Alkylresorcinol biosynthesis in plants: new insights from an ancient enzyme family?

Alkylresorcinols are members of an extensive family of bioactive compounds referred to as phenolic lipids, which occur primarily in plants, fungi, and bacteria. In plants, alkylresorcinols and their derivatives are thought to serve important roles as phytoanticipins and allelochemicals, although direct evidence for this is still somewhat lacking. Specialized type III polyketide synthases (referred to as 'alkylresorcinol synthases'), which catalyze the formation of 5-alkylresorcinols using fatty acyl-CoA starter units and malonyl-CoA extender units, have been characterized from several microbial species, however until very recently little has been known concerning their plant counterparts. Through the use of sorghum and rice EST and genomic data sets, significant inroads have now been made in this regard. Here we provide additional information concerning our recent report on the identification and characterization of alkylresorcinol synthases from Sorghum bicolor and Oryza sativa, as well as a brief consideration of the emergence of this intriguing subfamily of enzymes.

Alkylresorcinol synthases expressed in Sorghum bicolor root hairs play an essential role in the biosynthesis of the allelopathic benzoquinone sorgoleone.

Sorghum bicolor is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone, which likely accounts for many of the allelopathic properties of Sorghum spp. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells and involves the production of an alkylresorcinolic intermediate (5-[(Z,Z)-8',11',14'-pentadecatrienyl]resorcinol) derived from an unusual 16:3Delta(9,12,15) fatty acyl-CoA starter unit. This led to the suggestion of the involvement of one or more alkylresorcinol synthases (ARSs), type III polyketide synthases (PKSs) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. In an effort to characterize the enzymes responsible for the biosynthesis of the pentadecyl resorcinol intermediate, a previously described expressed sequence tag database prepared from isolated S. bicolor (genotype BTx623) root hairs was first mined for all PKS-like sequences. Quantitative real-time RT-PCR analyses revealed that three of these sequences were preferentially expressed in root hairs, two of which (designated ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units in recombinant enzyme studies. Furthermore, RNA interference experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 are likely to participate in the biosynthesis of sorgoleone in planta. The sequences of ARS1 and ARS2 were also used to identify several rice (Oryza sativa) genes encoding ARSs, which are likely involved in the production of defense-related alkylresorcinols.

Prehospital versus periprocedural abciximab in ST-elevation myocardial infarction treated by percutaneous coronary intervention.

OBJECTIVE: To investigate the potential benefit of an earliest possible out-of-hospital start of abciximab (ReoPro) therapy in ST-elevation myocardial infarction (STEMI; Lilly, Bad Homburg, Germany) and planned primary percutaneous intervention compared with periprocedural abciximab treatment on reperfusion and clinical outcome. METHODS: Randomization of one hundred and one patients with STEMI to prehospital or periprocedural abciximab treatment. Evaluation of thrombolysis in myocardial infarction (TIMI) flow, ST-segment resolution, myocardial blush grade, and maximal creatine kinase release before and after as well as clinical follow-up until 6 months after the index event. RESULTS: Prehospital abciximab (group 1) was initiated a median of 101 min (37-165 min) earlier compared with periprocedural treatment (group 2). Initial TIMI 3 flow (24 vs. 15%, P=NS), ST-segment resolution before percutaneous coronary intervention (PCI) (<30%: 33 vs. 46%, P=NS; >70%: 38 vs. 33%, P=NS), post-PCI myocardial blush grade 2 and 3 (72 vs. 75%, P=NS), maximal cardiac enzyme release (creatinine kinase MB median 77 U/l; range 33-137 vs. 74 U/l; range 39-143 U/l, P=NS), and 6 months follow-up (recurrent myocardial infarction or repeat coronary intervention, and PCI, need for coronary bypass surgery) did not differ significantly between both treatment groups. CONCLUSION: Prehospital intravenous administration of abciximab, although safe and feasible in a trained surrounding, does not add angiographic or clinical benefit to patients with STEMI.

A functional genomics investigation of allelochemical biosynthesis in Sorghum bicolor root hairs.

Sorghum is considered to be one of the more allelopathic crop species, producing phytotoxins such as the potent benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(Z,Z)-8',11',14'-pentadecatriene]-p-benzoquinone) and its analogs. Sorgoleone likely accounts for much of the allelopathy of Sorghum spp., typically representing the predominant constituent of Sorghum bicolor root exudates. Previous and ongoing studies suggest that the biosynthetic pathway for this plant growth inhibitor occurs in root hair cells, involving a polyketide synthase activity that utilizes an atypical 16:3 fatty acyl-CoA starter unit, resulting in the formation of a pentadecatrienyl resorcinol intermediate. Subsequent modifications of this resorcinolic intermediate are likely to be mediated by S-adenosylmethionine-dependent O-methyltransferases and dihydroxylation by cytochrome P450 monooxygenases, although the precise sequence of reactions has not been determined previously. Analyses performed by gas chromatography-mass spectrometry with sorghum root extracts identified a 3-methyl ether derivative of the likely pentadecatrienyl resorcinol intermediate, indicating that dihydroxylation of the resorcinol ring is preceded by O-methylation at the 3'-position by a novel 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase activity. An expressed sequence tag data set consisting of 5,468 sequences selected at random from an S. bicolor root hair-specific cDNA library was generated to identify candidate sequences potentially encoding enzymes involved in the sorgoleone biosynthetic pathway. Quantitative real time reverse transcription-PCR and recombinant enzyme studies with putative O-methyltransferase sequences obtained from the expressed sequence tag data set have led to the identification of a novel O-methyltransferase highly and predominantly expressed in root hairs (designated SbOMT3), which preferentially utilizes alk(en)ylresorcinols among a panel of benzene-derivative substrates tested. SbOMT3 is therefore proposed to be involved in the biosynthesis of the allelochemical sorgoleone.

Methylation of sulfhydryl groups: a new function for a family of small molecule plant O-methyltransferases.

In plants, type I and II S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) catalyze most hydroxyl group methylations of small molecules. A homology-based RT-PCR strategy using Catharanthus roseus (Madagascar periwinkle) RNA previously identified six new type I plant OMT family members. We now describe the molecular and biochemical characterization of a seventh protein. It shares 56-58% identity with caffeic acid OMTs (COMTs), but it failed to methylate COMT substrates, and had no activity with flavonoids. However, the in vitro incubations revealed unusually high background levels without added substrates. A search for the responsible component revealed that the enzyme methylated dithiothreitol (DTT), the reducing agent added for enzyme stabilization. Unexpectedly, product analysis revealed that the methylation occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34 compounds indicated a broad substrate range, with a preference for small hydrophobic molecules. Benzene thiol (Km 220 microm) and furfuryl thiol (Km 60 microm) were the best substrates (6-7-fold better than DTT). Small isosteric hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also methylated, but the activities were at least 5-fold lower than with thiols. The enzyme was named C. roseus S-methyltransferase 1 (CrSMT1). Models based on the COMT crystal structure suggest that S-methylation is mechanistically identical to O-methylation. CrSMT1 so far is the only recognized example of an S-methyltransferase in this protein family. Its properties indicate that a few changes in key residues are sufficient to convert an OMT into a S-methyltransferase (SMT). Future functional investigations of plant methyltransferases should consider the possibility that the enzymes may direct methylation at sulfhydryl groups.

Entrapment of pacemaker lead by a large net-like Eustachian valve within the right atrium.

During pacemaker implantation in a patient with permanent atrial fibrillation, it remained impossible to advance a passive fixation lead with fins through the right atrium. However, a lead with a retractable screw easily passed the right atrium and was positioned in the right ventricle. Transesophageal echocardiography revealed an extensive net-like perforated Eustachian valve within the right atrium that had caused entrapment of the anchor fins during lead implantation. Remnants of embryonal structures within the right atrium should be considered a rare possible barrier during pacemaker implantation.

Early versus periprocedural administration of abciximab for primary angioplasty: a pooled analysis of 6 studies.

BACKGROUND: The 2004 ACC/AHA guidelines on ST-elevation myocardial infarction state that it is reasonable to start treatment with abciximab as early as possible before primary percutaneous coronary intervention (PCI). We investigated the potential benefit of early use of abciximab by pooling data from all the available studies. METHODS: Six prospective studies were identified that had allocated 260 patients to receive early abciximab (either prehospital or soon after the patient arrived in hospital) and 342 to receive late abciximab (at the time of PCI). RESULTS: TIMI flow grade 2 or 3 was present in 42% of the early group compared with 29% in the late group (P = .001). After PCI, 59% of patients in the early group showed ST-resolution >or = 70%, compared with 41% in the late group (P = .003). The composite clinical outcomes death, new myocardial infarction, or repeat target vessel revascularization at 30 days occurred in 7.3% of the early group compared with 9.7% in the late group (odds ratio 0.73, 95% CI 0.41-1.32) and death alone occurred in 2.7% versus 4.7%, respectively (odds ratio 0.56, 95% CI 0.23-1.39). CONCLUSIONS: Early administration of abciximab improves epicardial patency (TIMI flow) before PCI and results in better myocardial tissue perfusion (ST-resolution) after the procedure. The promising effects on clinical outcomes need to be tested in larger studies.

Unusual pseudosubstrate specificity of a novel 3,5-dimethoxyphenol O-methyltransferase cloned from Ruta graveolens L.

A cDNA was cloned from Ruta graveolens cells encoding a novel O-methyltransferase (OMT) with high similarity to orcinol or chavicol/eugenol OMTs, but containing a serine-rich N-terminus and a 13 amino acid insertion between motifs IV and V. Expression in Escherichia coli revealed S-adenosyl-l-methionine-dependent OMT activity with methoxylated phenols only with an apparent Km of 20.4 for the prime substrate 3,5-dimethoxyphenol. The enzyme forms a homodimer of 84 kDa, and the activity was insignificantly affected by 2.0 mM Ca2+ or Mg2+, whereas Fe2+, Co2+, Zn2+, Cu2+ or Hg2+ were inhibitory (78-100%). Dithiothreitol (DTT) suppressed the OMT activity. This effect was examined further, and, in the presence of Zn2+ as a potential thiol methyltransferase (TMT) cofactor, the recombinant OMT methylated DTT to DTT-monomethylthioether. Sets of kinetic OMT experiments with 3,5-dimethoxyphenol at various Zn2+/DTT concentrations revealed the competitive binding of DTT with an apparent Ki of 52.0 microM. Thus, the OMT exhibited TMT activity with almost equivalent affinity to the thiol pseudosubstrate which is structurally unrelated to methoxyphenols.

An aldol switch discovered in stilbene synthases mediates cyclization specificity of type III polyketide synthases.

Stilbene synthase (STS) and chalcone synthase (CHS) each catalyze the formation of a tetraketide intermediate from a CoA-tethered phenylpropanoid starter and three molecules of malonyl-CoA, but use different cyclization mechanisms to produce distinct chemical scaffolds for a variety of plant natural products. Here we present the first STS crystal structure and identify, by mutagenic conversion of alfalfa CHS into a functional stilbene synthase, the structural basis for the evolution of STS cyclization specificity in type III polyketide synthase (PKS) enzymes. Additional mutagenesis and enzymatic characterization confirms that electronic effects rather than steric factors balance competing cyclization specificities in CHS and STS. Finally, we discuss the problematic in vitro reconstitution of plant stilbenecarboxylate pathways, using insights from existing biomimetic polyketide cyclization studies to generate a novel mechanistic hypothesis to explain stilbenecarboxylate biosynthesis.

Flavonoid methylation: a novel 4'-O-methyltransferase from Catharanthus roseus, and evidence that partially methylated flavanones are substrates of four different flavonoid dioxygenases.

Catharanthus roseus (Madagascar periwinkle) flavonoids have a simple methylation pattern. Characteristic are B-ring 5' and 3' methylations and a methylation in the position 7 of the A-ring. The first two can be explained by a previously identified unusual O-methyltransferase (CrOMT2) that performs two sequential methylations. We used a homology based RT-PCR strategy to search for cDNAs encoding the enzyme for the A-ring 7 position. Full-length cDNAs for three proteins were characterized (CrOMT5, CrOMT6, CrOMT7). The deduced polypeptides shared 59-66% identity among each other, with CrOMT2, and with CrOMT4 (a previously characterized protein of unknown function). The five proteins formed a cluster separate from all other OMTs in a relationship tree. Analysis of the genes showed that all C. roseus OMTs had a single intron in a conserved position, and a survey of OMT genes in other plants revealed that this intron was highly conserved in evolution. The three cDNAs were cloned for expression of His-tagged recombinant proteins. CrOMT5 was insoluble, but CrOMT6 and CrOMT7 could be purified by affinity chromatography. CrOMT7 was inactive with all compounds tested. The only substrates found for CrOMT6 were 3'-O-methyl-eriodictyol (homoeriodictyol) and the corresponding flavones and flavonols. The mass spectrometric analysis showed that the enzyme was not the expected 7OMT, but a B-ring 4'OMT. OMTs with this specificity had not been described before, and 3',4'-dimethylated flavonoids had not been found so far in C. roseus, but they are well-known from other plants. The identification of this enzyme activity raised the question whether methylation could be a part of the mechanisms channeling flavonoid biosynthesis. We investigated four purified recombinant 2-oxoglutarate-dependent flavonoid dioxygenases: flavanone 3beta-hydroxylase, flavone synthase, flavonol synthase, and anthocyanidin synthase. 3'-O-Methyl-eriodictyol was a substrate for all four enzymes. The activities were only slightly lower than with the standard substrate naringenin, and in some cases much higher than with eriodictyol. Methylation in the A-ring, however, strongly reduced or abolished the activities with all four enzymes. The results suggested that B-ring 3' methylation is no hindrance for flavonoid dioxygenases. These results characterized a new type of flavonoid O-methyltransferase, and also provided new insights into the catalytic capacities of key dioxygenases in flavonoid biosynthesis.

Covalent binding of chloroacetamide herbicides to the active site cysteine of plant type III polyketide synthases.

Chloroacetamide herbicides inhibit very-long-chain fatty acid elongase, and it has been suggested that covalent binding to the active site cysteine of the condensing enzyme is responsible [Pest Manage Sci 56 (2000), 497], but direct evidence was not available. The proposal implied that other condensing enzymes might also be targets, and therefore we have investigated four purified recombinant type III plant polyketide synthases. Chalcone synthase (CHS) revealed a high sensitivity to the chloroacetamide metazachlor, with 50% inhibition after a 10 min pre-incubation with 1-2 molecules per enzyme subunit, and the inactivation was irreversible. Stilbene synthase (STS) inactivation required 20-fold higher amounts, and 4-coumaroyltriacetic acid synthase and pyrone synthase revealed no response at the highest metazachlor concentrations tested. A similar spectrum of differential responses was detected with other herbicides that also inhibit fatty acid elongase (metolachlor and cafenstrole). The data indicate that type III polyketide synthases are potential targets of these herbicides, but each combination has to be investigated individually. The interaction of metazachlor with CHS was investigated by mass spectrometric peptide mapping, after incubation of the enzymes with the herbicides followed by tryptic digestion. A characteristic mass shift and MS/MS sequencing of the respective peptide showed that metazachlor was covalently bound to the cysteine of the active site, and the same was found with STS. This is the first direct evidence that the active site cysteine in condensing enzymes is the primary common target of these herbicides.

Stilbenecarboxylate biosynthesis: a new function in the family of chalcone synthase-related proteins.

Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.

A flavonol O-methyltransferase from Catharanthus roseus performing two sequential methylations.

Protein extracts from dark-grown cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) contained several O-methyltransferase (OMT) activities, including the 16-hydroxytabersonine O-methyltransferase (16HT-OMT) in indole alkaloid biosynthesis. This enzyme was enriched through several purification steps, including affinity chromatography on adenosine agarose. SDS-PAGE of the purified protein preparation revealed a protein band at the size expected for plant OMTs (38-43 kDa). Mass spectrometry indicated two dominant protein species of similar mass in this band, and sequences of tryptic peptides showed similarities to known OMTs. Homology-based RT-PCR identified cDNAs for four new OMTs. Two of these cDNAs (CrOMT2 and CrOMT4) encoded the proteins dominant in the preparation enriched for 16HT-OMT. The proteins were closely related (73% identity), but both shared only 48-53% identity with the closest relatives found in the public databases. The enzyme functions were investigated with purified recombinant proteins after cDNA expression in Escherichia coli. Unexpectedly, both proteins had no detectable 16HT-OMT activity, and CrOMT4 was inactive with all substrates investigated. CrOMT2 was identified as a flavonoid OMT that was expressed in dark-grown cell cultures and copurified with 16HT-OMT. It represented a new type of OMT that performs two sequential methylations at the 3'- and 5'-positions of the B-ring in myricetin (flavonol) and dihydromyricetin (dihydroflavonol). The resulting methylation pattern is characteristic for C. roseus flavonol glycosides and anthocyanins, and it is proposed that CrOMT2 is involved in their biosynthesis.

Predicting the substrates of cloned plant O-methyltransferases.

Plant O-methyltransferases (OMTs) have important roles in secondary metabolite biosynthesis. Sequencing projects and homology-based cloning strategies yield sequences for proteins with similarities to known OMTs, but the identification of the physiological substrates is not trivial. We investigated with a cDNA cloned from Catharanthus roseus the possibilities for predicting the substrates of OMTs, using the information from previous work and two newly identified motifs that were based on information from the crystal structures of two plant OMTs. The results, confirmed by functional analysis of the recombinant protein, indicated that a careful analysis of the deduced protein sequence can provide clues for predicting the substrates of cloned OMTs.