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PubMed1997

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.

Cells, Cultured↗

PubMed1983

Subcellular distribution of cholic acid:coenzyme a ligase and deoxycholic acid:Coenzyme a ligase activities in rat liver.

Cholic acid:CoA ligase (EC 6.2.1.7, choloyl-CoA synthetase) and deoxycholic acid:CoA ligase catalyze the synthesis of choloyl-CoA and deoxycholoyl-CoA from their respective bile acids in rat liver. A modification of the phase partition assay was introduced which yields significantly (3-fold) higher specific activities for cholic acid:CoA ligase than previously reported. An independent method of separating choloyl-CoA from the substrates by high-pressure liquid chromatography was also developed and validates the modification. Both enzymic activities were found to be localized predominantly in the endoplasmic reticulum of rat liver. The level of either ligase in other purified, active subcellular fractions is consistent with the level of contamination by endoplasmic reticulum, estimated by using marker enzymes. Hence, the ligase assay can be used as a sensitive enzymic marker for endoplasmic reticulum in rat liver. The kinetic parameters of both enzymic activities were determined by using purified rough endoplasmic reticulum from rat liver. While the apparent maximal velocities for the two substrates are similar, the Michaelis constant for deoxycholate is significantly lower than that for cholate. Taurocholate and deoxycholate are shown to be competitive inhibitors of cholic acid:CoA ligase. The inhibition constant of deoxycholate is similar to its Michaelis constant for the deoxycholoyl-CoA-synthesizing reaction, suggesting that the same enzyme is responsible for both ligase activities.

Animals↗

PubMed1991

Purification and characterization of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp.

The enzymes catalyzing the formation of coenzyme A (CoA) thioesters of benzoate and 2-aminobenzoate were studied in a denitrifying Pseudomonas sp. anaerobically grown with these aromatic acids and nitrate as sole carbon and energy sources. Three different rather specific aromatic acyl-CoA ligases, E1, E2, and E3, were found which catalyze the formation of CoA thioesters of benzoate, fluorobenzoates, and 2-aminobenzoate. ATP is cleaved into AMP and pyrophosphate. The enzymes were purified, their N-terminal amino acid sequences were determined, and their catalytic and molecular properties were studied. Cells anaerobically grown on benzoate and nitrate contain one CoA ligase (AMP forming) for benzoic acid (E1). It is a homodimer of Mr 120,000 which prefers benzoate as a substrate but shows some activity also with 2-aminobenzoate and fluorobenzoates, although with lower Km. Cells anaerobically grown on 2-aminobenzoate and nitrate contain three different CoA ligases for aromatic acids. The first one is identical with benzoate-CoA ligase (E1). The second enzyme is a 2-aminobenzoate-CoA ligase (E2). It is a monomer of Mr 60,000 which prefers 2-aminobenzoate but also activates benzoate, fluorobenzoates and, less effectively, 2-methylbenzoate, with lower affinities to the latter substrates. The enzymes E1 and E2 have similar activity levels; a third minor CoA ligase activity is due to a different 2-aminobenzoate-CoA ligase. The enzyme (E3) is a monomer of Mr, 65,000 which 2-aminobenzoate pathway (U. Altenschmidt, C. Eckerskorn, and G. Fuchs, Eur. J. Biochem. 194:647-653, 1990); apparently, it is not completely repressed under anaerobic conditions and therefore also is induced to a small extent by 2-aminobenzoate under anaerobic growth conditions.

Amino Acid Sequence↗

PubMed1995

Benzoate-coenzyme A ligase, encoded by badA, is one of three ligases able to catalyze benzoyl-coenzyme A formation during anaerobic growth of Rhodopseudomonas palustris on benzoate.

The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase. This enzyme, purified from Rhodopseudomonas palustris, is maximally active with 5 microM benzoate. To study the molecular basis for this reaction, the benzoate-coenzyme A ligase gene (badA) was cloned and sequenced. The deduced amino acid sequence of badA showed substantial similarity to other coenzyme A ligases, with the highest degree of similarity being that to 4-hydroxybenzoate-coenzyme A ligase (50% amino acid identity) from R. palustris. A badA mutant that was constructed had barely detectable levels of ligase activity when cell extracts were assayed at 10 microM benzoate. Despite this, the mutant grew at wild-type rates on benzoate under laboratory culture conditions (3 mM benzoate), and mutant cell extracts had high levels of ligase activity when assayed at a high concentration of benzoate (1 mM). This suggested that R. palustris expresses, in addition to BadA, a benzoate-activating enzyme(s) with a relatively low affinity for benzoate. A possible role of 4-hydroxybenzoate-coenzyme A ligase (encoded by hbaA) in this capacity was investigated by constructing a badA hbaA double mutant. Although the double mutant grew more slowly on benzoate than badA cells, growth rates were still significant, suggesting the involvement of a third enzyme in benzoate activation. Competition experiments involving the addition of a small amount of cyclohexanecarboxylate to ligase assay mixtures implicated cyclohexanecarboxylate-coenzyme A ligase as being this third enzyme. These results show that wild-type R. palustris cells synthesize at least three enzymes that can catalyze the initial step in anaerobic benzoate degradation during growth on benzoate. This observation supports previous suggestions that benzoyl-coenzyme A formation plays a central role in anaerobic aromatic compound biodegradation.

Acyl Coenzyme A↗

PubMed1997

Acyl-adenylate motif of the acyl-adenylate/thioester-forming enzyme superfamily: a site-directed mutagenesis study with the Pseudomonas sp. strain CBS3 4-chlorobenzoate:coenzyme A ligase.

4-Chlorobenzoate:coenzyme A (4-CBA:CoA) ligase catalyzes 4-chlorobenzoyl-coenzyme A formation in a two-step reaction consisting of the adenylation of 4-chlorobenzoate with adenosine 5'-triphosphate followed by acyl transfer from the 4-chlorobenzoyl adenosine 5'-monophosphate diester intermediate to coenzyme A. In this study, two core motifs present in the Pseudomonas sp. strain CBS3 4-CBA:CoA ligase (motif I, 161T-S-G-T-T-G-L-P-K-G170, and motif II, 302Y-G-T-T-E306) and conserved among the sequences representing the acyl-adenylate/thioester-forming enzyme family (to which the ligase belongs) were tested for their possible role in substrate binding and/or catalysis. The site-directed mutants G163I, G166I, P168A, K169M, and E306Q were prepared and then subjected to steady-state and transient kinetic studies. The results, which indicate reduced catalysis of the adenylation of 4-chlorobenzoate in the mutant enzymes, are interpreted within the context of the three-dimensional structure of the acyl-adenylate/thioester-forming enzyme family member, firefly luciferase.

Adenosine Triphosphate↗

PubMed1996

Determination of the chemical pathway for 4-chlorobenzoate:coenzyme A ligase catalysis.

4-Chlorobenzoate:coenzyme A ligase (4-CBA:CoA ligase) catalyzes the first step of the 4-CBA degradation pathway of Pseudomonas sp. strain CBS3. In this reaction, 4-CBA-CoA thioester synthesis is coupled to ATP cleavage. The studies described in this paper examine the intermediacy of 4-chlorobenzoyl-adenosine 5'phosphate diester (4-CBA-AMP) in the ligase reaction. The 4-CBA-AMP adduct was isolated from the ligase reaction mixture generated from magnesium adenosine 5-triphosphate (MgATP) and 4-CBA in the absence of CoA. The structure of the 4-CBA-AMP was verified by 1H- 13C-, and 31P-nuclear magnetic resonance analysis. Single-turnover reactions carried out with 14C-labeled 4-CBA in a rapid quench apparatus demonstrated formation of the enzyme. 4-CBA-AMP.MgPPi complex from the enzyme.4-CBA.MgATP complex at a rate of 135 s-1. The rate of ligand release from the enzyme.4-CBA-AMP.MgPPi complex was measured at 0.013 s-1. Single-turnover reactions of [14C]-4-CBA, MgATP, and CoA catalyzed by the ligase revealed that the 4-CBA-AMP intermediate formed reaches a maximum level of 25% of the starting 4-CBA within 10 ms and then declines with the formation of the 4-CBA-CoA. The rates of the adenylation and thioesterification partial reactions, determined by kinetic simulation of the rate data, are nearly equal (135 and 100 s-1). Substitution of CoA with the slow substrate pantetheine did not significantly alter the rate of the adenylation step but did reduce the rate of the thioesterification step to 2 s-1. The maximum level of 4-CBA-AMP reached during the single-turnover reaction of 4-CBA, MgATP, and pantetheine corresponded to one-half of the starting 4-CBA.

Adenosine Monophosphate↗

PubMed1992

Evidence of multiple forms of rat liver microsomal coenzyme A ligase catalysing the formation of 2-arylpropionyl-coenzyme A thioesters.

This study has demonstrated the involvement of multiple forms of rat hepatic microsomal CoA ligases in the formation of 2-arylpropionyl-CoA thioesters. In the presence of (-)R-ibuprofen (0.1 microM-1 mM) two enzymic processes were observed, one of which exhibited enantiospecificity and apparent high affinity for the R enantiomer (Km 0.06 microM) whilst the second, a low-affinity component was non-enantiospecific. An equivalent high-affinity isoform catalysing R-flurbiprofen-CoA formation at concentrations less than 100 microM was not demonstrated. However, at higher substrate concentrations formation of both R- and S-flurbiprofenyl-CoA thioesters occurred. Marked inter-individual variation was observed in the formation of S-ibuprofen-CoA and S-flurbiprofen-CoA in the rats studied.

Animals↗

PubMed1994

4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation.

Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.

Anaerobiosis↗

PubMed1986

Characterization of rat brain microsomal acyl-coenzyme A ligases: different enzymes for the synthesis of palmitoyl-coenzyme A and lignoceroyl-coenzyme A.

Palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by microsomal membranes but lignoceric acid solubilized with Triton WR-1339 was not an effective substrate even though the detergent dispersed the same amount of these fatty acids and was also not inhibitory to the enzyme [I. Singh, R. P. Singh, A. Bhushan, and A. K. Singh (1985) Arch. Biochem. Biophys. 236, 418-426]. This observation suggested that palmitoyl-CoA and lignoceroyl-CoA may be synthesized by two different enzymes. We have solubilized the acyl-CoA ligase activities for palmitic and lignoceric acid of rat brain microsomal membranes with Triton X-100 and resolved them into three separate peaks (fractions) by hydroxylapatite chromatography. Fraction A (palmitoyl-CoA ligase) had high specific activity for palmitic acid and Fraction C (lignoceroyl-CoA ligase) for lignoceric acid. Specific activity of palmitoyl-CoA ligase for palmitic acid was six times higher than in Fraction C and specific activity of lignoceroyl-CoA ligase for lignoceric acid was four times higher than in Fraction A. At higher concentrations of Triton X-100 (0.5%), lignoceroyl-CoA ligase loses activity whereas palmitoyl-CoA ligase does not. Lignoceroyl-CoA ligase lost 60% of activity at 0.6% Triton X-100. Palmitoyl-CoA ligase (T1/2 of 4.5 min) is more stable at 40 degrees C than lignoceroyl-CoA ligase (T1/2 of 1.5 min). The pH optimum of palmitoyl-CoA ligase was 7.7 and that of lignoceroyl-CoA ligase was 8.4. Similar to our results with intact membranes, palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by palmitoyl-CoA ligase whereas lignoceric acid when solubilized with Triton WR-1339 was not able to act as substrate for lignoceroyl-CoA ligase. Since solubilized enzyme activities for synthesis of palmitoyl-CoA and lignoceroyl-CoA from microsomal membranes can be resolved into different fractions by column chromatography and demonstrate different properties, we suggest that in microsomal membranes palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes.

Acyl Coenzyme A↗

PubMed2001

Structure and evolution of 4-coumarate:coenzyme A ligase (4CL) gene families.

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in general phenylpropanoid metabolism. 4CL is related to a larger class of prokaryotic and eukaryotic adenylate-forming enzymes and shares several conserved peptide motifs with these enzymes. In order to better characterize the nature of 4CL gene families in poplar, parsley, and tobacco, we used degenerate primers to amplify 4CL sequences from these species. In each species additional, divergent 4CL genes were found. Complete cDNA clones for the two new poplar 4CL genes were obtained, allowing examination of their expression patterns and determination of the substrate utilization profile of a xylem-specific isoform. Phylogenetic analysis of these genes and gene fragments confirmed previous results showing that 4CL proteins fall into two evolutionarily ancient subgroups . A comparative phylogenetic analysis of enzymes in the adenylate-forming superfamily showed that 4CLs, luciferases, and acetate CoA ligases each form distinct clades within the superfamily. According to this analysis, four Arabidopsis 4CL-like genes identified from the Arabidopsis Genome Project are only distantly related to bona fide 4CLs or are more closely related to fatty acid CoA ligases, suggesting that the three Arabidopsis 4CL genes previously characterized represent the extent of the 4CL gene family in this species.

Adenosine Monophosphate↗

PubMed1979

Kinetic characterization of long chain fatty acyl coenzyme A ligase from rat liver mitochondria.

Long chain fatty acyl coenzyme A ligase (EC 6.2.1.3) purified from rat liver mitochondria has been characterized with respect to several kinetic parameters. Many of the kinetic properties of the mitochondrial enzyme are similar to those of the purified microsomal enzyme with respect to palmitoyl-CoA formation, but there are distinct differences. The fatty acid and nucleotide specificities of the mitochondrial enzyme are similar to those of the microsomal enzyme, as are the apparent Km values for ATP and coenzyme A. On the other hand, the mitochondrial enzyme differs from the microsomal enzyme in that it has a lower pH optimum, is different in molecular weight, and does not show simple saturation kinetics with palmitate as substrate. Of particular interest is the evidence presented which indicates that the mitochondrial long chain fatty acyl-CoA ligase, unlike short and medium chain ligases, does not utilize an acyladenylate as an intermediate in the formation of fatty acyl-CoA.

Animals↗

PubMed2003

Cinnamate:coenzyme A ligase from the filamentous bacterium streptomyces coelicolor A3(2).

4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins. Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium Streptomyces coelicolor A3(2). The recombinant enzyme, produced in Escherichia coli with a histidine tag at its N-terminal end, showed distinct 4CL activity. The optimum pH and temperature of the reaction were pH 8.0 and 30 degrees C, respectively. The K(m) value for 4-coumarate and k(cat) were determined as 131 +/- 4 micro M and 0.202 +/- 0.007 s(-1), respectively. The K(m) value was comparable to those of plant 4CLs. The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs. The enzyme efficiently converted cinnamate (K(m), 190 +/- 2 micro M; k(cat), 0.475 +/- 0.012 s(-1)), which is a very poor substrate for plant 4CLs. Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs. The enzyme was therefore named ScCCL for S. coelicolor A3(2) cinnamate CoA ligase. To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated. Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala(294) and Ala(318) are involved in substrate recognition. Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme.

Amino Acid Sequence↗

PubMed2002

Differential substrate inhibition couples kinetically distinct 4-coumarate:coenzyme a ligases with spatially distinct metabolic roles in quaking aspen.

4-Coumarate:coenzyme A ligase (4CL) activates hydroxycinnamates for entry into phenylpropanoid branchways that support various metabolic activities, including lignification and flavonoid biosynthesis. However, it is not clear whether and how 4CL proteins with their broad substrate specificities fulfill the specific hydroxycinnamate requirements of the branchways they supply. Two tissue-specific 4CLs, Pt4CL1 and Pt4CL2, have previously been cloned from quaking aspen (Populus tremuloides Michx.), but whether they are catalytically adapted for the distinctive metabolic roles they are thought to support is not apparent from published biochemical data. Therefore, single- and mixed-substrate assays were conducted to determine whether the 4CLs from aspen exhibit clear catalytic identities under certain metabolic circumstances. Recombinant Pt4CL1 and Pt4CL2 exhibited the expected preference for p-coumarate in single-substrate assays, but strong competitive inhibition favored utilization of caffeate and p-coumarate, respectively, in mixed-substrate assays. The Pt4CL1 product, caffeoyl-CoA, predominated in mixed-substrate assays with xylem extract, and this was consistent with the near absence of Pt4CL2 expression in xylem tissue as determined by in situ hybridization. It is interesting that the Pt4CL2 product p-coumaroyl-CoA predominated in assays with developing leaf extract, although in situ hybridization revealed that both genes were coexpressed. The xylem extract and recombinant 4CL1 data allow us to advance a mechanism by which 4CL1 can selectively utilize caffeate for the support of monolignol biosynthesis in maturing xylem and phloem fibers. Loblolly pine (Pinus taeda), in contrast, possesses a single 4CL protein exhibiting broad substrate specificity in mixed-substrate assays. We discuss these 4CL differences in terms of the contrasts in lignification between angiosperm trees and their gymnosperm progenitors.

Acyl Coenzyme A↗

PubMed2004

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.

Base Sequence↗

PubMed2005

Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates.

Rhodopseudomonas palustris strain RCB100 degrades 3-chlorobenzoate (3-CBA) anaerobically. We purified from this strain a coenzyme A ligase that is active with 3-CBA and determined its N-terminal amino acid sequence to be identical to that of a cyclohexanecarboxylate-CoA ligase encoded by aliA from the R. palustris strain (CGA009) that has been sequenced. Strain CGA009 differs from strain RCB100 in that it does not use 3-CBA as a sole carbon source. The aliA gene from the 3-CBA degrading strain differed by a single nucleotide from the aliA gene from strain CGA009, causing the substitution of a serine for a threonine at position 208. Both AliA enzymes, purified as His-tagged fusion proteins, had comparable activities with cyclohexanecarboxylate. However, AliA from the 3-CBA degrading strain was 10-fold more active with 3-CBA (kcat/Km of 4.3 x 10(4) M(-1) s(-1)) than the enzyme from the sequenced strain (kcat/Km 0.32 x 10(4) M(-1) s(-1)). The CGA009 enzyme was not sufficiently active with 3-CBA to complement an RCB100 aliA mutant for growth on this compound. Here, whole genome sequence information enabled us to identify a single nucleotide among 5.4 million nucleotides that contributes to the substrate preference of a coenzyme A ligase.

Acyl Coenzyme A↗

PubMed1997

Structural Characterization of Modified Lignin in Transgenic Tobacco Plants in Which the Activity of 4-Coumarate:Coenzyme A Ligase Is Depressed.

Transgenic tobacco (Nicotiana tabacum L.) plants in which the activity of 4-coumarate:coenzyme A ligase is very low contain a novel lignin in their xylem. Details of changes in hydroxycinnamic acids bound to cell walls and in the structure of the novel lignin were identified by base hydrolysis, alkaline nitrobenzene oxidation, pyrolysis-gas chromatography, and 13C-nuclear magnetic resonance analysis. In the brownish tissue of the transgenic plants, the levels of three hydroxycinnamic acids, p-coumaric, ferulic, and sinapic, which were bound to cell walls, were apparently increased as a result of down-regulation of the expression of the gene for 4-coumarate:coenzyme A ligase. Some of these hydroxycinnamic acids were linked to cell walls via ester and ether linkages. The accumulation of hydroxycinnamic acids also induced an increase in the level of condensed units in the novel lignin of the brownish tissue. Our data indicate that the behavior of some of the incorporated hydroxycinnamic acids resembles lignin monomers in the brownish tissue, and their accumulation results in dramatic changes in the biosynthesis of lignin in transgenic plants.

Journal Article↗

PubMed2003

4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates.

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5'-tetraphosphate (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5''',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5',5'''-P(1),P(5)-pentaphosphate and dAp(4)dA from the appropriate substrates, p(4)A and dATP, respectively. Formation of Ap(3)A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p(4)A and diadenosine 5',5''',-P(1),P(4)-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.

Adenine Nucleotides↗

PubMed2000

3-Hydroxybenzoate:coenzyme A ligase from cell cultures of Centaurium erythraea: isolation and characterization.

In xanthone biosynthesis, 3-hydroxybenzoate:coenzyme A ligase (3HBL) supplies the starter substrate for the formation of an intermediate benzophenone. 3HBL from cell cultures of the medicinal plant Centaurium erythraea was purified to apparent homogeneity using a seven-step-procedure. The enzyme was an AMP-forming CoA ligase with a Km = 14.7 microM for 3-hydroxybenzoic acid, 8.5 microM for coenzyme A and 229 microM for ATP. The pH and temperature optima were 7.5 and 35 degrees C, respectively. In SDS-PAGE, two polypeptides of Mr 41,500 and 40,500 were detected. Both proteins were structurally related to each other as shown by tryptic digestion. Their N-termini were blocked. The difference in their apparent molecular masses could not be attributed to glycosylation. 3HBL had a native Mr of approx. 50,000 and is thus active as a monomer.

Adenosine Monophosphate↗