Acyl Glucuronide and Coenzyme A Thioester Metabolites of Carboxylic Acid-Containing Drug Molecules: Layering Chemistry with Reactive Metabolism and Toxicology.

Glucuronidation and CoA (coenzyme A) conjugation are common pathways for the elimination of carboxylic acid-containing drug molecules. In some instances, these biotransformations have been associated with toxicity (such as idiosyncratic hepatic injury, renal impairment, hemolytic anemia, gastrointestinal inflammation, and bladder cancer) attributed to, in part, the propensity of acyl glucuronides and acyl CoA thioesters to covalently modify biological macromolecules such as proteins and DNA. It is to be noted that, while acyl glucuronidation and CoA conjugation are indeed implicated in adverse effects, there are many safe drugs in the market that are cleared by these reactive pathways. It is therefore important that new molecular entities with carboxylic acid groups are evaluated for toxicity in a manner that is not unreasonably risk-averse. In the absence of truly predictable methods, therefore, the general approach is to apply a set of end points to generate a weight-of-evidence evaluation. In practice, the focus is to identify structural liabilities and provide structure-activity recommendations early in the program, at a stage where an attempt to improve reactive metabolism does not deoptimize other critical drug-quality criteria. This review will present a high-level overview of the chemistry of glucuronidation and CoA conjugation and provide a discussion of the possible mechanisms of adverse effects that have been associated with these pathways, as well as how such potential hazards are addressed while delivering a new chemical entity for clinical evaluation.

Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase.

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

A cysteine trapping assay for risk assessment of reactive acyl CoA metabolites.

Some drugs with carboxylic acid moieties can potentially cause rare but severe hepatotoxicity. The reactive chemical species generated by drug metabolism are thought to be one reason for this event. Although the phase II conjugation metabolism of carboxylic acids generally renders a compound more polar and inactive, it is also responsible for the formation of reactive metabolites.This study aimed to provide a new approach towards the risk assessment of carboxylic acids in the aspect of reactive acyl CoA metabolites.Although acyl CoA metabolites have been concerned, it is difficult to detect them because of their instability. We investigated the trapping agents for acyl CoA metabolites. We found that cysteine is a good trapping agent and developed an assay method for the reactivity of acyl CoA metabolites. We evaluated 17 drugs with carboxylic acid moieties, all drugs concerned with hepatotoxicity displayed reactive potential. With consideration of the exposure of each parent drug, the correlation between drug labels and the calculated risk of carboxylic drugs was improved.These evaluations can be conducted without radiochemical reagents or the authentic standards of metabolites. We believe that the method will be beneficial for drug discovery.

Chemoenzymatic Synthesis and Biological Evaluation for Bioactive Molecules Derived from Bacterial Benzoyl Coenzyme A Ligase and Plant Type III Polyketide Synthase.

Plant type III polyketide synthases produce diverse bioactive molecules with a great medicinal significance to human diseases. Here, we demonstrated versatility of a stilbene synthase (STS) from Pinus Sylvestris, which can accept various non-physiological substrates to form unnatural polyketide products. Three enzymes (4-coumarate CoA ligase, malonyl-CoA synthetase and engineered benzoate CoA ligase) along with synthetic chemistry was practiced to synthesize starter and extender substrates for STS. Of these, the crystal structures of benzoate CoA ligase (BadA) from Rhodopseudomonas palustris in an apo form or in complex with a 2-chloro-1,3-thiazole-5-carboxyl-AMP or 2-methylthiazole-5-carboxyl-AMP intermediate were determined at resolutions of 1.57 Å, 1.7 Å, and 2.13 Å, respectively, which reinforces its capacity in production of unusual CoA starters. STS exhibits broad substrate promiscuity effectively affording structurally diverse polyketide products. Seven novel products showed desired cytotoxicity against a panel of cancer cell lines (A549, HCT116, Cal27). With the treatment of two selected compounds, the cancer cells underwent cell apoptosis in a dose-dependent manner. The precursor-directed biosynthesis alongside structure-guided enzyme engineering greatly expands the pharmaceutical repertoire of lead compounds with promising/enhanced biological activities.

Analysis of 1-Deoxysphingoid Bases and Their N-Acyl Metabolites and Exploration of Their Occurrence in Some Food Materials.

Most common sphingolipids are comprised of "typical" sphingoid bases (sphinganine, sphingosine, and structurally related compounds) and are produced via the condensation of l-serine with a fatty acyl-CoA by serine palmitoyltransferase. Some organisms, including mammals, also produce "atypical" sphingoid bases that lack a 1-hydroxyl group as a result of the utilization of l-alanine or glycine instead of l-serine, resulting in the formation of 1-deoxy- or 1-desoxymethylsphingoid bases, respectively. Elevated production of "atypical" sphingolipids has been associated with human disease, but 1-deoxysphingoid bases have also been found to have potential as anticancer compounds, hence, the importance of knowing more about the occurrence of these compounds in food. Most of the "typical" and "atypical" sphingoid bases are found as the N-acyl metabolites (e.g., ceramides and 1-deoxyceramides) in mammals, but this has not been uniformly assessed in previous studies nor determined in consumed food. Therefore, we developed a method for the quantitative analysis of "typical" and "atypical" sphingoid bases and their N-acyl derivatives by reverse-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. On the basis of these analyses, there was considerable variability in the amounts and molecular subspecies of atypical sphingoid bases and their N-acyl metabolites found in different edible sources. These findings demonstrate that a broader assessment of the types of sphingolipids in foods is needed because some diets might contain sufficient amounts of atypical as well as typical sphingolipids that could have beneficial or possibly deleterious effects on human health.

The molecular mechanism of DHHC protein acyltransferases.

Protein S-acylation is a reversible lipidic posttranslational modification where a fatty acid chain is covalently linked to cysteine residues by a thioester linkage. A family of integral membrane enzymes known as DHHC protein acyltransferases (DHHC-PATs) catalyze this reaction. With the rapid development of the techniques used for identifying lipidated proteins, the repertoire of S-acylated proteins continues to increase. This, in turn, highlights the important roles that S-acylation plays in human physiology and disease. Recently, the first molecular structures of DHHC-PATs were determined using X-ray crystallography. This review will comment on the insights gained on the molecular mechanism of S-acylation from these structures in combination with a wealth of biochemical data generated by researchers in the field.

The peroxisomal zebrafish SCP2-thiolase (type-1) is a weak transient dimer as revealed by crystal structures and native mass spectrometry.

The SCP2 (sterol carrier protein 2)-thiolase (type-1) functions in the vertebrate peroxisomal, bile acid synthesis pathway, converting 24-keto-THC-CoA and CoA into choloyl-CoA and propionyl-CoA. This conversion concerns the ß-oxidation chain shortening of the steroid fatty acyl-moiety of 24-keto-THC-CoA. This class of dimeric thiolases has previously been poorly characterized. High-resolution crystal structures of the zebrafish SCP2-thiolase (type-1) now reveal an open catalytic site, shaped by residues of both subunits. The structure of its non-dimerized monomeric form has also been captured in the obtained crystals. Four loops at the dimer interface adopt very different conformations in the monomeric form. These loops also shape the active site and their structural changes explain why a competent active site is not present in the monomeric form. Native mass spectrometry studies confirm that the zebrafish SCP2-thiolase (type-1) as well as its human homolog are weak transient dimers in solution. The crystallographic binding studies reveal the mode of binding of CoA and octanoyl-CoA in the active site, highlighting the conserved geometry of the nucleophilic cysteine, the catalytic acid/base cysteine and the two oxyanion holes. The dimer interface of SCP2-thiolase (type-1) is equally extensive as in other thiolase dimers; however, it is more polar than any of the corresponding interfaces, which correlates with the notion that the enzyme forms a weak transient dimer. The structure comparison of the monomeric and dimeric forms suggests functional relevance of this property. These comparisons provide also insights into the structural rearrangements that occur when the folded inactive monomers assemble into the mature dimer.

In vitro reconstitution of Wnt acylation reveals structural determinants of substrate recognition by the acyltransferase human Porcupine.

Wnt proteins regulate a large number of processes, including cellular growth, differentiation, and tissue homeostasis, through the highly conserved Wnt signaling pathway in metazoans. Porcupine (PORCN) is an endoplasmic reticulum (ER)-resident integral membrane enzyme that catalyzes posttranslational modification of Wnts with palmitoleic acid, an unsaturated lipid. This unique form of lipidation with palmitoleic acid is a vital step in the biogenesis and secretion of Wnt, and PORCN inhibitors are currently in clinical trials for cancer treatment. However, PORCN-mediated Wnt lipidation has not been reconstituted in vitro with purified enzyme. Here, we report the first successful purification of human PORCN and confirm, through in vitro reconstitution with the purified enzyme, that PORCN is necessary and sufficient for Wnt acylation. By systematically examining a series of substrate variants, we show that PORCN intimately recognizes the local structure of Wnt around the site of acylation. Our in vitro assay enabled us to examine the activity of PORCN with a range of fatty acyl-CoAs with varying length and unsaturation. The selectivity of human PORCN across a spectrum of fatty acyl-CoAs suggested that the kink in the unsaturated acyl chain is a key determinant of PORCN-mediated catalysis. Finally, we show that two putative PORCN inhibitors that were discovered with cell-based assays indeed target human PORCN. Together, these results provide discrete, high-resolution biochemical insights into the mechanism of PORCN-mediated Wnt acylation and pave the way for further detailed biochemical and structural studies.

Lysine benzoylation is a histone mark regulated by SIRT2.

Metabolic regulation of histone marks is associated with diverse biological processes through dynamically modulating chromatin structure and functions. Here we report the identification and characterization of a histone mark, lysine benzoylation (Kbz). Our study identifies 22 Kbz sites on histones from HepG2 and RAW cells. This type of histone mark can be stimulated by sodium benzoate (SB), an FDA-approved drug and a widely used chemical food preservative, via generation of benzoyl CoA. By ChIP-seq and RNA-seq analysis, we demonstrate that histone Kbz marks are associated with gene expression and have physiological relevance distinct from histone acetylation. In addition, we demonstrate that SIRT2, a NAD+-dependent protein deacetylase, removes histone Kbz both in vitro and in vivo. This study therefore reveals a new type of histone marks with potential physiological relevance and identifies possible non-canonical functions of a widely used chemical food preservative.

The Causes and Consequences of Nonenzymatic Protein Acylation.

Thousands of protein acyl modification sites have now been identified in vivo. However, at most sites the acylation stoichiometry is low, making functional enzyme-driven regulation in the majority of cases unlikely. As unmediated acylation can occur on the surface of proteins when acyl-CoA thioesters react with nucleophilic cysteine and lysine residues, slower nonenzymatic processes likely underlie most protein acylation. Here, we review how nonenzymatic acylation of nucleophilic lysine and cysteine residues occurs; the factors that enhance acylation at particular sites; and the strategies that have evolved to limit protein acylation. We conclude that protein acylation is an unavoidable consequence of the central role of reactive thioesters in metabolism. Finally, we propose a hypothesis for why low-stoichiometry protein acylation is selected against by evolution and how it might contribute to degenerative processes such as aging.

Differences in substrate specificity of V. cholerae FabH enzymes suggest new approaches for the development of novel antibiotics and biofuels.

Vibrio cholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of ß-ketoacyl-acyl carrier protein synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH1 and six vcFabH2 structures determined in either apo form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate-binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally, the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production.

Structure-activity relationships of rationally designed AMACR 1A inhibitors.

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the 'racemisation' reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure-activity relationship study has been performed. This paper describes the first structure-activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC50 = 400-750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure-activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.

Biosynthesis of angelyl-CoA in Saccharomyces cerevisiae.

BACKGROUND: The angelic acid moiety represents an essential modification in many biologically active products. These products are commonly known as angelates and several studies have demonstrated their therapeutic benefits, including anti-inflammatory and anti-cancer effects. However, their availability for use in the development of therapeutics is limited due to poor extraction yields. Chemical synthesis has been achieved but its complexity prevents application, therefore microbial production may offer a promising alternative. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce angelyl-CoA, the CoA-activated form of angelic acid. RESULTS: For yeast-based production of angelyl-CoA we first expressed genes recently identified in the biosynthetic cluster ssf of Streptomyces sp. SF2575 in S. cerevisiae. Exogenous feeding of propionate and heterologous expression of a propionyl-CoA synthase from Streptomyces sp. were initially employed to increase the intracellular propionyl-CoA level, resulting in production of angelyl-CoA in the order of 5 mg/L. Substituting the Streptomyces sp. propionyl-CoA carboxylase with a carboxylase derived from Streptomyces coelicolor resulted in angelyl-CoA levels up to 6.4 mg/L. In vivo analysis allowed identification of important intermediates in the pathway, including methyl-malonyl-CoA and 3-hydroxyl-2-methyl-butyryl-CoA. Furthermore, methyl-malonate supplementation and expression of matB CoA ligase from S. coelicolor allowed for methyl-malonyl-CoA synthesis and supported, together with parts of the ssf pathway, angelyl-CoA titres of approximately 1.5 mg/L. Finally, feeding of angelic acid to yeasts expressing acyl-CoA ligases from plant species led to angelyl-CoA production rates of approximately 40 mg/L. CONCLUSIONS: Our results demonstrate the biosynthesis of angelyl-CoA in yeast from exogenously supplied carboxylic acid precursors. This is the first report on the activity of the ssf genes. We envision that our approach will provide a platform for a more sustainable production of the pharmaceutically important compound class of angelates.

Structure and substrate specificity of ß-ketoacyl-acyl carrier protein synthase III from Acinetobacter baumannii.

Originally annotated as the initiator of fatty acid synthesis (FAS), ß-ketoacyl-acyl carrier protein synthase III (KAS III) is a unique component of the bacterial FAS system. Novel variants of KAS III have been identified that promote the de novo use of additional extracellular fatty acids by FAS. These KAS III variants prefer longer acyl-groups, notably octanoyl-CoA. Acinetobacter baumannii, a clinically important nosocomial pathogen, contains such a multifunctional KAS III (AbKAS III). To characterize the structural basis of its substrate specificity, we determined the crystal structures of AbKAS III in the presence of different substrates. The acyl-group binding cavity of AbKAS III and co-crystal structure of AbKAS III and octanoyl-CoA confirmed that the cavity can accommodate acyl groups with longer alkyl chains. Interestingly, Cys264 formed a disulfide bond with residual CoA used in the crystallization, which distorted helices at the putative interface with acyl-carrier proteins. The crystal structure of KAS III in the alternate conformation can also be utilized for designing novel antibiotics.

Active site alanine preceding catalytic cysteine determines unique substrate specificity in bacterial CoA-acylating prenal dehydrogenase.

In detoxification and fermentation processes, acylating dehydrogenases catalyze the reversible oxidation of aldehydes to their corresponding acyl-CoA esters. Here, we characterize an enzyme from Aquincola tertiaricarbonis L108 responsible for prenal (3-methyl-2-butenal) to 3-methylcrotonyl-CoA oxidation. Enzyme kinetics demonstrate a preference for C5 substrates not yet observed in aldehyde dehydrogenases. Compared to acetaldehyde and acetyl-CoA, conversion of valeraldehyde and valeryl-CoA is > 100- and 8-fold more efficient, respectively. Enzyme variants with A254I, A254P, and A254G mutations indicate that active site Ala preceding the catalytic C255 is crucial for this unique specificity. These results shed new light on evolutionary adaptation of aldehyde dehydrogenases toward xenobiotics and structure-guided design of highly specific enzymes for production of biofuels, such as linear or iso-branched butanols and pentanols.

Comprehensive quantitative analysis of fatty-acyl-Coenzyme A species in biological samples by ultra-high performance liquid chromatography-tandem mass spectrometry harmonizing hydrophilic interaction and reversed phase chromatography.

Fatty acyl-Coenzyme A species (acyl-CoAs) are key biomarkers in studies focusing on cellular energy metabolism. Existing analytical approaches are unable to simultaneously detect the full range of short-, medium-, and long-chain acyl-CoAs, while chromatographic limitations encountered in the analysis of limited amounts of biological samples are an often overlooked problem. We report the systematic development of a UHPLC-ESI-MS/MS method which incorporates reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) separations in series, in an automated mode. The protocol outlined encompasses quantification of acyl-CoAs of varying hydrophobicity from C2 to C20 with recoveries in the range of 90-111 % and limit of detection (LOD) 1-5 fmol, which is substantially lower than previously published methods. We demonstrate that the poor chromatographic performance and signal losses in MS detection, typically observed for phosphorylated organic molecules, can be avoided by the incorporation of a 0.1% phosphoric acid wash step between injections. The methodological approach presented here permits a highly reliable, sensitive and precise analysis of small amounts of tissues and cell samples as demonstrated in mouse liver, human hepatic (HepG2) and skeletal muscle (LHCNM2) cells. The considerable improvements discussed pave the way for acyl-CoAs to be incorporated in routine targeted lipid biomarker profile studies.

Molecular Basis for Converting (2S)-Methylsuccinyl-CoA Dehydrogenase into an Oxidase.

Although flavoenzymes have been studied in detail, the molecular basis of their dioxygen reactivity is only partially understood. The members of the flavin adenosine dinucleotide (FAD)-dependent acyl-CoA dehydrogenase and acyl-CoA oxidase families catalyze similar reactions and share common structural features. However, both enzyme families feature opposing reaction specificities in respect to dioxygen. Dehydrogenases react with electron transfer flavoproteins as terminal electron acceptors and do not show a considerable reactivity with dioxygen, whereas dioxygen serves as a bona fide substrate for oxidases. We recently engineered (2S)-methylsuccinyl-CoA dehydrogenase towards oxidase activity by rational mutagenesis. Here we characterized the (2S)-methylsuccinyl-CoA dehydrogenase wild-type, as well as the engineered (2S)-methylsuccinyl-CoA oxidase, in detail. Using stopped-flow UV-spectroscopy and liquid chromatography-mass spectrometry (LC-MS) based assays, we explain the molecular base for dioxygen reactivity in the engineered oxidase and show that the increased oxidase function of the engineered enzyme comes at a decreased dehydrogenase activity. Our findings add to the common notion that an increased activity for a specific substrate is achieved at the expense of reaction promiscuity and provide guidelines for rational engineering efforts of acyl-CoA dehydrogenases and oxidases.

Comprehensive Analysis of Short-, Medium-, and Long-Chain Acyl-Coenzyme A by Online Two-Dimensional Liquid Chromatography/Mass Spectrometry.

Acyl-coenzyme A (CoA) is a pivotal metabolic intermediate in numerous biological processes. However, comprehensive analysis of acyl-CoAs is still challenging as the properties of acyl-CoAs greatly vary with different carbon chains. Here, we designed a two-dimensional liquid chromatography method coupled with high-resolution mass spectrometry (2D LC/HRMS) to cover all short-, medium-, and long-chain acyl-CoAs within one analytical run. Complex acyl-CoAs were separated into two fractions according to their acyl chains by the first dimensional prefractionation. Then, two fractions containing short-chain acyl-CoAs or medium- and long-chain acyl-CoAs were further separated by the two parallel columns in the second dimension. Nineteen representative standards were chosen to optimize the analytical conditions of the 2D LC/HRMS method. Resolution and sensitivity were demonstrated to be improved greatly, and lowly abundant acyl-CoAs and acyl-CoA isomers could be detected and distinguished. By using the 2D LC/HRMS method, 90 acyl-CoAs (including 21 acyl-dephospho-CoAs) were identified from liver extracts, which indicated that our method was one of the most powerful approaches for obtaining comprehensive profiling of acyl-CoAs so far. The method was further employed in the metabolomics study of malignant glioma cells with an isocitrate dehydrogenase 1 (IDH1) mutation to explore their metabolic differences. A total of 46 acyl-CoAs (including 2 acyl-dephospho-CoAs) were detected, and 12 of them were dysregulated in glioma cells with the IDH1 mutation. These results demonstrated the practicability and the superiority of the established method. Therefore, the 2D LC/HRMS method provides a robust and reproducible approach to the comprehensive analysis of acyl-CoAs in tissues, cells, and other biological samples.

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode.

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family.

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein.

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites.