatty acid synthase.

The role of the beta-ketoacyl synthase domains in dimerization of the 2505 residue subunits of the multifunctional animal FAS has been evaluated by a combination of crosslinking and characterization of several truncated forms of the protein. Polypeptides containing only the N-terminal 971 residues can form dimers, but polypeptides lacking only the N-terminal 422 residue beta-ketoacyl synthase domain cannot. FAS subunits can be crosslinked with spacer lengths as short as 6 A, via cysteine residues engineered near the N terminus of the full-length polypeptides. The proximity of the N-terminal beta-ketoacyl synthase domains and their essential role in dimerization is consistent with a revised model for the FAS in which a head-to-head arrangement of two coiled subunits facilitates functional interactions between the dimeric beta-ketoacyl synthase and the acyl carrier protein domains of either subunit.

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions.

Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the alpha-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), beta-ketoacyl-ACP synthase (KasB), l-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy.

Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases.

Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEII(srf)) and bacitracin (TEII(bac)). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4'-phosphopantetheine (4'PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEII(srf), a K(M) of 0.9 microM and a k(cat) of 95 min(-1) was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEII(srf). These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4'PP transferases that use not only free CoA, but also acyl-CoAs as 4'PP donors.

Purification and characterization of the fatty acid synthase from Bugula neritina.

The fatty acid synthase from Bugula neritina has been purified 100-fold using ammonium sulfate precipitation, ion-exchange and size exclusion chromatography. The purified enzyme has a molecular weight of approximately 382,000 Da, as judged by gel filtration. Polyacrylamide gel electrophoresis under denaturing conditions in the presence of SDS revealed one major protein band of approximately 190,000 Da suggesting that the enzyme is a homodimer. The size of the enzyme, together with the observation that the FAS activity is independent of the concentration of acyl carrier protein, indicate that the FAS from Bugula neritina is a type I. A detailed analysis of the products of the purified FAS indicated that palmitic acid is the primary product and longer chain fatty acids are not produced.

Inhibition of pigeon liver fatty acid synthetase by specific modification of lysine residues with 2,4,6-trinitrobenzenesulphonic acid.

Pigeon liver fatty acid synthetase was inactivated irreversibly by 2,4,6-trinitrobenzenesulphonic acid (TNBS). Biphasic inactivation of the enzyme was observed with the inhibitor. NADPH provided protection to the enzyme against inactivation by TNBS and the extent of protection increased with NADPH concentration indicating that the essential lysine residues are present at the NADPH binding site. The stoichiometric results with TNBS showed that 4 mol of lysine residues are modified per mole of fatty acid synthetase upon complete inactivation. The rapid reaction of two amino groups per enzyme molecule led to the loss of 60% of the enzyme activity. These approaches suggested that two lysine residues present at the active site are essential for the enzymatic activity of fatty acid synthetase.

Factors influencing the levels of fatty acid synthase complex activity in fowl.

There is a notable discrepancy between the FAS (fatty acid synthase) activity of four types of fowl (egg chicken, meat chicken, egg duck, and meat duck) with distinctively different body fat levels. There is a 14.8 fold difference per unit body weight between the maximum and minimum FAS activities. The three major factors affecting this discrepancy are liver weight per unit body weight, which is 2.3 times greater in meat ducks than in egg chickens, the amount of FAS protein per gram of liver, which is 1.85 times greater in meat ducks than in egg chickens, and the FAS specific activity in meat ducks, which is 3.5 times greater in meat ducks than in egg chickens. Within the same species of egg chickens, the abdomen fat per kg of body weight at 470 days after egg production is 66 times greater than 90 days before egg production and the liver FAS activity is increased 9.6 fold. The 9.6 fold FAS activity increase resulted from an increase in the specific activity, since the liver weight per kilogram of body weight remained constant at approx. 20 grams and the FAS weight per gram of liver also remained constant at approx. 4.5 mg. This shows that the control of the basic FAS activity level which is closely related to the level of body fat does not mainly arise from genetic control. For the same kind of fowl, the control of the basic FAS activity level occurs after gene expression. It is suggested that control may be imposed in the folding phase when new peptides give rise to functional proteins.

Inactivation of enoyl-CoA reductase in pigeon liver fatty acid synthetase by pyridoxal 5'-phosphate: evidence for the presence of one lysine residue at the active site.

Pigeon liver fatty acid synthetase (FAS) was rapidly inactivated by pyridoxal 5'-phosphate (PLP). Assays of the partial activities of the PLP-treated synthetase showed that only the enoyl-CoA reductase was decreased significantly. The inactivation of both the overall activity and enoyl-CoA reductase activity of FAS by PLP could be reversed by dialysis or dilution but not by reduction with sodium borohydride. Malonyl-CoA and acetyl-CoA did not protect the enzyme, whereas NADPH provided 68% protection against PLP-inactivation indicating that PLP modified lysine residues present at or near the co-enzyme binding site. PLP-treated enzyme after reduction with sodium borohydride exhibited fluorescence with a maximum at 397 nm (irradiation at 325 nm). Stoichiometric analysis showed that modification of four lysine residues per enzyme molecule resulted in complete inactivation of the overall and enoyl-CoA reductase activities of FAS. NADPH prevented the inactivation by protecting two of these lysine residues from modification, suggesting the presence of two essential lysine residues per enzyme molecule. These results are consistent with the hypothesis that each subunit of the enzyme contains an enoyl-CoA reductase domain in which a lysine residue, at or near the active site, interacts with NADPH.