- 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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- In vitro studies of maleidride-forming enzymes
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In vitro assays of enzymes involved in the biosynthesis of maleidrides from polyketides in fungi were performed. The results show that the enzymes are closely related to primary metabolism enzymes of the citric acid cycle in terms of stereochemical preferences, but with an expanded substrate selectivity. A key citrate synthase can react both saturated and unsaturated acyl CoA substrates to give solely anti substituted citrates. This undergoes anti-dehydration to afford an unsaturated precursor which is cyclised in vitro by ketosteroid-isomerase-like enzymes to give byssochlamic acid. This journal is
- Yin, Sen,Friedrich, Steffen,Hrupins, Vjaceslavs,Cox, Russell J.
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p. 14922 - 14931
(2021/05/19)
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- Coenzyme A-Conjugated Cinnamic Acids – Enzymatic Synthesis of a CoA-Ester Library and Application in Biocatalytic Cascades to Vanillin Derivatives
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We present a bioorthogonal method for the ligation of coenzyme A (CoA) with cinnamic acids. The reaction, which is the initial step in the biosynthesis of a multitude of bioactive secondary metabolites, is catalyzed by a promiscuous plant ligase and yields CoA conjugates with different functionalization in high purity and without formation of by-products. Its applicability in biosynthetic cascades is shown for the direct transformation of cinnamic acids into natural benzaldehydes (like vanillin) or artificial derivatives (e. g. ethylvanillin). (Figure presented.).
- Dippe, Martin,Bauer, Anne-Katrin,Porzel, Andrea,Funke, Evelyn,Müller, Anna O.,Schmidt, Jürgen,Beier, Maria,Wessjohann, Ludger A.
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supporting information
p. 5346 - 5350
(2019/11/29)
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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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- 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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- 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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- Extending carbon chain length of 1-butanol pathway for 1-hexanol synthesis from glucose by engineered Escherichia coli
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An Escherichia coli strain was engineered to synthesize 1-hexanol from glucose by extending the coenzyme A (CoA)-dependent 1-butanol synthesis reaction sequence catalyzed by exogenous enzymes. The C4-acyl-CoA intermediates were first synthesized via acetyl-CoA acetyltransferase (AtoB), 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt), and trans-enoyl-CoA reductase (Ter) from various organisms. The butyryl-CoA synthesized was further extended to hexanoyl-CoA via β-ketothiolase (BktB), Hbd, Crt, and Ter. Finally, hexanoyl-CoA was reduced to yield 1-hexanol by aldehyde/alcohol dehydrogenase (AdhE2). Enzyme activities for the C6 intermediates were confirmed by assays using HPLC and GC. 1-Hexanol was secreted to the fermentation medium under anaerobic conditions. Furthermore, co-expressing formate dehydrogenase (Fdh) from Candida boidinii increased the 1-hexanol titer. This demonstration of 1-hexanol production by extending the 1-butanol pathway provides the possibility to produce other medium chain length alcohols using the same strategy.
- Dekishima, Yasumasa,Lan, Ethan I.,Shen, Claire R.,Cho, Kwang Myung,Liao, James C.
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supporting information; experimental part
p. 11399 - 11401
(2011/10/04)
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- A three enzyme pathway for 2-amino-3-hydroxycyclopent-2-enone formation and incorporation in natural product biosynthesis
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A number of natural products contain a 2-amino-3-hydroxycyclopent-2-enone five membered ring, termed C5N, which is condensed via an amide linkage to a variety of polyketide-derived polyenoic acid scaffolds. Bacterial genome mining indicates three tandem ORFs that may be involved in C5N formation and subsequent installation in amide linkages. We show that the protein products of three tandem ORFs (ORF33-35) from the ECO-02301 biosynthetic gene cluster in Streptomyces aizunenesis NRRL-B-11277, when purified from Escherichia coli, demonstrate the requisite enzyme activities for C5N formation and amide ligation. First, succinyl-CoA and glycine are condensed to generate 5-aminolevulinate (ALA) by a dedicated PLP-dependent ALA synthase (ORF34). Then ALA is converted to ALA-CoA through an ALA-AMP intermediate by an acyl-CoA ligase (ORF35). ALA-CoA is unstable and has a half-life of ～10 min under incubation conditions for off-pathway cyclization to 2,5-piperidinedione. The ALA synthase can compete with the nonenzymatic decomposition route and act in a novel second transformation, cyclizing ALA-CoA to C5N. C 5N is then a substrate for the third enzyme, an ATP-dependent amide synthetase (ORF33). Using octatrienoic acid as a mimic of the C56 polyenoic acid scaffold of ECO-02301, formation of the octatrienyl-C 5N product was observed. This three enzyme pathway is likely the general route to the C5N ring system in other natural products, including the antibiotic moenomycin.
- Zhang, Wenjun,Bolla, Megan L.,Kahne, Daniel,Walsh, Christopher T.
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experimental part
p. 6402 - 6411
(2010/07/04)
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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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p. 2106 - 2114
(2007/10/03)
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- 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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p. 329 - 337
(2007/10/03)
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