Publications

• Structural basis for specificity and flexibility in a plant 4-coumarate:CoA ligase.

  Li Z., Nair S.K.

  Plant 4-coumarate:CoA ligase (4CL) serves as a central catalyst in the phenylpropanoid pathway that provides precursors for numerous metabolites and regulates carbon flow. Here, we present several high-resolution crystal structures of Nicotiana tabacum 4CL isoform 2 (Nt4CL2) in complex with Mg(2+) ... >> More

  Plant 4-coumarate:CoA ligase (4CL) serves as a central catalyst in the phenylpropanoid pathway that provides precursors for numerous metabolites and regulates carbon flow. Here, we present several high-resolution crystal structures of Nicotiana tabacum 4CL isoform 2 (Nt4CL2) in complex with Mg(2+) and ATP, with AMP and coenzyme A (CoA), and with three different hydroxycinnamate-AMP intermediates: 4-coumaroyl-AMP, caffeoyl-AMP, and feruloyl-AMP. The Nt4CL2-Mg(2+)-ATP structure is captured in the adenylate-forming conformation, whereas the other structures are in the thioester-forming conformation. These structures represent a rare example of an ANL enzyme visualized in both conformations, and also reveal the binding determinants for both CoA and the hydroxycinnamate substrate. Kinetic studies of structure-based variants were used to identify residues crucial to catalysis, ATP binding, and hydroxycinnamate specificity. Lastly, we characterize a deletion mutant of Nt4CL2 that possesses the unusual sinapinate-utilizing activity. These studies establish a molecular framework for the engineering of this versatile biocatalyst. << Less

  Structure 23:2032-2042(2015) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase.

  Wang Y., Yi H., Wang M., Yu O., Jez J.M.

  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 h ... >> More

  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 Å of each other provides the basis for enhanced in vivo synthesis of resveratrol. << Less

  J. Am. Chem. Soc. 133:20684-20687(2011) [PubMed] [EuropePMC]

• The biosynthetic pathway of major avenanthramides in oat.

  Li Z., Chen Y., Meesapyodsuk D., Qiu X.

  Avenanthramides are a group of <i>N</i>-cinnamoylanthranilic acids, with health-promoting properties mainly found in oat (<i>Avena sativa</i> L.). However, the biosynthetic mechanism for the main three types of avenanthramides (Avn-A, Avn-B and Avn-C) is not completely understood. In the present s ... >> More

  Avenanthramides are a group of <i>N</i>-cinnamoylanthranilic acids, with health-promoting properties mainly found in oat (<i>Avena sativa</i> L.). However, the biosynthetic mechanism for the main three types of avenanthramides (Avn-A, Avn-B and Avn-C) is not completely understood. In the present study, we report molecular identification and functional characterization of three different types of genes from oat encoding 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl-CoA:hydroxyanthranilate <i>N</i>-hydroxycinnamoyl transferase (HHT) and a caffeoyl-CoA <i>O</i>-methyltransferase (CCoAOMT) enzymes, all involved in the biosynthesis of these avenanthramides. In vitro enzymatic assays using the proteins expressed in <i>Escherichia coli</i> showed that oat 4CL could convert <i>p</i>-coumaric acid, caffeic acid and ferulic acid to their CoA thioesters. Oat HHTs were only responsible for the biosynthesis of Avn-A and Avn-C using hydroxyanthranilic acid as an acyl acceptor and <i>p</i>-coumaroyl-CoA and caffeoyl-CoA as an acyl donor, respectively. Avn-B was synthesized by a CCoAOMT enzyme through the methylation of Avn-C. Collectively, these results have elucidated the molecular mechanisms for the biosynthesis of three major avenanthramides in vitro and paved the way for metabolic engineering of the biosynthetic pathway in heterologous systems to produce nutraceutically important compounds and make possible genetic improvement of this nutritional trait in oat through marker-assisted breeding. << Less

  Metabolites 9:0-0(2019) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Synthesis of p-coumaroyl coenzyme a with a partially purified p-coumarate:CoA ligase from cell suspension cultures of soybean (Glycine max).

  Lindl T., Kreuzaler F., Hahlbrock K.

  Biochim Biophys Acta 302:457-464(1973) [PubMed] [EuropePMC]

• Functional characterization of evolutionarily divergent 4-coumarate:coenzyme a ligases in rice.

  Gui J., Shen J., Li L.

  4-Coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) is a key enzyme in the phenylpropanoid metabolic pathways for monolignol and flavonoid biosynthesis. 4CL has been much studied in dicotyledons, but its function is not completely understood in monocotyledons, which display a different monolignol com ... >> More

  4-Coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) is a key enzyme in the phenylpropanoid metabolic pathways for monolignol and flavonoid biosynthesis. 4CL has been much studied in dicotyledons, but its function is not completely understood in monocotyledons, which display a different monolignol composition and phenylpropanoid profile. In this study, five members of the 4CL gene family in the rice (Oryza sativa) genome were cloned and analyzed. Biochemical characterization of the 4CL recombinant proteins revealed that the rice 4CL isoforms displayed different substrate specificities and catalytic turnover rates. Among them, Os4CL3 exhibited the highest turnover rate. No apparent tissue-specific expression of the five 4CLs was observed, but significant differences in their expression levels were detected. The rank in order of transcript abundance was Os4CL3 > Os4CL5 > Os4CL1 > Os4CL4 > Os4CL2. Suppression of Os4CL3 expression resulted in significant lignin reduction, shorter plant growth, and other morphological changes. The 4CL-suppressed transgenics also displayed decreased panicle fertility, which may be attributed to abnormal anther development as a result of disrupted lignin synthesis. This study demonstrates that the rice 4CLs exhibit different in vitro catalytic properties from those in dicots and that 4CL-mediated metabolism in vivo may play important roles in regulating a broad range of biological events over the course of rice growth and development. << Less

  Plant Physiol. 157:574-586(2011) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

  Klempien A., Kaminaga Y., Qualley A., Nagegowda D.A., Widhalm J.R., Orlova I., Shasany A.K., Taguchi G., Kish C.M., Cooper B.R., D'Auria J.C., Rhodes D., Pichersky E., Dudareva N.

  Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family th ... >> More

  Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway. << Less

  Plant Cell 24:2015-2030(2012) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.