- Isolation from bovine liver mitochondria and characterization of three distinct carboxylic acid: CoA ligases with activity toward xenobiotics.
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A mitochondrial freeze/thaw lysate was fractionated on a DEAE-cellulose column into four distinct acyl-CoA ligase fractions. First to elute was a 50 kDa short-chain ligase that activated only short-chain fatty acids. Next to elute were three ligases that had activity toward both medium-chain fatty acids and xenobiotic carboxylic acids; these were termed xenobiotic/medium-chain ligases (X-ligases) and labeled XL-I, XL-II, and XL-III, respectively, based on order of elution. The molecular weight of X-ligases I, II, and III were ca. 55,000, 55,500 and 53,000, respectively. Form XL-III showed no pH optimum; the rate increased steadily with pH beginning from pH 7.0. XL-I and XL-II showed the same behavior with benzoate as substrate, but with medium-chain fatty acids, both forms had a pH optimum at 8.8. The three X-ligases differed in substrate specificity. XL-I was the predominant nicotinic acid activating form and had the lowest Km for benzoate. Form XL-II was the only form with measurable salicylate activity, although it was extremely low. XL-III was the only 2,4,6,8-decatetraenoic acid activating form and also was the predominant medium-chain fatty acid-activating form. By comparison of substrate specificities, it was concluded that the two previously reported ligase preparations were mixtures of the three forms. When the ligase rates were compared to previously determined N-acyltransferase rates toward benzoyl-CoA and phenylacetyl-CoA, the data showed that ligase activities are 100-fold lower, and thus the ligase is rate limiting for the conjugation of both of these xenobiotics.
- Vessey,Hu
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- Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids.
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It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.
- Oku, Hirosuke,Futamori, Naoya,Masuda, Kenichi,Shimabukuro, Yumiko,Omine, Tomoyo,Iwasaki, Hironori
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- Comparison of acyl-CoA synthetic activities and enantioselectivity toward 2-arylpropanoic acids in firefly luciferases
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Measurement of thioesterification activities for dodecanoic acid (C12) and ketoprofen was done using five firefly luciferases, from Pyrocoelia miyako (PmL), Photinus pyralis (PpL), Luciola cruciata (LcL), Hotaria parvura (HpL), and Luciola mingrelica (LmL). Among these, PmL, PpL, and LcL showed the expected thioesterification activities toward both substrates. All the enzymes exhibited (R)-enantioselectivity toward ketoprofen, which had same tendency as firefly luciferase from Luciola lateralis (LUC-H). HpL and LmL, however, did not accept ketoprofen, although they had thioesterification activity toward C12. These results indicate that the substrate acceptance of luciferases for the thioesterification reaction varies dramatically relying on the origin of firefly. Hence we focused primarily on PmL and investigated the effect of pH on enzymatic activity. In addition, by determining the kinetic parameters at various pH values, we verified that the kcat parameter contributed to the preferential enantioselectivity of this enzyme.
- Kato, Dai-Ichiro,Yokoyama, Keisuke,Hiraishi, Yoshihiro,Takeo, Masahiro,Negoro, Seiji
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- Purification and Gene cloning of an enantioselective thioesterification enzyme from Brevibacterium Ketoglutamicum KU1073, a deracemization bacterium of 2-(4-Chlorophenoxy)propanoic acid
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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 Km and kcat values for (S)-CPPA were 0:92 ± 0:17mM 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 (C8). 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.
- Kato, Dai-Ichiro,Yoshida, Hiromitsu,Takeo, Masahiro,Negoro, Seiji,Ohta, Hiromichi
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- Identification of an α-Oxoamine Synthase and a One-Pot Two-Step Enzymatic Synthesis of α-Amino Ketones
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Alb29, an α-oxoamine synthase involved in albogrisin biosynthesis in Streptomyces albogriseolus MGR072, was characterized and responsible for the incorporation of l-glutamate to acyl-coenzyme A substrates. Combined with Alb29 and Mgr36 (an acyl-coenzyme A ligase), a one-pot enzymatic system was established to synthesize seven α-amino ketones. When these α-amino ketones were fed into the alb29 knockout strain Δalb29, respectively, the albogrisin analogs with different side chains were observed.
- Zhou, Ting,Gao, Du,Li, Jia-Xin,Xu, Min-Juan,Xu, Jun
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supporting information
p. 37 - 41
(2020/12/21)
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- Repurposing the 3-Isocyanobutanoic Acid Adenylation Enzyme SfaB for Versatile Amidation and Thioesterification
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Genome mining of microbial natural products enables chemists not only to discover the bioactive molecules with novel skeletons, but also to identify the enzymes that catalyze diverse chemical reactions. Exploring the substrate promiscuity and catalytic mechanism of those biosynthetic enzymes facilitates the development of potential biocatalysts. SfaB is an acyl adenylate-forming enzyme that adenylates a unique building block, 3-isocyanobutanoic acid, in the biosynthetic pathway of the diisonitrile natural product SF2768 produced by Streptomyces thioluteus, and this AMP-ligase was demonstrated to accept a broad range of short-chain fatty acids (SCFAs). Herein, we repurpose SfaB to catalyze amidation or thioesterification between those SCFAs and various amine or thiol nucleophiles, thereby providing an alternative enzymatic approach to prepare the corresponding amides and thioesters in vitro.
- Zhu, Mengyi,Wang, Lijuan,He, Jing
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supporting information
p. 2030 - 2035
(2020/11/30)
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- A KAS-III Heterodimer in Lipstatin Biosynthesis Nondecarboxylatively Condenses C8 and C14 Fatty Acyl-CoA Substrates by a Variable Mechanism during the Establishment of a C22 Aliphatic Skeleton
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β-Ketoacyl-acyl carrier protein synthase-III (KAS-III) and its homologues are thiolase-fold proteins that typically behave as homodimers functioning in diverse thioester-based reactions for C-C, C-O, or C-N bond formation. Here, we report an exception obs
- Zhang, Daozhong,Zhang, Fang,Liu, Wen
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supporting information
p. 3993 - 4001
(2019/03/12)
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- A versatile biosynthetic approach to amide bond formation
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The development of versatile and sustainable catalytic strategies for amide bond formation is a major objective for the pharmaceutical sector and the wider chemical industry. Herein, we report a biocatalytic approach to amide synthesis which exploits the diversity of Nature's amide bond forming enzymes, N-acyltransferases (NATs) and CoA ligases (CLs). By selecting combinations of NATs and CLs with desired substrate profiles, non-natural biocatalytic pathways can be built in a predictable fashion to allow access to structurally diverse secondary and tertiary amides in high yield using stoichiometric ratios of carboxylic acid and amine coupling partners. Transformations can be performed in vitro using isolated enzymes, or in vivo where reactions rely solely on cofactors generated by the cell. The utility of these whole cell systems is showcased through the preparative scale synthesis of a key intermediate of Losmapimod (GW856553X), a selective p38-mitogen activated protein kinase inhibitor.
- Philpott, Helena K.,Thomas, Pamela J.,Tew, David,Fuerst, Doug E.,Lovelock, Sarah L.
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supporting information
p. 3426 - 3431
(2018/08/07)
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- Screening and Engineering the Synthetic Potential of Carboxylating Reductases from Central Metabolism and Polyketide Biosynthesis
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Carboxylating enoyl-thioester reductases (ECRs) are a recently discovered class of enzymes. They catalyze the highly efficient addition of CO2 to the double bond of α,β-unsaturated CoA-thioesters and serve two biological functions. In primary metabolism of many bacteria they produce ethylmalonyl-CoA during assimilation of the central metabolite acetyl-CoA. In secondary metabolism they provide distinct α-carboxyl-acyl-thioesters to vary the backbone of numerous polyketide natural products. Different ECRs were systematically assessed with a diverse library of potential substrates. We identified three active site residues that distinguish ECRs restricted to C4 and C5-enoyl-CoAs from highly promiscuous ECRs and successfully engineered a selected ECR as proof-of-principle. This study defines the molecular basis of ECR reactivity, allowing for predicting and manipulating a key reaction in natural product diversification.
- Peter, Dominik M.,Schada Von Borzyskowski, Lennart,Kiefer, Patrick,Christen, Philipp,Vorholt, Julia A.,Erb, Tobias J.
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supporting information
p. 13457 - 13461
(2015/11/09)
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- Synthesis of coenzyme A thioesters using methyl acyl phosphates in an aqueous medium
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Regioselective S-acylation of coenzyme A (CoA) is achieved under aqueous conditions using various aliphatic and aromatic carboxylic acids activated as their methyl acyl phosphate monoesters. Unlike many hydrophobic activating groups, the anionic methyl acyl phosphate mixed anhydride is more compatible with aqueous solvents, making it useful for conducting acylation reactions in an aqueous medium.
- Pal, Mohan,Bearne, Stephen L.
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p. 9760 - 9763
(2015/01/08)
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- Establishing a toolkit for precursor-directed polyketide biosynthesis: Exploring substrate promiscuities of acid-CoA ligases
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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. (Chemical Presented).
- Go, Maybelle Kho,Chow, Jeng Yeong,Cheung, Vivian Wing Ngar,Lim, Yan Ping,Yew, Wen Shan
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experimental part
p. 4568 - 4579
(2012/08/28)
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- Enantiodifferentiation of ketoprofen by Japanese firefly luciferase from Luciola lateralis
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Recently, we found that firefly luciferase exhibited (R)-enantioselective thioesterification activity toward 2-arylpropanoic acids. In the case of Japanese firefly luciferase from Luciola lateralis (LUC-H), the E-value for ketoprofen was approximately 20. In this study, we used a spectrophotometric method to measure the catalytic activity of LUC-H. Using this method allowed us to judge the reaction efficiency easily. Our results confirmed that LUC-H exhibits enantioselective thioesterification activity toward a series of 2-arylpropanoic acids. The highest activity was observed with ketoprofen. We also observed high enzymatic activity of LUC-H toward long-chain fatty acids. These results were reasonable because LUC-H is homologous with long-chain acyl-CoA synthetase. To obtain further information about the enantiodifferentiation mechanism of the LUC-H catalyzed thioesterification of ketoprofen, we determined the kinetic parameters of the reaction relative to each of its three substrates: ketoprofen, ATP, and coenzyme A (CoASH). We found that whereas the affinities of each compound are not affected by the chirality of ketoprofen, enantiodifferentiation is achieved by a chirality-dependent difference in the kcat parameter.
- Kato, Dai-Ichiro,Tatsumi, Tomohiro,Bansho, Asami,Teruya, Keisuke,Yoshida, Hiromitsu,Takeo, Masahiro,Negoro, Seiji
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experimental part
p. 140 - 146
(2012/01/19)
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- Aminoacyl-coenzyme A synthesis catalyzed by a CoA ligase from Penicillium chrysogenum
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Coenzyme A ligases play an important role in metabolism by catalyzing the activation of carboxylic acids. In this study we describe the synthesis of aminoacyl-coenzyme As (CoAs) catalyzed by a CoA ligase from Penicillium chrysogenum. The enzyme accepted medium-chain length fatty acids as the best substrates, but the proteinogenic amino acids l-phenylalanine and l-tyrosine, as well as the non-proteinogenic amino acids d-phenylalanine, d-tyrosine and (R)- and (S)-β-phenylalanine were also accepted. Of these amino acids, the highest activity was found for (R)-β-phenylalanine, forming (R)-β-phenylalanyl-CoA. Homology modeling suggested that alanine 312 is part of the active site cavity, and mutagenesis (A312G) yielded a variant that has an enhanced catalytic efficiency with β-phenylalanines and d-α-phenylalanine.
- Koetsier, Martijn J.,Jekel, Peter A.,Wijma, Hein J.,Bovenberg, Roel A.L.,Janssen, Dick B.
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experimental part
p. 893 - 898
(2012/03/12)
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