Toluene monooxygenase from the fungus Cladosporium sphaerospermum

Assimilation of toluene by Cladosporium sphaerospermum is initially catalyzed by toluene monooxygenase (TOMO). TOMO activity was induced by adding toluene to a glucose-pregrown culture of C. sphaerospermum. The corresponding microsomal enzyme needed NADPH and O(2) to oxidize toluene and glycerol, EDTA, DTT, and PMSF for stabilization. TOMO activity was maximal at 35 degrees C and pH 7.5 and was inhibited by carbon monoxide, Metyrapone, and cytochrome c. TOMO preferred as substrates also other aromatic hydrocarbons with a short aliphatic side chain. Its reduced carbon monoxide difference spectrum showed a maximum at 451 nm. A substrate-induced Type I spectrum was observed on addition of toluene. These results indicated that TOMO is a cytochrome P450. TOMO and its corresponding reductase were eventually purified by a simultaneous purification revealing apparent molecular masses of 58 and 78 kDa, respectively.     

Acetogenic anthraquinones: biosynthetic convergence and chemical evidence of enzymatic cooperation in nature

Phenylanthraquinones belong to the quite rare class of fully unsymmetric biaryls, consisting of two different molecular portions, an anthraquinone part, chrysophanol, and a phenyl part, 2,4-dihydroxy-6-methoxyacetophenone, linked together by phenol-oxidative coupling. The biosynthesis of these two moieties, from eight and four acetate units, respectively, bears some unique features: Chrysophanol is the first example of an acetogenic natural product that is, in an organism-specific manner, formed via more than one folding mode: In eukaryotes, like, e.g., in fungi, in higher plants, and in insects, it is formed via folding mode F, while in prokaryotes it originates through mode S. It has, more recently, even been found to be synthesized by a third pathway, which we have named mode S′. It is thus the first example of biosynthetic convergence in polyketide biosynthesis. The monocyclic “southern” portion of the molecule, which is much simpler (arising from only four acetate units and without decarboxylation), unexpectedly does not show the anticipated randomization of the C₂-labeling in the aromatic ring, but has clearly localized C₂ units, excluding any symmetric intermediate like, e.g., 2,4,6-trihydroxyacetophenone. This is confirmed by competitive feeding experiments with specifically ¹³C₂-labeled acetophenones, showing the O-methylation to be the decisive symmetry-preventing step, which hints at a close collaboration of the participating enzymes. The results make knipholone an instructive example of structure, function, and evolution of polyketide synthases and O-methyltransferases, and their cooperation.

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.     

Monolignol biosynthesis in microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.)

Microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.) contained coniferaldehyde 5-hydroxylase activity and immunodetectable caffeic acid 3-O-methyltransferase (COMT), and catalyzed the S-adenosyl L-methionine (SAM) dependent methylation of caffeic acid, caffeyl aldehyde and caffeyl alcohol. When supplied with NADPH and SAM, the microsomes converted caffeyl aldehyde to coniferaldehyde, 5-hydroxyconiferaldehyde, and traces of sinapaldehyde. Coniferaldehyde was a better precursor of sinapaldehyde than was 5-hydroxyconiferaldehyde. The alfalfa microsomes could not metabolize 4-coumaric acid, 4-coumaraldehyde, 4-coumaroyl CoA, or ferulic acid. No metabolism of monolignol precursors was observed in microsomal preparations from transgenic alfalfa down-regulated in COMT expression. In most microsomal preparations, the level of the metabolic conversions was independent of added recombinant COMT. Taken together, the data provide only limited support for the concept of metabolic channeling in the biosynthesis of S monolignols via coniferaldehyde.     

PksS from Bacillus subtilis is a cytochrome P450 involved in bacillaene metabolism

As part of the pksX gene cluster of Bacillus subtilis strain 168, pksS has been preliminarily annotated as a cytochrome P450 homolog that hydroxylates the polyketide product of this cluster, which was recently shown to be involved in the biosynthesis of bacillaene and dihydrobacillaene. Here we report that there is a frame-shift error in the reported sequence for pksS, and that we have successfully cloned, overexpressed, and purified the protein encoded by the corrected sequence. By utilizing electronic absorption spectrophotometry, we have observed that the ferrous CO complex of PksS absorbs maximally near 450 nm, which confirms the annotation that this protein is a cytochrome P450. We have also established a cell-free system derived from crude cytosolic B. subtilis protein extracts which provides reductase activity essential to sustaining the putative catalytic cycle of PksS. Using LC-MS analysis we have collected data which suggests that the substrate for PksS is dihydrobacillaene.     

The biology of insecticidal activity and resistance

Identifying insecticide resistance mechanisms is paramount for pest insect control, as the understandings that underpin insect control strategies must provide ways of detecting and managing resistance. Insecticide resistance studies rely heavily on detailed biochemical and genetic analyses. Although there have been many successes, there are also many examples of resistance that still challenge us. As a precursor to rational pest insect control, the biology of the insect, within the contexts of insecticide modes of action and insecticide metabolism, must be well understood. It makes sense to initiate this research in the best model insect system, Drosophila melanogaster, and translate these findings and methodologies to other insects. Here we explore the usefulness of the D. melanogaster model in studying metabolic-based insecticide resistances, target-site mediated resistances and identifying novel insecticide targets, whilst highlighting the importance of having a more complete understanding of insect biology for insecticide studies.     

Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO₂) the recalcitrant fused-ring high molecular weight (?4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30 years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.     

Functional study of Capsicum annuum fatty acid desaturase 1 cDNA clone induced by Tobacco mosaic virus via microarray and virus-induced gene silencing

A series of microarray analyses employing the expressed sequence tags (ESTs) of hot pepper was conducted in an effort to elucidate the molecular mechanisms inherent to hypersensitive response (HR) by viral or bacterial pathogens. There were 2535 ESTs exhibiting differential expression (over 2-fold changes) among about 5000 ESTs during viral or bacterial response. Further, via virus-induced gene silencing (VIGS) and TMV-infection studies, we were able to isolate several ESTs, which may be relevant to defense response against TMV. Of these ESTs, Capsicum annuum fatty acid desaturase 1 (CaFAD1) showed the distinct phenotype against TMV infection and thus was subjected to further study. CaFAD1-silenced plants showed weaker resistance against TMV-P0 infection compared to TRV2 control plants. Also the suppression of FAD1 expression caused blocking of cell death induced by Bcl2-associated X (Bax) protein in tobacco plants. Therefore, this report presents that both microarray and VIGS approaches are feasible in hot pepper plants and the TMV-induced CaFAD1 plays a role in HR response.     

N-Methyltransferase involved in gramine biosynthesis in barley: cloning and characterization

The indole alkaloid gramine occurs in leaves of certain barley (Hordeum vulgare L.) cultivars but not in others. A gene sequence in barley that earlier was characterized as a jasmonate-induced O-methyltransferase (MT) (EC 2.1.1.6, GenBank accession U54767) was here found to be absent in some barley cultivars and breeding lines that all lacked gramine. The cDNA was cloned and expressed in Escherichia coli and the recombinant protein purified. The purified recombinant protein methylated two substrates in the pathway to gramine: 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI) at a high rate, with Km-values of 77 microM and 184 microM, respectively. In contrast, the protein did not exhibit any detectable methylation with the earlier suggested substrate for O-methylation, caffeic acid. A number of cultivars and breeding lines of barley were analyzed for presence of the U54767 gene sequence and MT protein and the enzyme activity in vitro with MAMI or caffeic acid as substrates. The results showed a clear relationship between the presence of the MT gene, the MT protein and N-methyltransferase activity, and confirmed the identification of the gene as coding for an N-methyltransferase (NMT, EC 2.1.1) and being involved in gramine biosynthesis.     

Lipid metabolism during aging of high-alpha-linolenate-phenotype potato tubers

Previous studies demonstrated that high levels of alpha-linolenate in cell membranes of potato tubers (achieved by overexpressing fatty acid desaturases) enhances lipid peroxidation, oxidative stress, and tuber metabolic rate, effectively accelerating the physiological age of tubers. This study details the changes in lipid molecular species of microsomal and mitochondrial membranes from wild-type (WT) and high-alpha-linolenate tubers during aging. The microsomal and mitochondrial polar lipids of high-alpha-linolenate tubers were dominated by 18:3/18:3 and 16:0/18:3 molecular species. Relative to WT tubers, high-alpha-linolenate tubers had a substantially higher 16:0/18:n to 18:n/18:n molecular species ratio in mitochondria and microsomes, potentially reflecting a compensatory response to maintain membrane biophysical properties in the face of increased unsaturation. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for 53 and 37% of polar lipids, respectively, in mitochondria from younger WT and high-alpha-linolenate tubers. The relative proportions of these phospholipids (PL) did not change during aging of WT tubers. In contrast, PE increased to dominate the PL pool of mitochondria during aging of high-alpha-linolenate tubers. While aging effected an increase in mitochondrial 18:3-bearing PCs and PEs in WT tubers, the concentration of 18:3-bearing PCs fell with a concomitant increase in 18:3-bearing PEs during aging of high-alpha-linolenate tubers. These age- and high-alpha-linolenate-induced changes had no effect on the respiration rate and functional integrity of isolated mitochondria. Differential increases in the respiration rates of WT and high-alpha-linolenate tubers during aging were therefore a consequence of unsaturation-dependent alterations in the microenvironments of cells. Microsomal 18:3-bearing PCs, PEs, digalactosyldiacylglycerols (DGDG), and monogalactosyldiacylglycerols all increased in WT tubers during aging. In contrast, a selective loss of 18:3-bearing PCs and DGDGs from microsomes of high-alpha-linolenate tubers likely reflects a greater susceptibility of membranes to peroxidative catabolism during aging. Aging resulted in an increase in sterol/PL ratio in microsomes from WT tubers, due primarily to a decline in PL. In high-alpha-linolenate tubers, the increase in sterol/PL ratio during aging was due to increases in Delta 5-avenasterol and stigmasterol, indicating membrane rigidification and likely contributing to increased membrane permeability. Age-induced changes in 18:3-bearing lipids in membranes of transformed tubers are discussed relative to the development of oxidative stress and accelerated aging.     

Effect of monoterpenes on lipid oxidation in maize

The monoterpenes 1,8-cineole, thymol, geraniol, menthol and camphor strongly inhibited the root growth of Zea mays L. seedlings. They induced an oxidative stress as measured by the production of malondialdehyde, conjugated dienes and peroxides. This oxidative stress depended on the length of the exposure and on the monoterpene applied. The total fatty acid content was measured and fatty acid composition was analyzed. Unsaturated fatty acids increased in the treated samples. The alcoholic and non-alcoholic monoterpenes appeared to have different modes of action.Abbreviations MDA Malondialdehyde - TFA Total fatty acid content - FA Fatty acid - IC₈₀ Concentration causing 80% inhibition     

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella)

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b₅ on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.     

Fatty acid desaturase-6 (Fad6) is required for salt tolerance in Arabidopsis thaliana

Fatty acid desaturases play important role in plant responses to abiotic stresses including cold, high temperature, drought, and osmotic stress. In this work, we provide the evidence that Fad6, a chloroplast desaturase, is required for salt tolerance during the early seedling development of Arabidopsis. Expression of Fad6 was responsive to salt and osmotic stress. Compared with the wild-type plants, the fad6 mutant showed reduced tolerance to salt stress, and accumulated more Na⁺ and less K⁺ under high NaCl stress condition. Furthermore, cellular oxidative damage was more severe in fad6 when treated with high concentrations of NaCl, as indicated by increased electrolyte leakage rate and malondialdehyde production, as well as by decreased activities of anti-oxidative enzymes. All these results suggest that Fad6 is required for salt resistance in Arabidopsis.     

A fatty acid elongase ELO with novel activity from Dictyostelium discoideum

Fatty acid elongation was examined in the cellular slime mold, Dictyostelium discoideum. Profiling of the total fatty acid content of D. discoideum indicated that fatty acid elongation is active. Orthologs of the fatty acid elongase ELO family were identified in the D. discoideum genome and the cDNA for one, eloA, was cloned and functionally characterized by expression in yeast. EloA is a highly active ELO with strict substrate specificity for monounsaturated fatty acids, in particular 16:1^Δ9 to produce the unusual 18:1^Δ11 fatty acid. This is the first report on fatty acid elongation in a cellular slime mold.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: Assessment of algal oils for biodiesel production

The objective of this study was to document and compare the lipid class and fatty acid composition of the green microalga Chlorella zofingiensis cultivated under photoautotrophic and heterotrophic conditions. Compared with photoautotrophic cells, a 900% increase in lipid yield was achieved in heterotrophic cells fed with 30 g L⁻¹ of glucose. Furthermore heterotrophic cells accumulated predominantly neutral lipids (NL) that accounted for 79.5% of total lipids with 88.7% being triacylglycerol (TAG); whereas photoautotrophic cells contained mainly the membrane lipids glycolipids (GL) and phospholipids (PL). Together with the much higher content of oleic acid (C18:1) (35.2% of total fatty acids), oils from heterotrophic C. zofingiensis appear to be more feasible for biodiesel production. Our study highlights the possibility of using heterotrophic algae for producing high quality biodiesel.     

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.     

Partial molar volumes of acyl carrier proteins are related to their states of acylation

Acyl carrier protein (ACP), an abundant protein in every cell, plays a central role in a number of metabolic processes requiring acyl group transfer. Conformational flexibility while crucial for its function remains substantially unaddressed. By dual polarization interferometry we establish correlation between the chain length of aliphatic groups covalently linked to Escherichia coli and Plasmodium falciparum ACP and their respective partial molar volumes in solution which helps to subserve the aforesaid goal.     

Drosophila melanogaster CYP6A8, an insect P450 that catalyzes lauric acid (omega-1)-hydroxylation

Only a handful of P450 genes have been functionally characterized from the approximately 90 recently identified in the genome of Drosophila melanogaster. Cyp6a8 encodes a 506-amino acid protein with 53.6% amino acid identity with CYP6A2. CYP6A2 has been shown to catalyze the metabolism of several insecticides including aldrin and heptachlor. CYP6A8 is expressed at many developmental stages as well as in adult life. CYP6A8 was produced in Saccharomyces cerevisiae and enzymatically characterized after catalytic activity was reconstituted with D. melanogaster P450 reductase and NADPH. Although several saturated or non-saturated fatty acids were not metabolized by CYP6A8, lauric acid (C12:0), a short-chain unsaturated fatty acid, was oxidized by CYP6A8 to produce 11-hydroxylauric acid with an apparent V(max) of 25 nmol/min/nmol P450. This is the first report showing that a member of the CYP6 family catalyzes the hydroxylation of lauric acid. Our data open new prospects for the CYP6 P450 enzymes, which could be involved in important physiological functions through fatty acid metabolism.     

Molecular basis for cytokinin biosynthesis

Cytokinins (CKs) are a group of phytohormones that play a crucial role in the regulation of plant growth and development. Identification of the enzymes and the corresponding genes that are involved in CK metabolism allowed us to understand how plants synthesize CKs and adjust CK activity to optimal levels. A major accomplishment toward these goals was the identification of genes for the first enzyme in the CK biosynthetic pathway, adenosine phosphate-isopentenyltransferase (IPT). In Arabidopsis thaliana and Agrobacterium tumefaciens, detailed analyses of IPTs were conducted through not only enzymatic characterization but also molecular structural approaches. These studies revealed the molecular basis for the Agrobacterium-origin of IPT used for the efficient biosynthesis of trans-zeatin that promotes tumorigenesis in host plants. Another landmark in CK research was the identification of CYP735A as an enzyme that converts iP-nucleotide to tZ-nucleotide. Furthermore, the identification of a CK-activating enzyme, LOG, which catalyzes a novel activation pathway, is a remarkable recent achievement in CK research. Collectively, these advances have revealed the complexity of the entire metabolic scheme for CK biosynthesis.