3-Hydroxybenzoate:coenzyme A ligase and 4-coumarate: coenzyme A ligase from cultured cells of Centaurium erythraea

3-Hydroxybenzoate:coenzyme A ligase, an enzyme involved in xanthone biosynthesis, was detected in cell-free extracts from cultured cells of Centaurium erythraea Rafn. The enzyme was separated from 4-coumarate:coenzyme A ligase by fractionated ammonium sulphate precipitation and hydrophobic interaction chromatography. The CoA ligases exhibited different substrate specificities. 3-Hydroxybenzoate:coenzyme A ligase activated 3-hydroxybenzoic acid most efficiently and lacked affinity for cinnamic acids. In contrast, 4-coumarate:CoA ligase mainly catalyzed the activation of 4-coumaric acid but did not act on benzoic acids. The two enzymes were similar with respect to their relative molecular weight, their pH and temperature optima, their specific activity and the changes in their activity during cell culture growth. Received: 23 September 1996 / Accepted: 28 November 1996     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

Identification of 4-coumarate:coenzyme A ligase (4CL) substrate recognition domains

4-coumarate:CoA ligase (4CL), the last enzyme of the general phenylpropanoid pathway, provides precursors for the biosynthesis of a large variety of plant natural products. 4 CL catalyzes the formation of CoA thiol esters of 4-coumarate and other hydroxycinnamates in a two step reaction involving the formation of an adenylate intermediate. 4 CL shares conserved peptide motifs with diverse adenylate-forming enzymes such as firefly luciferases, non-ribosomal peptide synthetases, and acyl:CoA synthetases. Amino acid residues involved in 4 CL catalytic activities have been identified, but domains involved in determining substrate specificity remain unknown. To address this question, we took advantage of the difference in substrate usage between the Arabidopsis thaliana 4 CL isoforms At4CL1 and At4CL2. While both enzymes convert 4-coumarate, only At4CL1 is also capable of converting ferulate. Employing a domain swapping approach, we identified two adjacent domains involved in substrate recognition. Both substrate binding domain I (sbd I) and sbd II of At4CL1 alone were sufficient to confer ferulate utilization ability upon chimeric proteins otherwise consisting of At4CL2 sequences. In contrast, sbd I and sbd II of At4CL2 together were required to abolish ferulate utilization in the context of At4CL1. Sbd I corresponds to a region previously identified as the substrate binding domain of the adenylation subunit of bacterial peptide synthetases, while sbd II centers on a conserved domain of so far unknown function in adenylate-forming enzymes (GEI/LxIxG). At4CL1 and At4CL2 differ in nine amino acids within sbd I and four within sbd II, suggesting that these play roles in substrate recognition.     

Stable and specific expression of 4-coumarate:coenzyme A ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco

The ability of 4-coumarate:coenzyme A ligase promoter from Populus tomentosa (Pto4CL1p) to drive expression of the GUS reporter gene and 4-coumarate:coenzyme A ligase gene in tobacco has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Intense GUS histochemical staining was detected in the xylem of stem in transgenic tobacco plants carrying the 1140 bp Pto4CL1p promoter. To further investigate the regulation function of the tissue-specific expression promoter, Pto4CL1p, a binary vector containing Pto4CL1p promoter fused with 4CL1 gene was transferred into tobacco. The activity of the 4CL1 enzyme doubled in the stems of transgenic tobacco but did not increase in the leaves. The content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

苯丙氨酸代谢途径关键酶:PAL、C4H、4CL研究新进展

主要阐述了苯丙氨酸代谢途径中三种关键酶:苯丙氨酸解氨酶(PAL)、肉桂酸4-羟基化酶(C4H)、4-香豆酸辅酶A连接酶(4CL)的研究进展,希望能为研究植物次生代谢途径的研究工作者提供一些帮助。     

Enzymatic synthesis and purification of aromatic coenzyme a esters

Two recombinant His-tagged proteins, a plant 4-coumarate:coenzyme A ligase (EC 6.2.1.12) and a bacterial benzoate:coenzyme A ligase (EC 6.2.1.25), were expressed in Escherichia coli and purified in a single step using Ni-chelating chromatography. Purified enzymes were used to synthesize cinnamoyl-coenzyme A (CoA), p-coumaroyl-CoA, feruloyl-CoA, caffeoyl-CoA, and benzoyl-CoA. Conversions up to 95% were achieved. Using a rapid solid-phase extraction procedure, the target CoA esters were isolated with yields of up to 80%. Structures were confirmed by analytical comparison with chemically synthesized reference compounds and electrospray ionization-mass spectrometry. The recombinant enzymes were stable for several months at -80 degrees C, thus providing a reliable and facile method to produce these delicate biological intermediates.     

Positive selection drives adaptive diversification of the 4‐coumarate: CoA ligase (4CL) gene in angiosperms

Lignin and flavonoids play a vital role in the adaption of plants to a terrestrial environment. 4‐Coumarate: coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for both lignin and flavonoids biosynthesis. However, very little is known about how such essential enzymatic functions evolve and diversify. Here, we analyze 4CL sequence variation patterns in a phylogenetic framework to further identify the evolutionary forces that lead to functional divergence. The results reveal that lignin‐biosynthetic 4CLs are under positive selection. The majority of the positively selected sites are located in the substrate‐binding pocket and the catalytic center, indicating that nonsynonymous substitutions might contribute to the functional evolution of 4CLs for lignin biosynthesis. The evolution of 4CLs involved in flavonoid biosynthesis is constrained by purifying selection and maintains the ancestral role of the protein in response to biotic and abiotic factors. Overall, our results demonstrate that protein sequence evolution via positive selection is an important evolutionary force driving adaptive diversification in 4CL proteins in angiosperms. This diversification is associated with adaption to a terrestrial environment.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K_m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ～85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ～64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Purification and substrate specifities of hydroxycinnamate: CoA ligase from anthocyanin-containing and anthocyanin-free carrot cells

Callus cells of Daucus carota L. have different phenylpropanoid pathways depending on the medium composition. Cells propagated on a medium with gibberellic acid do not accumulate cyanidin but incorporate [¹⁴C]phenylalanine into chlorogenic acid at a high rate. Cells grown on a medium free of gibberellic acid accumulate cyanidin in very large amounts. We here describe partial purification of hydroxycinnamate: CoA ligase, and its properties in these two cell lines. The enzymes extracted from the two cell populations had different substrate specifities: for that from anthocyanin-containing cells, p-coumaric acid was the best substrate, and caffeic acid and ferulic acid were also activated. With enzyme from anthocyanin-free cells, the lowest K_m values were obtained for caffeic acid, while ferulic acid had higher values, and p-coumaric acid was nearly inactive. The enzyme did not separate into isoenzymes during purification. Only on polyacrylamide gels the partially purified enzyme from anthocyanin-containing cells separated into three peaks, and that from anthocyanin-free cells, into only two peaks. This difference is discussed in the context of the lack of activity with p-coumaric acid in anthocyanin-free cells.Abbreviations GA₃ gibberellic acid     

Interaction of salicylate and ibuprofen with the carboxylic acid: CoA ligases from bovine liver mitochondria

Neither salicylate nor ibuprofen was a substrate or inhibitor of the long-chain fatty acid: CoA ligase. In contrast, all three xenobiotic-metabolizing medium-chain fatty acid:CoA ligases (XL-I, XL-II, and XL-III) had activity toward salicylate. The K_m value for salicylate was similar for all three forms (2 to 3 μM), but XL-II and XL-III had higher activity at V_max. For ibuprofen, only XL-III catalyzed its activation, and it had a K_m for ibuprofen of 36 μM. Studies of salicylate inhibition of XL-I, XL-II, and XL-III revealed that it inhibited the benzoate activity of all three forms with K₁ values of ca. 2 μM, which is in agreement with the K_m values obtained with salicylate as substrate. Kinetic analysis revealed that salicylate conjugation by all three forms is characterized by substrate inhibition when salicylate exceeds ca. 20 μM. Substrate inhibition was more extensive with XL-I and XL-III. Previous work on the ligases employed assay concentrations of salicylate in the range of 0.1 to 1.0 mM, which are clearly inhibitory, particularly toward XL-I and XL-III. Thus, activity was not properly measured in previous studies, which accounts for the fact that salicylate conjugation was only found with one form, which is most likely XL-II since it has the highest V_max activity and shows the least amount of substrate inhibition. Studies with ibuprofen indicated that it inhibited XL-I, XL-II, and XL-III, with K₁ values being in the range of 75–125 μM. The short-chain ligase was inhibited by both salicylate and ibuprofen with K₁ values of 93 and 84 μM, respectively. It was concluded that pharmacological doses of salicylate, but not ibuprofen, will affect the metabolism of medium-chain fatty acids and carboxylic acid xenobiotics and that the previously described mitochondrial ibuprofen:CoA ligase activity is attributable to XL-III. © 1996 John Wiley & Sons, Inc.     

Differential Properties of 4-Coumarate: CoA Ligase Related to Growth Suppression by Chalcone in Maize and Rice

Lignin and related metabolites have diverse and important functions for plant growth and development. 4-Coumarate: CoA ligase (4CL, EC 6.2.1.12) is one of the key enzymes in phenylpropanoid metabolism and lignin biosynthesis. In a previous study, maize (Zea maize L. cv. Yellowcorn) growth was suppressed to a greater extent by root-applied chalcone than rice (Oryza sativa L. cv. Nipponbare). The objective of this study is to clarify the relationship between the growth suppression and 4CL properties. In crude extracts, total 4CL activity and total protein content of rice were higher 1.8- and 2.7-fold than that of maize, respectively. After a gel-filtration chromatography, a single peak of 4CL activity from maize and rice was evident coincidently for both species. After anion-exchange chromatography, a single peak of 4CL activity was also apparent for both species; however, the peak of maize did not coincide with that of rice. The enzyme activity of maize and rice exhibited similar order of substrate specificities when using p-coumaric, cinnamic, caffeic, ferulic and sinapic acids substrates. Chalcone inhibited 4CL activity in maize more strongly than in rice, and 4CL kinetic data in the presence and absence of chalcone exhibited uncompetitive inhibition in both maize and rice. These results suggest that total activity and the inhibitory property of 4CL contributes to differences in growth suppression by chalcone between maize and rice, although further efforts are needed to clarify the potential of 4CL as a novel action site of the growth suppression.     

烟草4CL蛋白免疫荧光定位研究

4-香豆酸辅酶A连接酶(4CL)是维管植物木质素生物合成途径的关键酶,应用原核表达系统获得了毛白杨可溶性4CL1融合蛋白,以Ni2 -Agrose亲和柱层析纯化得到的SDS-PAGE电泳纯的毛白杨4CL1融合蛋白为抗原,免疫家兔获得毛白杨4CL1多克隆抗体,Western blotting鉴定表明兔抗毛白杨4CL1多克隆抗体具有高度特异性,免疫荧光定位发现普通烟草4CL1蛋白特异性地在木质部表达.为进一步应用木质部特异表达启动子定向调控木质素生物合成奠定了理论基础.     

Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate

The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate+CoA+ATP phenylacetyl-CoA+AMP+PP_i and requires Mg²⁺. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mol min^-1 x mg^-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mol min^-1 mg^-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 ±2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn²⁺ can substitute for Mg²⁺. The apparent K _m values for phenylacetate, CoA, and ATP are 60, 150, and 290 M, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzye was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.     

Two Divergent Members of 4-Coumarate： Coenzyme A Ligase from Salvia miltiorrhiza Bunge： cDNA Cloning and Functional Study

4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen （Salvia miltiorrhiza Bunge）, a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs （Sm4CL1 and Sm4CL2） encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction （RT-PCR） with degenerate primers followed by 5′/3′rapid amplification of cDNA ends （RACE） （Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164）. Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were （72.20±4.10） and （6.50±1.45） μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.     

Molecular cloning and functional analysis of (R)-3-hydroxyacyl-acyl carrier protein:coenzyme A transacylase from Pseudomonas mendocina LZ

An inactive (R)-3-hydroxyacyl-acyl carrier protein:coenzyme A transacylase (PhaG(Pm)) was cloned from a newly isolated Proteobacteria Pseudomonas mendocina LZ. It is the first characterized native inactive PhaG protein. Sequence analysis indicated that there were only two sites where the amino acid sequence differed between this inactive protein and the functional PhaG(Pp) from P. putida. The differences were located at position 78 and in the region 109-113 in the amino acid sequence. Mutagenesis was carried out to investigate these two sites. A recombinant strain harboring a S78C PhaG(Pp) mutant accumulated polyhydroxyalkanoates (PHA) at 11.9% of the cellular dry weight, as compared to the 21.6% PHA produced by the recombinant harboring the wild-type PhaG(Pp). On the other hand, the changes in the amino acid region 109-113 of PhaG(Pp) to its corresponding region of PhaG(Pm) resulted in negligible PHA accumulation. This demonstrated that region 109-113 in PhaG is relatively important for transacylase activity, while position 78 just plays a supporting role for the enzyme. Furthermore, 3-D structural models of PhaG(Pp) and PhaG(Pm) developed by computational prediction revealed that the variation in amino acids at 109-113 leads to the destruction of the PhaG catalytic center, resulting in the loss of enzyme activity.     

Reinvestigation of coenzyme Q10 isolation from Sporidiobolus johnsonii

There is considerable current interest in coenzyme Q10 (CoQ10) from a medical perspective. CoQ10 has been shown to alleviate the side effects of statin drugs, for instance, and so there is a push to find naturally high producers of the compound. Sporidiobolus johnsonii (S. johnsonii) has been reported to produce CoQ10 in studies that used only standards on thin‐layer chromatography (TLC) and also suggested the production of coenzyme Q9 (CoQ9). This work set out to verify CoQ9/CoQ10 production in S. johnsonii and quantify as appropriate. We show that S. johnsonii produces CoQ10 but found no evidence for CoQ9 biosynthesis. The specific production of CoQ10 was noted at 10 mg/g dry cell weight (DCW) in media supplemented with 4‐hydroxybenzoic acid (HBA). This makes S. johnsonii a naturally high CoQ10 producer. New methods for extraction and purification of CoQ10 are also discussed, and identification of a closely eluting side product under normal phase isolation is reported.