ThnL, a B12-dependent radical S-adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis.

Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.

Design and synthesis of Coenzyme A analogues as Aurora kinase A inhibitors: An exploration of the roles of the pyrophosphate and pantetheine moieties.

Coenzyme A (CoA) is a highly selective inhibitor of the mitotic regulatory enzyme Aurora A kinase, with a novel mode of action. Herein we report the design and synthesis of analogues of CoA as inhibitors of Aurora A kinase. We have designed and synthesised modified CoA structures as potential inhibitors, combining dicarbonyl mimics of the pyrophosphate group with a conserved adenosine headgroup and different length pantetheine-based tail groups. An analogue with a -SH group at the end of the pantotheinate tail showed the best IC50, probably due to the formation of a covalent bond with Aurora A kinase Cys290.

Efficient one-pot enzymatic synthesis of dephospho coenzyme A.

Dephospho coenzyme A (depCoA) is the last intermediate for CoA biosynthesis, and it can be used as a transcription initiator to prepare CoA-linked RNA by in vitro transcription. However, commercially available depCoA is expensive. We hereby describe a simple and efficient enzymatic synthesis of depCoA in a single-step from commercially available and inexpensive oxidized pantethine (Ox-Pan) and ATP. A plasmid (pCoaDAa) was constructed to co-express and co-purify two enzymes pantothenate kinase (PanK/coaA) and phosphopantetheine adenylyltransferase (PPAT/coaD). Starting from Ox-Pan and ATP, two different synthetic routes of one-pot reaction catalyzed by PanK and PPAT, followed by a simple column purification step, afforded depCoA and its oxidized dimer (Ox-depCoA) with high yields and purity. The simplicity and low cost of our method should make depCoA easily accessible to a broad scientific community, and promote research on CoA-related areas in biology and biomedicine.

Experimentally validated novel inhibitors of Helicobacter pylori phosphopantetheine adenylyltransferase discovered by virtual high-throughput screening.

Helicobacter pylori is a major etiologic agent associated with the development and maintenance of human gastritis. The goal of this study was to develop novel antibiotics against H. pylori, and we thus targeted H. pylori phosphopantetheine adenylyltransferase (HpPPAT). PPAT catalyzes the penultimate step in coenzyme A biosynthesis. Its inactivation effectively prevents bacterial viability, making it an attractive target for antibacterial drug discovery. We employed virtual high-throughput screening and the HpPPAT crystal structure to identify compounds in the PubChem database that might act as inhibitors of HpPPAT. d-amethopterin is a potential inhibitor for blocking HpPPAT activity and suppressing H. pylori viability. Following treatment with d-amethopterin, H. pylori exhibited morphological characteristics associated with cell death. d-amethopterin is a mixed inhibitor of HpPPAT activity; it simultaneously occupies the HpPPAT 4'-phosphopantetheine- and ATP-binding sites. Its binding affinity is in the micromolar range, implying that it is sufficiently potent to serve as a lead compound in subsequent drug development. Characterization of the d-amethopterin and HpPPAT interaction network in a docked model will allow us to initiate rational drug optimization to improve the inhibitory efficacy of d-amethopterin. We anticipate that novel, potent, and selective HpPPAT inhibitors will emerge for the treatment of H. pylori infection.

Discovery of small molecule vanin inhibitors: new tools to study metabolism and disease.

Vanins are enzymes with pantetheinase activity and are presumed to play a role in the recycling of pantothenic acid (vitamin B5) from pantetheine. Pantothenic acid is an essential nutrient required to synthesize coenzyme A, a cofactor involved in many biological processes such as fatty acid synthesis and oxidation of pyruvate to fuel the citric acid cycle. Hydrolysis of pantetheine also liberates cysteamine, a known antioxidant. Vanin-1 is highly expressed in liver and is under transcriptional control of PPAR-α and nutritional status, suggesting a role in energy metabolism. The lack of potent and specific inhibitors of vanins has hampered detailed investigation of their function. We hereby report the design, synthesis, and characterization of a novel pantetheine analogue, RR6, that acts as a selective, reversible, and competitive vanin inhibitor at nanomolar concentration. Oral administration of RR6 in rats completely inhibited plasma vanin activity and caused alterations of plasma lipid concentrations upon fasting, thereby illustrating its potential use in chemical biology research.

Substrate recognition by ß-ketoacyl-ACP synthases.

ß-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a ß-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.

Crystal structure of phosphopantetheine adenylyltransferase from Enterococcus faecalis in the ligand-unbound state and in complex with ATP and pantetheine.

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) to form dephospho-CoA (dPCoA) and pyrophosphate in the Coenzyme A (CoA) biosynthetic pathway. Importantly, PPATs are the potential target for developing antibiotics because bacterial and mammalian PPATs share little sequence homology. Previous structural studies revealed the mechanism of the recognizing substrates and products. The binding modes of ATP, ADP, Ppant, and dPCoA are highly similar in all known structures, whereas the binding modes of CoA or 3'-phosphoadenosine 5'-phosphosulfate binding are novel. To provide further structural information on ligand binding by PPATs, the crystal structure of PPAT from Enterococcus faecalis was solved in three forms: (i) apo form, (ii) binary complex with ATP, and (iii) binary complex with pantetheine. The substrate analog, pantetheine, binds to the active site in a similar manner to Ppant. The new structural information reported in this study including pantetheine as a potent inhibitor of PPAT will supplement the existing structural data and should be useful for structure-based antibacterial discovery against PPATs.

Enzymatic extender unit generation for in vitro polyketide synthase reactions: structural and functional showcasing of Streptomyces coelicolor MatB.

In vitro experiments with modular polyketide synthases (PKSs) are often limited by the availability of polyketide extender units. To determine the polyketide extender units that can be biocatalytically accessed via promiscuous malonyl-CoA ligases, structural and functional studies were conducted on Streptomyces coelicolor MatB. We demonstrate that this adenylate-forming enzyme is capable of producing most CoA-linked polyketide extender units as well as pantetheine- and N-acetylcysteamine-linked analogs useful for in vitro PKS studies. Two ternary product complex structures, one containing malonyl-CoA and AMP and the other containing (2R)-methylmalonyl-CoA and AMP, were solved to 1.45 Å and 1.43 Å resolution, respectively. MatB crystallized in the thioester-forming conformation, making extensive interactions with the bound extender unit products. This first structural characterization of an adenylate-forming enzyme that activates diacids reveals the molecular details for how malonate and its derivatives are accepted. The orientation of the α-methyl group of bound (2R)-methylmalonyl-CoA, indicates that it is necessary to epimerize α-substituted extender units formed by MatB before they can be accepted by PKS acyltransferase domains. We demonstrate the in vitro incorporation of methylmalonyl groups ligated by MatB to CoA, pantetheine, or N-acetylcysteamine into a triketide pyrone by the terminal module of the 6-deoxyerythronolide B synthase. Additionally, a means for quantitatively monitoring certain in vitro PKS reactions using MatB is presented.

Synthetic chain terminators off-load intermediates from a type I polyketide synthase.

Modular biocatalysis is responsible for the generation of countless bioactive products and its mining remains a major focus for drug discovery purposes. One of the enduring hurdles is the isolation of biosynthetic intermediates in a readily-analysed form. We prepared a series of nonhydrolysable pantetheine and N-acetyl cysteamine mimics of the natural (methyl)malonyl extender units recruited for polyketide formation. Using these analogues as competitive substrates, we were able to trap and off-load diketide and triketide species directly from an in vitro reconstituted type I polyketide synthase, the 6-deoxyerythronolide B synthase 3 (DEBS3). The putative intermediates, which were extracted in organic solvent and characterised by LC-HR-ESI-MS, are the first of their kind and prove that small-molecule chain terminators can be used as convenient probes of the biosynthetic process.

Current medical aspects of pantethine.

Pantethine, the stable disulfide form of pantetheine, is the major precursor of coenzyme A, which plays a central role in the metabolism of lipids and carbohydrates. Coenzyme A is a cofactor in over 70 enzymatic pathways, including fatty acid oxidation, carbohydrate metabolism, pyruvate degradation, amino acid catabolism, haem synthesis, acetylcholine synthesis, phase II detoxification, acetylation, etc. Pantethine has beneficial effects in vascular disease, it able to decrease the hyperlipidaemia, moderate the platelet function and prevent the lipid-peroxidation. Moreover its neuro-endocrinological regulating role, its good influence on cataract and cystinosis are also proved. This molecule is a well-tolerated therapeutic agent; the frequency of its side-effect is very low and mild. Based on these preclinical and clinical data, it could be recommended using this compound as adjuvant therapy.

Differential gene expression in mouse liver associated with the hepatoprotective effect of clofibrate.

Pretreatment of mice with the peroxisome proliferator clofibrate (CFB) protects against acetaminophen (APAP)-induced hepatotoxicity. Previous studies have shown that activation of the nuclear peroxisome proliferator activated receptor-alpha (PPARalpha) is required for this effect. The present study utilizes gene expression profile analysis to identify potential pathways contributing to PPARalpha-mediated hepatoprotection. Gene expression profiles were compared between wild type and PPARalpha-null mice pretreated with vehicle or CFB (500 mg/kg, i.p., daily for 10 days) and then challenged with APAP (400 mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment and hybridized to Affymetrix Mouse Genome MGU74 v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring software. Our analysis identified 53 genes of interest including vanin-1, cell cycle regulators, lipid-metabolizing enzymes, and aldehyde dehydrogenase 2, an acetaminophen binding protein. Vanin-1 could be important for CFB-mediated hepatoprotection because this protein is involved in the synthesis of cysteamine and cystamine. These are potent antioxidants capable of ameliorating APAP toxicity in rodents and humans. HPLC-ESI/MS/MS analysis of liver extracts indicates that enhanced vanin-1 gene expression results in elevated cystamine levels, which could be mechanistically associated with CFB-mediated hepatoprotection.

Isothermal unfolding studies on the apo and holo forms of Plasmodium falciparum acyl carrier protein. Role of the 4'-phosphopantetheine group in the stability of the holo form of Plasmodium falciparum acyl carrier protein.

The unfolding pathways of the two forms of Plasmodium falciparum acyl carrier protein, the apo and holo forms, were determined by guanidine hydrochloride-induced denaturation. Both the apo form and the holo form displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provides values for the conformational stability of the two proteins. Although both forms have the same amino acid sequence, and they have similar secondary structures, it was found that the - DeltaG of unfolding of the holo form was lower than that of the apo form at all the temperatures at which the experiments were done. The higher stability of the holo form can be attributed to the number of favorable contacts that the 4'-phosphopantetheine group makes with the surface residues by virtue of a number of hydrogen bonds. Furthermore, there are several hydrophobic interactions with 4'-phosphopantetheine that firmly maintain the structure of the holo form. We show here for the first time that the interactions between 4'-phosphopantetheine and the polypeptide backbone of acyl carrier protein stabilize the protein. As Plasmodium acyl carrier protein has a similar secondary structure to the other acyl carrier proteins and acyl carrier protein-like domains, the detailed biophysical characterization of Plasmodium acyl carrier protein can serve as a prototype for the analysis of the conformational stability of other acyl carrier proteins.

Solution structures of conformationally equilibrium forms of holo-acyl carrier protein (PfACP) from Plasmodium falciparum provides insight into the mechanism of activation of ACPs.

Acyl Carrier Protein (ACP) from the malaria parasite, Plasmodium falciparum (PfACP) in its holo form is found to exist in two conformational states in solution. Unique 3D solution structures of holo-PfACP have been determined for both equilibrium conformations, using high-resolution NMR methods. Twenty high-resolution solution structures for each of the two forms of holo-PfACP have been determined on the basis of 1226 and 1218 unambiguously assigned NOEs (including NOEs between 4'-phosphopantetheine prosthetic group (4'-PP) and protein), 55 backbone dihedral angles and 26 hydrogen bonds. The atomic rmsd values of the determined structures of two equilibrium forms, about the mean coordinates of the backbone and heavy atoms, are 0.48 +/- 0.09 and 0.92 +/- 0.10 and 0.49 +/- 0.08 and 0.97 +/- 0.11 A, respectively. The interaction of 4'-PP with the polypeptide backbone is reported here for the first time for any of the ACPs. The structures of holo-PfACP consist of three well-defined helices that are tightly packed. The structured regions of the molecule are stabilized by extensive hydrophobic interactions. The difference between the two forms arises from a reorientation of the 4'-PP group. The enthalpy difference between the two forms, although small, implies that a conformational switch is essential for the activation of holo-ACP. Sequence and structures of holo-PfACP have been compared with those of the ACPs from type I and type II fatty acid biosynthesis pathways (FAS), in particular with the ACP from rat and the butyryl-ACP from E. coli. The PfACP structure, thus determined has several novel features hitherto not seen in other ACPs.

Modular synthesis of pantetheine and phosphopantetheine.

[structure: see text] D-Pantetheine and D-phosphopantetheine, precursors to coenzyme A, have been synthesized though a linear sequence from three modules (M1-M3) in 9 and 10 steps, respectively. These routes provide access to analogues of coenzyme A containing modified cystamines, beta-alanines, and pantoic acid residues. All three modules were joined using conventional methods of peptide synthesis. The chiral component, M3, was derived from D-pantolactone.

Mechanistic studies on phosphopantothenoylcysteine decarboxylase: trapping of an enethiolate intermediate with a mechanism-based inactivating agent.

Phosphopantothenoylcysteine decarboxylase (PPC-DC) catalyzes the decarboxylation of the cysteine moiety of 4'-phosphopantothenoylcysteine (PPC) to form 4'-phosphopantetheine (PPantSH); this reaction forms part of the biosynthesis of coenzyme A. The enzyme is a member of the larger family of cysteine decarboxylases including the lantibiotic-biosynthesizing enzymes EpiD and MrsD, all of which use a tightly bound flavin cofactor to oxidize the thiol moiety of the substrate to a thioaldehyde. The thioaldehyde serves to delocalize the charge that develops in the subsequent decarboxylation reaction. In the case of PPC-DC enzymes the resulting enethiol is reduced to a thiol giving net decarboxylation of cysteine, while in EpiD and MrsD it is released as the final product of the reaction. In this paper, we describe the characterization of the novel cyclopropyl-substituted product analogue 4'-phospho-N-(1-mercaptomethyl-cyclopropyl)-pantothenamide (PPanDeltaSH) as a mechanism-based inhibitor of the human PPC-DC enzyme. This inhibitor alkylates the enzyme on Cys(173), resulting in the trapping of a covalently bound enethiolate intermediate. When Cys(173) is exchanged for the weaker acid serine by site-directed mutagenesis the enethiolate reaction intermediate also accumulates. This suggests that Cys(173) serves as an active site acid in the protonation of the enethiolate intermediate in PPC-DC enzymes. We propose that this protonation step is the key mechanistic difference between the oxidative decarboxylases EpiD and MrsD (which have either serine or threonine at the corresponding position in their active sites) and PPC-DC enzymes, which also reduce the intermediate in an overall simple decarboxylation reaction.

Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues.

The class II PHA (polyhydroxyalkanoate) synthases [PHA(MCL) synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHA(MCL)s using CoA thioesters of medium-chain-length 3-hydroxy fatty acids (C6-C14) as a substrate. Only recently PHA(MCL) synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHA(MCL) synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the alpha/beta-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the alpha/beta-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved gamma-turns of the alpha/beta-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved residue Trp-398 was replaced by Phe and Ala, respectively, which caused inactivation of the enzyme indicating an essential role of this residue. In the threading model this residue was found to be surface-exposed. No evidence for post-translational modification by 4-phosphopantetheine was obtained. Overall, these data suggested that in class II PHA synthases the conserved histidine which was found as general base catalyst in the catalytic triad of enzymes related to the alpha/beta-hydrolase superfamily, was functionally replaced by His-453 which is conserved among all PHA synthases.

4'-phosphopantetheine and coenzyme A biosynthesis in plants.

Coenzyme A is required for many synthetic and degradative reactions in intermediary metabolism and is the principal acyl carrier in prokaryotic and eukaryotic cells. Coenzyme A is synthesized in five steps from pantothenate, and recently the CoaA biosynthetic genes in bacteria and human have all been identified and characterized. Coenzyme A biosynthesis in plants is not fully understood, and to date only the AtHAL3a (AtCoaC) gene of Arabidopsis thaliana has been cloned and identified as 4'-phosphopantothenoylcysteine (PPC) decarboxylase (Kupke, T., Hernández-Acosta, P., Steinbacher, S., and Culiáñez-Macià, F. A. (2001) J. Biol. Chem. 276, 19190-19196). Here, we demonstrate the cloning of the four missing genes, purification of the enzymes, and identification of their functions. In contrast to bacterial PPC synthetases, the plant synthetase is not CTP-but ATP-dependent. The complete biosynthetic pathway from pantothenate to coenzyme A was reconstituted in vitro by adding the enzymes pantothenate kinase (AtCoaA), 4'-phosphopantothenoylcysteine synthetase (AtCoaB), 4'-phosphopantothenoylcysteine decarboxylase (AtCoaC), 4'-phosphopantetheine adenylyltransferase (AtCoaD), and dephospho-coenzyme A kinase (AtCoaE) to a mixture containing pantothenate, cysteine, ATP, dithiothreitol, and Mg2+.

Selectivity of the yersiniabactin synthetase adenylation domain in the two-step process of amino acid activation and transfer to a holo-carrier protein domain.

The adenylation (A) domain of the Yersinia pestis nonribosomal peptide synthetase that biosynthesizes the siderophore yersiniabactin (Ybt) activates three molecules of L-cysteine and covalently aminoacylates the phosphopantetheinyl (P-pant) thiols on three peptidyl carrier protein (PCP) domains embedded in the two synthetase subunits, two in cis (PCP1, PCP2) in subunit HMWP2 and one in trans (PCP3) in subunit HMWP1. This two-step process of activation and loading by the A domain is analogous to the operation of the aminoacyl-tRNA synthetases in ribosomal peptide synthesis. Adenylation domain specificity for the first step of reversible aminoacyl adenylate formation was assessed with the amino acid-dependent [(32)P]-PP(i)-ATP exchange assay to show that S-2-aminobutyrate and beta-chloro-L-alanine were alternate substrates. The second step of A domain catalysis, capture of the bound aminoacyl adenylate by the P-pant-SH of the PCP domains, was assayed both by catalytic release of PP(i) and by covalent aminoacylation of radiolabeled substrates on either the PCP1 fragment of HMWP2 or the PCP3-thioesterase double domain fragment of HMWP1. There was little selectivity for capture of each of the three adenylates by PCP3 in the second step, arguing against any hydrolytic proofreading of incorrect substrates by the A domain. The holo-PCP3 domain accelerated PP(i) release and catalytic turnover by 100-200-fold over the leak rate (<1 min(-1)) of aminoacyl adenylates into solution while PCP1 in trans had only about a 5-fold effect. Free pantetheine could capture cysteinyl adenylate with a 25-50-fold increase in k(cat) while CoA was 10-fold less effective. The K(m) of free pantetheine (30-50 mM) was 3 orders of magnitude larger than that of PCP3-TE (10-25 microM), indicating a net 10(4) greater catalytic efficiency for transfer to the P-pant arm of PCP3 by the Ybt synthetase A domain, relative to P-pant alone.

Dibromopropanone cross-linking of the phosphopantetheine and active-site cysteine thiols of the animal fatty acid synthase can occur both inter- and intrasubunit. Reevaluation of the side-by-side, antiparallel subunit model.

The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal beta-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.