- Unexpected Reactions of α,β-Unsaturated Fatty Acids Provide Insight into the Mechanisms of CYP152 Peroxygenases
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CYP152 peroxygenases catalyze decarboxylation and hydroxylation of fatty acids using H2O2 as cofactor. To understand the molecular basis for the chemo- and regioselectivity of these unique P450 enzymes, we analyze the activities of three CYP152 peroxygenases (OleTJE, P450SPα, P450BSβ) towards cis- and trans-dodecenoic acids as substrate probes. The unexpected 6S-hydroxylation of the trans-isomer and 4R-hydroxylation of the cis-isomer by OleTJE, and molecular docking results suggest that the unprecedented selectivity is due to OleTJE’s preference of C2?C3 cis-configuration. In addition to the common epoxide products, undecanal is the unexpected major product of P450SPα and P450BSβ regardless of the cis/trans-configuration of substrates. The combined H218O2 tracing experiments, MD simulations, and QM/MM calculations unravel an unusual mechanism for Compound I-mediated aldehyde formation in which the active site water derived from H2O2 activation is involved in the generation of a four-membered ring lactone intermediate. These findings provide new insights into the unusual mechanisms of CYP152 peroxygenases.
- Jiang, Yuanyuan,Li, Shengying,Li, Zhong,Peng, Wei,Tang, Dandan,Wang, Binju,You, Cai,Zhao, Yue
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supporting information
p. 24694 - 24701
(2021/10/14)
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- (2R)- and (2S)- 2-hydroxy- hexanoyl and octanoyl-L-homoserine lactones: New highly potent Quorum Sensing modulators with opposite activities
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The synthesis and the QS modulation activity of diastereoisomerically pure 2-hydroxy-N-acyl-L-homoserine lactones (2-OH-AHLs) are unveiled. (2R)- and (2S)- 2-hydroxy-N-hexanoyl-L-homoserine lactone and 2-hydroxy-N-octanoyl-L-homoserine lactone have been identified as very potent QS agonists and antagonists on the Vibrio fischeri-quorum sensing system with opposite activities depending on the configuration of the carbon atom with the hydroxyl group. Flexible molecular docking showed that the (2R)–OH configuration in the antagonist isomer induces new hydrogen bonds with Tyr70 and Asp79, two importantly conserved residues in the LuxR protein family, while the (2S)–OH agonist configuration exhibits a binding mode comparable to the natural ligand 3-oxo-hexanoyl-L-homoserine lactone (OHHL). For the analogs with long alkyl chain 3a and 3b and aromatic analogs, all are antagonists with no effect of the configuration at C-2.
- Jeanneau, Erwann,Queneau, Yves,Soulère, Laurent,Zhang, Qiang
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- P450Jα: A New, Robust and α-Selective Fatty Acid Hydroxylase Displaying Unexpected 1-Alkene Formation
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Oxyfunctionalization of fatty acids (FAs) is a key step in the design of novel synthetic pathways for biobased/biodegradable polymers, surfactants and fuels. Here, we show the isolation and characterization of a robust FA α-hydroxylase (P450Jα) which catalyses the selective conversion of a broad range of FAs (C6:0-C16:0) and oleic acid (C18:1) with H2O2 as oxidant. Under optimized reaction conditions P450Jα yields α-hydroxy acids all with >95 % regioselectivity, high specific activity (up to 15.2 U mg?1) and efficient coupling of oxidant to product (up to 85 %). Lauric acid (C12:0) turned out to be an excellent substrate with respect to productivity (TON=394 min?1). On preparative scale, conversion of C12:0 reached 83 % (0.9 g L?1) when supplementing H2O2 in fed-batch mode. Under similar conditions P450Jα allowed further the first biocatalytic α-hydroxylation of oleic acid (88 % conversion on 100 mL scale) at high selectivity and in good yields (1.1 g L?1; 79 % isolated yield). Unexpectedly, P450Jα displayed also 1-alkene formation from shorter chain FAs (≤C10:0) showing that oxidative decarboxylation is more widely distributed across this enzyme family than reported previously.
- Armbruster, Julia,Steinmassl, Mathilde,Müller Bogotá, Christina A.,Berg, Gabriele,Nidetzky, Bernd,Dennig, Alexander
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p. 15910 - 15921
(2020/10/29)
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- Light-Driven Kinetic Resolution of α-Functionalized Carboxylic Acids Enabled by an Engineered Fatty Acid Photodecarboxylase
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Chiral α-functionalized carboxylic acids are valuable precursors for a variety of medicines and natural products. Herein, we described an engineered fatty acid photodecarboxylase (CvFAP)-catalyzed kinetic resolution of α-amino acids and α-hydroxy acids, which provides the unreacted R-configured substrates with high yields and excellent stereoselectivity (ee up to 99 %). This efficient light-driven process requires neither NADPH recycling nor prior preparation of esters, which were required in previous biocatalytic approaches. The structure-guided engineering strategy is based on the scanning of large amino acids at hotspots to narrow the substrate binding tunnel. To the best of our knowledge, this is the first example of asymmetric catalysis by an engineered CvFAP.
- Xu, Jian,Hu, Yujing,Fan, Jiajie,Arkin, Mamatjan,Li, Danyang,Peng, Yongzhen,Xu, Weihua,Lin, Xianfu,Wu, Qi
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supporting information
p. 8474 - 8478
(2019/05/24)
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- Preparative Asymmetric Synthesis of Canonical and Non-canonical a-amino Acids through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids
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Chemical and biocatalytic synthesis of non-canonical a-amino acids (ncAAs) from renewable feedstocks and using mild reaction conditions has not efficiently been solved. Here, we show the development of a three-step, scalable and modular one-pot biocascade for linear conversion of renewable fatty acids (FAs) into enantiopure l-a-amino acids. In module 1, selective a-hydroxylation of FAs is catalyzed by the P450 peroxygenase P450CLA. By using an automated H2O2 supplementation system, efficient conversion (46 to >99%; TTN>3300) of a broad range of FAs (C6:0 to C16:0) into valuable a-hydroxy acids (a-HAs; >90% a-selective) is shown on preparative scale (up to 2.3 gL1 isolated product). In module 2, a redox-neutral hydrogen borrowing cascade (alcohol dehydrogenase/amino acid dehydrogenase) allowed further conversion of a-HAs into l-a-AAs (20 to 99%). Enantiopure l-a-AAs (e.e. >99%) including the pharma synthon l-homo-phenylalanine can be obtained at product titers of up to 2.5 gL1. Based on renewables and excellent atom economy, this biocascade is among the shortest and greenest synthetic routes to structurally diverse and industrially relevant ncAAs.
- Dennig, Alexander,Blaschke, Fabio,Gandomkar, Somayyeh,Tassano, Erika,Nidetzky, Bernd
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supporting information
p. 1348 - 1358
(2019/10/28)
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- Preparative Asymmetric Synthesis of Canonical and Non-canonical α-amino Acids Through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids
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Chemical and biocatalytic synthesis of non-canonical α-amino acids (ncAAs) from renewable feedstocks and using mild reaction conditions has not efficiently been solved. Here, we show the development of a three-step, scalable and modular one-pot biocascade for linear conversion of renewable fatty acids (FAs) into enantiopure l-α-amino acids. In module 1, selective α-hydroxylation of FAs is catalyzed by the P450 peroxygenase P450CLA. By using an automated H2O2 supplementation system, efficient conversion (46 to >99%; TTN>3300) of a broad range of FAs (C6:0 to C16:0) into valuable α-hydroxy acids (α-HAs; >90% α-selective) is shown on preparative scale (up to 2.3 g L?1 isolated product). In module 2, a redox-neutral hydrogen borrowing cascade (alcohol dehydrogenase/amino acid dehydrogenase) allowed further conversion of α-HAs into l-α-AAs (20 to 99%). Enantiopure l-α-AAs (e.e. >99%) including the pharma synthon l-homo-phenylalanine can be obtained at product titers of up to 2.5 g L?1. Based on renewables and excellent atom economy, this biocascade is among the shortest and greenest synthetic routes to structurally diverse and industrially relevant ncAAs. (Figure presented.).
- Dennig, Alexander,Blaschke, Fabio,Gandomkar, Somayyeh,Tassano, Erika,Nidetzky, Bernd
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supporting information
(2019/02/09)
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- Structural and catalytic properties of the peroxygenase P450 enzyme CYP152K6 from Bacillus methanolicus
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The CYP152 family of cytochrome P450 enzymes (P450s or CYPs) are bacterial peroxygenases that use hydrogen peroxide to drive hydroxylation and decarboxylation of fatty acid substrates. We have expressed and purified a novel CYP152 family member – CYP152K6 from the methylotroph Bacillus methanolicus MGA3. CYP152K6 was characterized using spectroscopic, analytical and structural methods. CYP152K6, like its peroxygenase counterpart P450SPα (CYP152B1) from Sphingomonas paucimobilis, does not undergo significant fatty acid-induced perturbation to the heme spectrum, with the exception of a minor Soret shift observed on binding dodecanoic acid. However, CYP152K6 purified from an E. coli expression system was crystallized and its structure was determined to 1.3 ? with tetradecanoic acid bound. No lipids were present in conditions used for crystallogenesis, and thus CYP152K6 must form a complex by incorporating the fatty acid from E. coli cells. Turnover studies with dodecanoic acid revealed several products, with 2-hydroxydodecanoic acid as the major product and much smaller quantities of 3-hydroxydodecanoic acid. Secondary turnover products were undec-1-en-1-ol, 2-hydroxydodec-2-enoic acid and 2,3-dihydroxydodecanoic acid. This is the first report of a 2,3-hydroxylated fatty acid product made by a peroxygenase P450, with the dihydroxylated product formed by CYP152K6-catalyzed 3-hydroxylation of 2-hydroxydodecanoic acid, but not by 2-hydroxylation of 3-hydroxydodecanoic acid.
- Girvan, Hazel M.,Poddar, Harshwardhan,McLean, Kirsty J.,Nelson, David R.,Hollywood, Katherine A.,Levy, Colin W.,Leys, David,Munro, Andrew W.
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- Succinct synthesis of saturated hydroxy fatty acids and: In vitro evaluation of all hydroxylauric acids on FFA1, FFA4 and GPR84
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Saturated hydroxy fatty acids make up a class of underexplored lipids with potentially interesting biological activities. We report a succinct and general synthetic route to saturated hydroxy fatty acids hydroxylated at position 6 or higher, and exemplify this with the synthesis of hydroxylauric acids. All regioisomers of hydroxylauric acids were tested on free fatty acid receptors FFA1, FFA4 and GPR84. The results show that the introduction of a hydroxy group and its position have a high impact on receptor activity.
- Kaspersen, Mads Holmgaard,Jenkins, Laura,Dunlop, Julia,Milligan, Graeme,Ulven, Trond
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supporting information
p. 1360 - 1365
(2017/07/07)
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- Role of the hydrophobic moiety of tumor promoters. Synthesis and activity of 2-alkylated benzolactams
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The size and position of a hydrophobic moiety on a benzolactam skeleton, which reproduces the active conformation and biological activity of teleocidins, play an important role in the appearance of the activity. Compounds with alkyl groups of various sizes and shapes at the 2-position of benzolactam were synthesized. Structure-activity results indicate that a hydrophobic substituent at the C-2 position plays a critical role in the appearance of biological activities, as in the case of substitution at C-9.
- Endo, Yasuyuki,Yokoyama, Akihiro
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- Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cδ by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation
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Phorbol esters (12-O-tetradecanoylphorbol 13-acetate; TPA) and teleocidins are known to be potent tumor promoters and to activate protein kinase C (PKC) by binding competitively to the enzyme. The relationship between the chemical structures and the activities of these compounds has attracted much attention because of the marked structural dissimilarities. The benzolactam 5, with an eight-membered lactam ring and benzene ring instead of the nine-membered lactam ring and indole ring of teleocidins, reproduces the active ring conformation and biological activities of teleocidins. Herein we describe the synthesis of benzolactams with hydrophobic substituents at various positions. Structure-activity data indicate that the existence of a hydrophobic region between C-2 and C-9 and the steric factor at C-8 play critical roles in the appearance of biological activities. We also computationally simulated the docking of teleocidin and the modified benzolactam molecules to the Cys2 domain structure observed in the crystalline complex of PKCδ with phorbol 13-acetate. Teleocidin and benzolactams fitted well into the same cavity as phorbol 13-acetate. Of the three functional groups hydrogenbonding to the protein, two hydrogen-bonded with protein atoms in common with phorbol 13-acetate, but the third one hydrogen-bonded with a different protein atom from that in the case of phorbol 13-acetate. The model explains well the remarkable difference in activity between 5 and its analogue having a bulky substituent at C-8.
- Endo, Yasuyuki,Takehana, Shunji,Ohno, Michihiro,Driedger, Paul E.,Stabel, Silvia,Mizutani, Miho Y.,Tomioka, Nobuo,Itai, Akiko,Shudo, Koichi
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p. 1476 - 1496
(2007/10/03)
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- Role of the hydrophobic moiety of tumor promoters. Synthesis and activity of benzolactams with alkyl substituents at various positions
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We synthesized benzolactams with hydrophobic substituents at various positions as analogs of (-)-benzolactam-V8-310 ((-)-BL-V8-310, 1) which reproduces the active conformation and biological activity of teleocidins. Structure-activity data indicate that the existence of a hydrophobic region between C-2 and C-9, and the steric factor at C-8 play critical roles in the appearance of biological activities.
- Endo, Yasuyuki,Ohno, Michihiro,Takehana, Shunji,Driedger, Paul E.,Stabel, Silvia,Shudo, Koichi
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p. 424 - 426
(2007/10/03)
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- Enantioselective α hydroxylation of carboxylic acids with molecular oxygen catalyzed by the α oxidation enzyme system of young pea leaves (Pisum sativum): A substrate selectivity study
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The substrate selectivity of the α oxidation of carboxylic acids 1 by crude homogenate of young pea leaves was investigated. Saturated fatty acids with 7 to 16 carbon atoms and oleic acid were transformed to the enantiomerically pure (R)-2-hydroxy acids 2 in the presence of molecular oxygen.
- Adam, Waldemar,Lazarus, Michael,Saha-Moeller, Chantu R.,Schreier, Peter
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p. 2287 - 2292
(2007/10/03)
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- Synthesis of S-Methyl 2-Hydrxyalkanethioates, 2-Hydroxyalkanoic Acids and Related Compounds via the Addition Reaction of Tris(methylthio)methanide Ion to Alkanals
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In connection with the studies on biological activities on myrmicacin and related compounds, the synthetic method for 2-hydroxyalkanoic acids and the corresponding 1,2-diols was studied.The addition reaction of tris(methylthio)methyllithium to the aldehydes (propanal-dodecanal) gave the corresponding 1,1,-tris(methylthio)-2-alkanols 1a-j.Treatment of 1 with mercury(II) chloride-mercury(II) oxide in water-acetone afforded S-methyl 2-hydroxyalkanethioates 2a-j, and in methanol methyl 2-hydroxyalkanoates 3c-j were obtained.Reduction of the thioates 2 with lithium aluminium hydride gave 1,2-diols 4c-j and saponification produced the corresponding 2-hydroxyalkanoic acids 5c-j.
- Orito, Kazuhiko,Seki, Yoshikatsu,Suginome, Hiroshi,Iwadare, Tsukasa
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p. 2013 - 2017
(2007/10/02)
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- New potential immunoenhancing compounds. Synthesis and pharmacological evaluation of new long-chain 2-amido-2-deoxy-D-glucose derivatives
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A series of long-chain fatty acids and the corresponding 2-hydroxy, 2-oxo, 3-hydroxy acid glucosamides were evaluated as immunomodulating compounds. In a preliminary screening, 2-[(2-ethoxycarbonyloxy)tetradecanoyl-amino]-2-deoxy-D-glucose (2b) and 2-(3-hydroxydodecanoyl-amino)-2-deoxy-D-glucose (5a) resulted to be the most effective in enhancing the glucosamine activity. The findings of in vitro-ex vivo tests (unidirectional mixed lymphocyte culture reaction and primary antibody production) and in vivo tests (delayed type hypersensitivity, protection against bacterial or fungal infection and against Sarcoma 180 or Lewis lung carcinoma transplants) were very encouraging and allowed to assume for the two substances a protective activity, presumably through the ability of activating phagocytic and NK cells.
- Valcavi,Albertoni,Brandt,Corsi,Farina,Foresta,Pascucci,Ramacci
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p. 1190 - 1195
(2007/10/02)
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- REACTIONS OF HALOGENOMAGNESIUM ALCOHOLATES WITH ACID ANHYDRIDES
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A method was developed for the synthesis of the esters of 2-acyloxy carboxylic acids.The method is based on the reaction of the halogenomagnesium alcoholates of the esters of aliphatic 2-hydroxycarboxylic acids with acid anhydrides.
- Kashinskii, V. N.
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p. 1880 - 1882
(2007/10/02)
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