- Effect of flexible linker length on the activity of fusion protein 4-coumaroyl-CoA ligase::stilbene synthase
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In order to elucidate the effect of flexible linker length on the catalytic efficiency of fusion proteins, two short flexible peptide linkers of various lengths were fused between Arabidopsis thaliana 4-coumaroyl-CoA ligase (4CL) and Polygonum cuspidatum stilbene synthase (STS) to generate fusion proteins 4CL-(GSG)n-STS (n ≤ 5) and 4CL-(GGGGS)n-STS (n ≤ 4). The fusion proteins were expressed in both Escherichia coli and Saccharomyces cerevisiae, and their bioactivities were tested in vitro and in vivo using purified proteins and engineered strains, respectively. The catalytic efficiency of the fusions decreased gradually with the increase of GSG or GGGGS repeats. In both engineered S. cerevisiae and E. coli in vivo experiments, the capacity of resveratrol production decreased gradually with increasing linker length. In silico analysis showed that the prediction of homology models of fusion proteins was consistent with the in vitro and in vivo results.
- Guo, Huili,Yang, Yadong,Xue, Feiyan,Zhang, Hong,Huang, Tiran,Liu, Wenbin,Liu, Huan,Zhang, Fenqiang,Yang, Mingfeng,Liu, Chunmei,Lu, Heshu,Zhang, Yansheng,Ma, Lanqing
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- Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase
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To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 A of each other provides the basis for enhanced in vivo synthesis of resveratrol.
- Wang, Yechun,Yi, Hankuil,Wang, Melissa,Yu, Oliver,Jez, Joseph M.
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- Facile enzymic synthesis of caffeoyl CoA
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p-Hydroxycinnamic acid:CoA ligase (EC 6.2.1.12) was extracted from aspen xylem and its activity with several cinnamic acids was determined. The highest activity was found with p-hydroxycinnamic acid and the extract had reasonable activity with ferulic, ca
- Meng, Huabin,Campbell, Wilbur H.
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- Cloning and functional characterization of two 4-coumarate: CoA ligase genes from selaginella moellendorffii
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Selaginella is an extant lycopodiophyte genus, which is representative of an ancient lineage of tracheophytes. The important evolutionary status makes it a valuable resource for the study of metabolic evolution in vascular plants. 4-coumarate: CoA ligase
- Liu, Xin-Yan,Wang, Ping-Ping,Wu, Yi-Feng,Cheng, Ai-Xia,Lou, Hong-Xiang
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- 4-Coumarate:coenzyme A ligase isoform 3 from Piper nigrum (Pn4CL3) catalyzes the CoA thioester formation of 3,4-methylenedioxycinnamic and piperic acids
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Black pepper, dried green fruit of Piper nigrum L., is a household spice most popular in the world. Piperine, the pungency compound of black pepper, is proposed to partially arise from phenylpropanoid pathway. In the biosynthesis of piperine, 4-coumarate:CoA ligase (4CLs) must play a pivotal role in activating intermediate acids to corresponding CoA thioesters to serve as substrates. Based on transcriptome data, we isolated three P. nigrum 4CL isoforms (Pn4CL1, -2, and -3) from unripe peppercorn. These Pn4CLs were expressed in E. coli for in vitro enzyme assay with putative substrates, namely cinnamic, coumaric, ferulic, piperonylic, 3,4-methylenedioxycinnamic (3,4-MDCA), and piperic acids. Phylogenetic analysis and substrate usage study indicated that Pn4CL1, active towards coumaric and ferulic acids, belongs to class I 4CL for lignin synthesis. Pn4CL2 was a typical cinnamate-specific coumarate:CoA ligase-like (CLL) protein. The Pn4CL3, as class II enzyme, exhibited general 4CL activity towards coumaric and ferulic acids. However, Pn4CL3 was also active towards piperonylic acid, 3,4-MDCA, and piperic acid. Pn4CL3 possessed ~2.6 times higher catalytic efficiency (kcat/KM) towards 3,4-MDCA and piperic acid than towards coumaric and ferulic acids, suggesting its specific role in piperine biosynthesis. Different substrate preference among the Pn4CL isoforms can be explained by 3-dimensional protein structure modeling, which demonstrated natural variants in amino acid residues of binding pocket to accommodate different substrates. Quantitative PCR analysis of these isoforms indicated that Pn4CL1 transcript level was highest in the roots whereas Pn4CL2 in the fruits and Pn4CL3 in the leaves.
- Jin, Zhehao,Wungsintaweekul, Juraithip,Kim, Sang-Hoon,Kim, Jeong-Han,Shin, Yongho,Ro, Dae-Kyun,Kim, Soo-Un
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- Chemoenzymatic synthesis and biological evaluation for bioactive molecules derived from bacterial benzoyl coenzyme a ligase and plant type III polyketide synthase
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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 ?, 1.7 ?, and 2.13 ?, 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.
- Adhikari, Kamal,Chen, Chun-Liang,Li, Tsung-Lin,Li, Yi-Shan,Lin, Kuan-Hung,Lo, I.-Wen,Rattinam, Rajesh,Wang, Yung-Lin,Wu, Chang-Jer,Zadeh, Saeid Malek
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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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- 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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- Characterization and functional analysis of 4-coumarate:CoA ligase genes in mul-berry
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A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. n
- Wang, Chuan-Hong,Yu, Jian,Cai, Yu-Xiang,Zhu, Pan-Pan,Liu, Chang-Ying,Zhao, Ai-Chun,Lü, Rui-Hua,Li, Meng-Jiao,Xu, Feng-Xiang,Yu, Mao-De
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- FERULOYL-CoA:MONOLIGNOL TRANSFERASE
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The invention relates to nucleic acids encoding a feruloyl-CoA:monolignol transferase and the feruloyl-CoA:monolignol transferase enzyme that enables incorporation of monolignol ferulates, for example, including p-coumaryl ferulate, coniferyl ferulate, and sinapyl ferulate, into the lignin of plants.
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Paragraph 0159
(2013/08/15)
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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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- Identification of a 4-coumarate:CoA ligase gene family in the moss, Physcomitrella patens
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Since the early evolution of land plants from primitive green algae, phenylpropanoid compounds have played an important role. In the biosynthesis of phenylpropanoids, 4-coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role at the divergence point from general phenylpropanoid metabolism to several major branch pathways. Although higher plant 4CLs have been extensively studied, little information is available on the enzymes from bryophytes. In Physcomitrella patens, we have identified a 4CL gene family consisting of four members, taking advantage of the available EST sequences and a draft sequence of the P. patens genome. The encoded proteins of three of the genes display similar substrate utilization profiles with highest catalytic efficiency towards 4-coumarate. Interestingly, the efficiency with cinnamate as substrate is in the same range as with caffeate and ferulate. The deduced proteins of the four genes share sequence identities between 78% and 86%. The intron/exon structures are pair wise similar. Pp4CL2 and Pp4CL3 each consists of four exons and three introns, whereas Pp4CL1 and Pp4CL4 are characterized each by five exons and four introns. Pp4CL1, Pp4CL2 and Pp4CL3 are expressed in both gametophore and protonema tissue of P. patens, unlike Pp4CL4 whose expression could not be demonstrated under the conditions employed. Phylogenetic analysis suggests an early evolutionary divergence of Pp4CL gene family members. Using Streptomyces coelicolor cinnamate:CoA ligase (ScCCL) as an outgroup, the P. patens 4CLs are clearly separated from the spermatophyte proteins, but are intercalated between the angiosperm 4CL class I and class II. A comparison of three P. patens subspecies from diverse geographical locations shows high sequence identities for the four 4CL isoforms.
- Silber, Martina V.,Meimberg, Harald,Ebel, Juergen
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p. 2449 - 2456
(2008/12/23)
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- Combinatorial mutasynthesis of flavonoid analogues from acrylic acids in microorganisms
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Flavonoids are plant secondary metabolites often used as nutraceutical supplements, but a growing number of unnatural flavonoids are being investigated as therapeutic agents. Cultures of Saccharomyces cerevisiae expressing recombinant flavonoid enzymes, including 4-coumaroyl: CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3β-hydroxylase (FHT), produced novel flavanones and dihydroflavonois when fed with a number of aromatic acrylic acids. The flavonoid network also exhibited broad substrate specificity by converting muconic acid into a unique polypropanoid.
- Chemler, Joseph A.,Yan, Yajun,Leonard, Effendi,Koffas, Mattheos A. G.
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p. 1855 - 1858
(2008/02/02)
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- Biosynthesis of curcuminoids and gingerols in turmeric (Curcuma longa) and ginger (Zingiber officinale): Identification of curcuminoid synthase and hydroxycinnamoyl-CoA thioesterases
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Members of the Zingiberaceae such as turmeric (Curcuma longa L.) and ginger (Zingiber officinale Rosc.) accumulate at high levels in their rhizomes important pharmacologically active metabolites that appear to be derived from the phenylpropanoid pathway. In ginger, these compounds are the gingerols; in turmeric these are the curcuminoids. Despite their importance, little is known about the biosynthesis of these compounds. This investigation describes the identification of enzymes in the biosynthetic pathway leading to the production of these bioactive natural products. Assays for enzymes in the phenylpropanoid pathway identified the corresponding enzyme activities in protein crude extracts from leaf, shoot and rhizome tissues from ginger and turmeric. These enzymes included phenylalanine ammonia lyase, polyketide synthases, p-coumaroyl shikimate transferase, p-coumaroyl quinate transferase, caffeic acid O-methyltransferase, and caffeoyl-CoA O-methyltransferase, which were evaluated because of their potential roles in controlling production of certain classes of gingerols and curcuminoids. All crude extracts possessed activity for all of these enzymes, with the exception of polyketide synthases. The results of polyketide synthase assays showed detectable curcuminoid synthase activity in the extracts from turmeric with the highest activity found in extracts from leaves. However, no gingerol synthase activity could be identified. This result was explained by the identification of thioesterase activities that cleaved phenylpropanoid pathway CoA esters, and which were found to be present at high levels in all tissues, especially in ginger tissues. These activities may shunt phenylpropanoid pathway intermediates away from the production of curcuminoids and gingerols, thereby potentially playing a regulatory role in the biosynthesis of these compounds.
- Ramirez-Ahumada, Maria del Carmen,Timmermann, Barbara N.,Gang, David R.
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p. 2017 - 2029
(2008/02/12)
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- Characterization in vitro and in vivo of the putative multigene 4-coumarate:CoA ligase network in Arabidopsis: Syringyl lignin and sinapate/sinapyl alcohol derivative formation
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A recent in silico analysis revealed that the Arabidopsis genome has 14 genes annotated as putative 4-coumarate:CoA ligase isoforms or homologues. Of these, 11 were selected for detailed functional analysis in vitro, using all known possible phenylpropanoid pathway intermediates (p-coumaric, caffeic, ferulic, 5-hydroxyferulic and sinapic acids), as well as cinnamic acid. Of the 11 recombinant proteins so obtained, four were catalytically active in vitro, with fairly broad substrate specificities, confirming that the 4CL gene family in Arabidopsis has only four members. This finding is in agreement with our previous phylogenetic analyses, and again illustrates the need for comprehensive characterization of all putative 4CLs, rather than piecemeal analysis of selected gene members. All 11 proteins were expressed with a C-terminal His 6-tag and functionally characterized, with one, At4CL1, expressed in native form for kinetic property comparisons. Of the 11 putative His 6-tagged 4CLs, isoform At4CL1 best utilized p-coumaric, caffeic, ferulic and 5-hydroxyferulic acids as substrates, whereas At4CL2 readily transformed p-coumaric and caffeic acids into the corresponding CoA esters, while ferulic and 5-hydroxyferulic acids were converted quite poorly. At4CL3 also displayed broad substrate specificity efficiently converting p-coumaric, caffeic and ferulic acids into their CoA esters, whereas 5-hydroxyferulic acid was not as effectively utilized. By contrast, while At4CL5 is the only isoform capable of ligating sinapic acid, the two preferred substrates were 5-hydroxyferulic and caffeic acids. Indeed, both At4CL1 and At4CL5 most effectively utilized 5-hydroxyferulic acid with kenz ~ 10-fold higher than that for At4CL2 and At4CL3. The remaining seven 4CL-like homologues had no measurable catalytic activity (at ~100 μg protein concentrations), again bringing into sharp focus both the advantages to, and the limitations of, current database annotations, and the need to unambiguously demonstrate true enzyme function. Lastly, although At4CL5 is able to convert both 5-hydroxyferulic and sinapic acids into the corresponding CoA esters, the physiological significance of the latter observation in vitro was in question, i.e. particularly since other 4CL isoforms can effectively convert 5-hydroxyferulic acid into 5-hydroxyferuloyl CoA. Hence, homozygous lines containing T-DNA or enhancer trap inserts (knockouts) for 4cl5 were selected by screening, with Arabidopsis stem sections from each mutant line subjected to detailed analyses for both lignin monomeric compositions and contents, and sinapate/sinapyl alcohol derivative formation, at different stages of growth and development until maturation. The data so obtained revealed that this "knockout" had no significant effect on either lignin content or monomeric composition, or on the accumulation of sinapate/sinapyl alcohol derivatives. The results from the present study indicate that formation of syringyl lignins and sinapate/sinapyl alcohol derivatives result primarily from methylation of 5-hydroxyferuloyl CoA or derivatives thereof rather than sinapic acid ligation. That is, no specific physiological role for At4CL5 in direct sinapic acid CoA ligation could be identified. How the putative overlapping 4CL metabolic networks are in fact organized in planta at various stages of growth and development will be the subject of future inquiry.
- Costa, Michael A.,Bedgar, Diana L.,Moinuddin, Syed G.A.,Kim, Kye-Won,Cardenas, Claudia L.,Cochrane, Fiona C.,Shockey, Jay M.,Helms, Gregory L.,Amakura, Yoshiaki,Takahashi, Hironobu,Milhollan, Jessica K.,Davin, Laurence B.,Browse, John,Lewis, Norman G.
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p. 2072 - 2091
(2008/02/03)
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- QUINOLIZIDINE ALKALOIDS AND THE ENZYMATIC SYNTHESIS OF THEIR CINNAMIC AND HYDROXYCINNAMIC ACID ESTERS IN LUPINUS ANGUSTIFOLIUS AND L. LUTEUS
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The accumulation pattern of quinolizidine alkaloids during development of seedings and young plants of Lupinus angustifolius and L. luteus are described.The enzymes catalysing the syntheses of the cinnamic acid O-ester of 13-hydroxylupanine and hydroxycinnamic acid O-esters of lupinine (4-coumaroyl- and feruloyllupinine) are characterized and classified as cinnamoyl-CoA:13-hydroxylupanine O-cinnamoyltransferase (EC 2.3.1.-) and hydroxy cinnamoyl-CoA:lupinine O-hydroxycinnamoyltransferase (EC 2.3.1.-). Key Word Index - Lupinus angustifolius; L. luteus; Fabaceae; quinolizidine alkaloids; hydroxycinnamic acid esters; acyltransferase; hydroxycinnamoyl-coenzyme A; biosynthesis.
- Strack, Dieter,Becher, Andrea,Brall, Sabine,Witte, Ludger
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p. 1493 - 1498
(2007/10/02)
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- SYNTHESE DE PARAHYDROXYTHIOCINNAMATES DE S-PHENYLE PRECURSEURS D'ESTERS DE S-COA
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S-phenyl 4-hydroxycinnamates are synthesized in good yield from the corresponding acids activated by iminium or pyridinium salts.Prior protection of the phenolic group is unnecessary; competitive Michael addition are generally unobserved.By exchange reaction with CoASH, S-phenyl esters give S-CoA cinnamoylesters which are involved in lignin biosynthesis process.
- Duran,Elisabeth,Duran, Hubert,Cazaux, Louis,Gorrichon, Liliane,Tisnes, Pierre,Sarni, Farid
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p. 143 - 148
(2007/10/02)
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