Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme.

Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.

Isolation and characterization of mutants corresponding to the MENA, MENB, MENC and MENE enzymatic steps of 5'-monohydroxyphylloquinone biosynthesis in Chlamydomonas reinhardtii.

Phylloquinone (PhQ), or vitamin K1 , is an essential electron carrier (A1 ) in photosystem I (PSI). In the green alga Chlamydomonas reinhardtii, which is a model organism for the study of photosynthesis, a detailed characterization of the pathway is missing with only one mutant deficient for MEND having been analyzed. We took advantage of the fact that a double reduction of plastoquinone occurs in anoxia in the A1 site in the mend mutant, interrupting photosynthetic electron transfer, to isolate four new phylloquinone-deficient mutants impaired in MENA, MENB, MENC (PHYLLO) and MENE. Compared with the wild type and complemented strains for MENB and MENE, the four men mutants grow slowly in low light and are sensitive to high light. When grown in low light they show a reduced photosynthetic electron transfer due to a specific decrease of PSI. Upon exposure to high light for a few hours, PSI becomes almost completely inactive, which leads in turn to lack of phototrophic growth. Loss of PhQ also fully prevents reactivation of photosynthesis after dark anoxia acclimation. In silico analyses allowed us to propose a PhQ biosynthesis pathway in Chlamydomonas that involves 11 enzymatic steps from chorismate located in the chloroplast and in the peroxisome.

Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

Xanthomonas campestris†RpfB is a fatty Acyl-CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase.

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coli FadD, in the E. coli ß-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.

FadD3 is an acyl-CoA synthetase that initiates catabolism of cholesterol rings C and D in actinobacteria.

The cholesterol catabolic pathway occurs in most mycolic acid-containing actinobacteria, such as Rhodococcus jostii RHA1, and is critical for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl-CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild-type and accumulated 3aα-H-4α(3'-propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP), consistent with the catabolism of half the steroid molecule. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3(Mtb) catalysed the ATP-dependent CoA thioesterification of HIP and its hydroxylated analogues, 5α-OH HIP and 1ß-OH HIP. The apparent specificity constant (k(cat) /K(m) ) of FadD3(Mtb) for HIP was 7.3 ± 0.3 × 10(5) M(-1) s(-1) , 165 times higher than for 5α-OH HIP, while the apparent K(m) for CoASH was 110 ± 10 µM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3(Mtb) did not detectably transform a metabolite with a partially degraded C17 side-chain. Overall, these results indicate that FadD3 is a HIP-CoA synthetase that initiates catabolism of steroid rings C and D after side-chain degradation is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degradation enzymes.

Establishing a toolkit for precursor-directed polyketide biosynthesis: exploring substrate promiscuities of acid-CoA ligases.

Polyketides are chemically diverse and medicinally important biochemicals that are biosynthesized from acyl-CoA precursors by polyketide synthases. One of the limitations to combinatorial biosynthesis of polyketides has been the lack of a toolkit that describes the means of delivering novel acyl-CoA precursors necessary for polyketide biosynthesis. Using five acid-CoA ligases obtained from various plants and microorganisms, we biosynthesized an initial library of 79 acyl-CoA thioesters by screening each of the acid-CoA ligases against a library of 123 carboxylic acids. The library of acyl-CoA thioesters includes derivatives of cinnamyl-CoA, 3-phenylpropanoyl-CoA, benzoyl-CoA, phenylacetyl-CoA, malonyl-CoA, saturated and unsaturated aliphatic CoA thioesters, and bicyclic aromatic CoA thioesters. In our search for the biosynthetic routes of novel acyl-CoA precursors, we discovered two previously unreported malonyl-CoA derivatives (3-thiophenemalonyl-CoA and phenylmalonyl-CoA) that cannot be produced by canonical malonyl-CoA synthetases. This report highlights the utility and importance of determining substrate promiscuities beyond conventional substrate pools and describes novel enzymatic routes for the establishment of precursor-directed combinatorial polyketide biosynthesis.

Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an outer membrane fatty acid transfer complex.

CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM.

E. coli propionyl-CoA synthetase is regulated in vitro by an intramolecular disulfide bond.

The E. coli propionyl-CoA synthetase (PCS) was cloned, expressed, purified, and analyzed. Kinetic analyses suggested that the enzyme preferred propionate as substrate but would also use acetate. The purified, stored protein had relatively low activity but was activated up to about 10-fold by incubation with dithiothreitol (DTT). The enzyme activation by DTT was reversed by diamide. This suggests that the protein contains a regulatory disulfide bond and that the reduction to two sulfhydryl groups activates PCS while the oxidation to a disulfide leads to its inactivation. This idea was tested by sequential mutagenesis of the 9 Cys in the protein to Ala. It was revealed that the C128A and C315A mutants had wildtype enzyme activity but were no longer activated by DTT or inhibited by diamide. The data obtained indicate that two Cys residues could be involved in redox-regulated system through formation of an intramolecular disulfide bridge in PCS.

Purification and gene cloning of an enantioselective thioesterification enzyme from Brevibacterium ketoglutamicum KU1073, a deracemization bacterium of 2-(4-chlorophenoxy)propanoic acid.

We succeeded in the purification and gene cloning of a new enzyme, α-methyl carboxylic acid deracemizing enzyme 1 (MCAD1) from Brevibacterium ketoglutamicum KU1073, which catalyzes the (S)-enantioselective thioesterification reaction of 2-(4-chlorophenoxy)propanoic acid (CPPA). The cloned gene of MCAD1 contained an ORF of 1,623 bp, encoding a polypeptide of 540 amino acids. In combination with cofactors ATP, coenzyme A (CoASH), and Mg(2+), MCAD1 demonstrated perfect enantioselectivity toward CPPA. The optimal pH and temperature for reaction were found to be 7.25 and 30 °C. Under these conditions, the K(m) and k(cat) values for (S)-CPPA were 0.92 ± 0.17 mM and 0.28 ± 0.026 s(-1) respectively. The results for substrate specificity revealed that MCAD1 had highest activity toward fatty acid tails with a medium chain-length (C(8)). This result indicates that MCAD1 should be classified into a family of medium-chain acyl-CoA synthetase. This novel activity has never been reported for this family.

Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum.

Enzymatic activation of PAA (phenylacetic acid) to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and POA (phenoxyacetic acid) act as acyl donors in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a PCL (phenylacetate-CoA ligase), was cloned in Escherichia coli as a maltose-binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA into phenylacetyl-CoA in an ATP- and magnesium-dependent reaction. PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with k(cat)/K(m) values of 0.23+/-0.06 and 7.8+/-1.2 mM(-1).s(-1) for PAA and POA respectively. Surprisingly, PCL was very efficient in catalysing the conversion of trans-cinnamic acids to the corresponding CoA thioesters [k(cat)/K(m)=(3.1+/-0.4)x10(2) mM(-1).s(-1) for trans-cinnamic acid]. Of all the substrates screened, medium-chain fatty acids, which also occur as the side chains of the natural penicillins F, DF, H and K, were the best substrates for PCL. The high preference for fatty acids could be explained by a homology model of PCL that was constructed on the basis of sequence similarity with the Japanese firefly luciferase. The results suggest that PCL has evolved from a fatty-acid-activating ancestral enzyme that may have been involved in the beta-oxidation of fatty acids.

Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) beta-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA beta-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in beta-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell.

A minimal biochemical route towards de novo formation of synthetic phospholipid membranes.

All living cells consist of membrane compartments, which are mainly composed of phospholipids. Phospholipid synthesis is catalyzed by membrane-bound enzymes, which themselves require pre-existing membranes for function. Thus, the principle of membrane continuity creates a paradox when considering how the first biochemical membrane-synthesis machinery arose and has hampered efforts to develop simplified pathways for membrane generation in synthetic cells. Here, we develop a high-yielding strategy for de novo formation and growth of phospholipid membranes by repurposing a soluble enzyme FadD10 to form fatty acyl adenylates that react with amine-functionalized lysolipids to form phospholipids. Continuous supply of fresh precursors needed for lipid synthesis enables the growth of vesicles encapsulating FadD10. Using a minimal transcription/translation system, phospholipid vesicles are generated de novo in the presence of DNA encoding FadD10. Our findings suggest that alternate chemistries can produce and maintain synthetic phospholipid membranes and provides a strategy for generating membrane-based materials.

Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae.

Long chain acyl-CoA synthetase (ACSL; fatty acid CoA ligase: AMP forming; EC 6.2.1.3) catalyzes the formation of acyl-CoA through a process, which requires fatty acid, ATP and coenzymeA as substrates. In the yeast Saccharomyces cerevisiae the principal ACSL is Faa1p (encoded by the FAA1 gene). The preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. Our previous work has shown Faa1p is a principal component of a fatty acid transport/activation complex that also includes the fatty acid transport protein Fat1p. In the present work hexameric histidine tagged Faa1p was purified to homogeneity through a two-step process in the presence of 0.1% eta-dodecyl-beta-maltoside following expression at 15 degrees C in Escherichia coli. In order to further define the role of this enzyme in fatty acid transport-coupled activation (vectorial acylation), initial velocity kinetic studies were completed to define the kinetic parameters of Faa1p in response to the different substrates and to define mechanism. These studies showed Faa1p had a Vmax of 158.2 nmol/min/mg protein and a Km of 71.1 microM oleate. When the concentration of oleate was held constant at 50 microM, the Km for CoA and ATP were 18.3 microM and 51.6 microM respectively. These initial velocity studies demonstrated the enzyme mechanism for Faa1p was Bi Uni Uni Bi Ping Pong.

Propanil and swep inhibit 4-coumarate:CoA ligase activity in vitro.

4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) in the phenylpropanoid pathway in plants has attracted interest as a novel target for developing effective plant growth inhibitors (PGIs). In a previous study in which the 4CL inhibitory activity of 28 existing herbicides was investigated using an optimized in vitro screening assay, 4CL activity was found to be strongly inhibited by propanil and swep at 100 microM. Here, further experimental evidence is provided to substantiate the previous result. Using 4-coumaric acid as substrate, tobacco 4CL activity was inhibited by propanil or swep in a concentration-dependent manner, with 50% inhibition concentrations (I(50)) of 39.6 and 6 microM respectively. These herbicides also exhibited uncompetitive inhibition towards 4-coumaric acid. Furthermore, 4CLs from several plant species were inhibited by the herbicides within a range from 1 to 50 microM. It is proposed that these herbicides have another site of action as a result of the inhibition of 4CL in the phenylpropanoid pathway, and this enzyme represents a new target site for the development of PGI.

Purification and characterization of benzoate-CoA ligase from Magnetospirillum sp. strain TS-6 capable of aerobic and anaerobic degradation of aromatic compounds.

Benzoate-CoA ligase (EC 6.2.1.25), the initial enzyme of anaerobic benzoate degradation, was purified and characterized from Magnetospirillum sp. strain TS-6 grown under both anaerobic and aerobic conditions. The enzyme purified from anaerobically grown cells was a homodimer with a relative molecular mass of 120 kDa. The specific activity for benzoyl-CoA synthesis was 13.4 micromol min(-1) mg(-1) protein. The enzyme purified from aerobically grown cells was concluded to be the same gene product as the anaerobic enzyme. The benzoate-CoA ligase gene consisting of 1587 nucleotides was cloned and sequenced, and its induction under aerobic and anaerobic conditions during growth on benzoate was confirmed by quantitative reverse transcription PCR. These results indicate that a single benzoate-CoA ligase is expressed and benzoate is converted into benzoyl-CoA under both aerobic and anaerobic conditions in Magnetospirillum sp.

An in vitro screening assay to discover novel inhibitors of 4-coumarate:CoA ligase.

4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) exists only in plants and plays an important role in the phenylpropanoid pathway. Identification of inhibitors targeting 4CL provides a novel approach for developing effective plant growth inhibitors (PGIs). The full-length gene of tobacco 4CL (Nt4CL1) was cloned and expressed in Escherichia coli Cast & Chalm. The recombinant 4CL protein was extracted and purified by several purification steps including gel-filtration and anion-exchange chromatography. 4CL activity assay was miniaturized and optimized using a 96-well microplate and a reader. Among 28 existing herbicides, propanil and swep strongly inhibited in vitro 4CL enzyme activity, and they were selected for further studies. The process of this assay can be developed into a high-throughput screening system of PGI targeting 4CL in the phenylpropanoid pathway.

Further purification, characterization and salt activation of acyl-CoA synthetase from Escherichia coli.

Acyl-CoA synthetase was further purified from Escherichia coli in good yield and fold purification by affinity chromatography on CoA-Sepharose 4B. The molecular weight of the active form of the purified enzyme was estimated as 45 000 by Sephadex G-100 and 47 000 by Sephadex G-200. Sedimentation equilibrium ultracentrifugation analysis revealed a molecular weight of 50 000. The sedimentation coefficient was calculated as 4.4 S. An absorption maximum at 276 nm was observed in the ultraviolet light absorption spectrum. The molar extinction coefficient was 9.2 X 10(4). Kinetic constants were determined for trans fatty acids. All ions tested, including chaotropic and lyotropic ions, stimulated or inhibited acyl-CoA synthetase activity depending on their concentrations in the assay system. In a series of chaotropes, the lower concentration required to maximally activate acyl-CoA synthetase in increasing order of potency of chaotropic ions. The inhibitory effect of chaotrope on the enzyme activity was reversible. These data suggest that salts have a common mode of action and influence acyl-CoA synthetase activity primarily through their effect on the solution structure.

Isolation and characterization of the multiple charge isoforms of acyl-CoA synthetase from Escherichia coli.

The three fractions of acyl-CoA synthetase differing in isoelectric pH were isolated from Escherichia coli by isoelectric focusing and characterized. They had the same molecular weight and identical immunochemical properties. The three fractions differed appreciably in pH-velocity profiles. These fractions had distinguishable thermal stabilities and peptide patterns obtained after limited proteolysis. Apparent Km and Vmax values for fatty acids were also significantly different in these fractions, although the specificity ranged from C8 to C18 fatty acids with maximum activity for lauric acid in all fractions.

Acyl-CoA synthetases in guinea-pig liver mitochondria. Purification and characterization of a distinct propionyl-CoA synthetase.

Guinea-pig liver mitochondria contain three soluble ATP-dependent acyl-CoA synthetases: (a) a medium-chain acyl-CoA synthetase, (b) a salicylate activating enzyme, and (c) a propionyl-CoA synthetase. A complete separation of these enzymes has been accomplished and the resulting preparation of propionyl-CoA synthetase (Spec. act. 4 units/mg protein) accepts acetate, propionate and butyrate as substrates with a high preference for propionate.

Occurrence and characterization of a dehydratase enzyme in the leek icosanoyl-CoA synthase complex.

The presence of a beta-hydroxyacyl-CoA dehydratase involved in the icosanoyl-CoA synthase (EC 2.3.1.119) complex of leek epidermis has been demonstrated using antibodies raised against the purified beta-hydroxyacyl-CoA dehydratase from rat liver. In a first step the leek icosanoyl-CoA synthase activity was measured in the presence of different amounts of this antibody, the results obtained showed a 75% inhibition of the activity using a 8:1 IgG/microsomal protein ratio, whereas only a weak diminution of the activity occurred using pre-immune IgG. The analysis of the reaction products after incubation in the presence of increasing IgG amounts showed a decrease of the fatty acids (the final product) and an accumulation of beta-hydroxy fatty acids using immune IgG, whereas no change occurred in the presence of pre-immune IgG. Moreover, the beta-hydroxyacyl-CoA dehydratase activity was strongly inhibited, whereas in the same conditions, the beta-ketoacyl-CoA reductase and the (trans-2-3) enoyl-CoA reductase activities were not affected. The protein fractions that eluted from the DEAE and Ultrogel columns containing the leek icosanoyl-CoA synthase activity were able to specifically bind the anti beta-hydroxyacyl-CoA dehydratase from rat liver. The cross-reactivity was demonstrated. In immunoblotting experiments using the same antiserum after SDS-PAGE of the purified leek icosanoyl-CoA synthase, only one of the four protein bands constituting the leek icosanoyl-CoA synthase was detected. This protein, having an apparent molecular mass of 65 kDa, could be the dehydratase component of the elongation complex.