Genetic and Biochemical Characterization of a Novel Monoterpene ɛ-Lactone Hydrolase from Rhodococcus erythropolis DCL14

A monoterpene -lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.     

Rhodococcus erythropolis DCL14 contains a novel degradation pathway for limonene

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (-)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1, 2-monooxygenase activity, a cofactor-independent limonene-1, 2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S, 4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R, 4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the beta-oxidation pathway.     

Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the β-oxidation pathway.     

Alternate substrate inhibitors of an alpha-chymotrypsin: enantioselective interaction of aryl-substituted enol lactones

Four enol lactones, bearing phenyl or 1-naphthyl substituents on the alpha or beta positions [3-phenyl-6-methylenetetrahydro-2-pyranone (alpha Ph6H, IIc), 3-(1-naphthyl)-6-methylenetetrahydro-2-pyranone (alpha Np6H, IId), 4-phenyl-6-methylenetetrahydro-2-pyranone (beta Ph6H, IIIc), and 4-(1-naphthyl)-6-methylenetetrahydro-2-pyranone (beta Np6H, IIId)], available as pure R and S enantiomers, have been studied as alternate substrate inhibitors of chymotrypsin. Kinetic constants for substrate binding (Ks) and acylation (ka) were determined by a competitive substrate assay, using succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide; the deacylation rate constant (kd) was determined by the proflavin displacement assay. All lactones undergo rapid acylation (ka varies from 17 to 170 min-1) that shows little enantioselectivity; there is, however, pronounced enantioselectivity in substrate binding for three of the lactones [Ks(R/S) = 40-110]. In each case it is the enantiomer with the S configuration that has the higher affinity. In all cases, deacylation rates are slow, and in two cases, acyl enzymes with half-lives of 4.0 and 12.5 h at pH 7.2, 25 degrees C, are obtained (for beta Ph6H and alpha Np6H, respectively). In these cases, high deacylation enantioselectivity is observed [kd(S/R) = 60-70], and the lactone more weakly bound as a substrate (R enantiomer) gives the more stable acyl enzyme. Two hypotheses, involving hindrance of the attack of water or an exchange of the ester and ketone carbonyl groups in the acyl enzyme, are advanced as possible explanations for the high stability of these acyl enzymes.     

Ophirasterol, a new C31 sterol from the marine sponge Topsentia ophiraphidites

Analysis of the sterol fraction obtained from the Colombian Caribbean sponge Topsentia ophiraphidites revealed that this sponge is a rich source of C30 and C31 sterols. Among them, a new C31 sterol, named ophirasterol, was isolated, and its structure was established as (22E,24R)-24-(1-buten-2-yl)cholesta-5,22-dien-3beta-ol (1) by spectral means and comparison with synthetic C-24 epimers with known configuration. Other isolated C30 and C31 sterols were the known 24-ethyl-24-methyl-22-dehydrocholesterol (2), 24-isopropyl-22-dehydrocholesterol (3), 24-isopropylcholesterol (4), 24-ethyl-24-methylcholesterol (5), 24-isopropenyl-25-methyl-22-dehydrocholesterol (6) and 24-isopropenyl-25-methylcholesterol (7), and 24-isopropenyl-22-dehydrocholesterol (8).     

3-(4-Methyl-3-pentenyl)-2(5H)-furanone, alpha,alpha-acariolide and 4-(4-methyl-3-pentenyl)-2(5H)-furanone, alpha,beta-acariolide: new monoterpene lactones from the astigmatid mites, Schwiebea araujoae and Rhizoglyphus sp. (Astigmata: Acaridae)

A new monoterpene lactone from the acarid mite, Schwiebea araujoae, was elucidated without its isolation by GC/FT-IR and GC/MS analyses to be 3-(4-methyl-3-pentenyl)-2(5H)-furanone (1) and tentatively named as alpha,alpha-acariolide. The structure of 1 was identified by its synthesis from alpha-bromo-gamma-butyrolactone via 4 reaction steps. The synthesized compound gave the same GC/MS and GC/FT-IR spectra as those of the natural product. The other monoterpene lactone was likewise elucidated from the unidentified Rhizoglyphus mite to be 4-(4-methyl-3-pentenyl)-2(5H)-furanone (2) and named as alpha,beta-acariolide; it was also identified by its synthesis in 5 reaction steps from the same butyrolactone as the starting material. GC/MS and GC/FT-IR spectra of the preparation were identical to those of the natural product.     

Kinetics and mechanism of human leukocyte elastase inactivation by ynenol lactones

Human leukocyte elastase (HLE), a serine protease involved in inflammation and tissue degradation, can be irreversibly inactivated in a time- and concentration-dependent manner by ynenol lactones. Ynenol lactones that are alpha-unsubstituted do not inactivate but are alternate substrate inhibitors that are hydrolyzed by the enzyme. Ynenol lactones that are both substituted alpha to to the lactone carbonyl and unsubstituted at the acetylene terminus are rapid inactivators of HLE and inactivate pancreatic elastase and trypsin more slowly. 3-Benzyl-5(E)-(prop-2-ynylidene)tetrahydro-2-furanone inactivates HLE with biphasic kinetics and an apparent second-order rate of up to 22,000 M-1 s-1 (pH 7.8, 25 degrees C). The rate of inactivation is pH-dependent and is slowed by a competitive inhibitor. The partition ratio is 1.6 +/- 0.1. Rapid removal of ynenol lactone during the course of inactivation yields a mixture of acyl and inactivated enzyme species, which then shows a partial recovery of activity that is time- and pH-dependent. Inactivation is not reversible with hydroxylamine. The enzyme is not inactivated if the untethered allenone is added exogenously. All of these results are consistent with a mechanism involving enzyme acylation at serine-195 by the ynenol lactone, isomerization of the acyl enzyme to give a tethered allenone, and capture of a nucleophile (probably histidine-57) to inactivate the enzyme. Substitution at the acetylene terminus of ynenol lactones severely reduces their ability to inactivate HLE, because allenone formation is slowed and/or nucleophile capture is hindered. Chemical competence of each of these steps has been demonstrated [Spencer, R.W., Tam, T.F., Thomas, E.M., Robinson, V.J.,& Krantz, A. (1986) J. Am. Chem. Soc. 108, 5589-5597].     

Purification and characterization of 4-methylmuconolactone methyl-isomerase, a novel enzyme of the modified 3-oxoadipate pathway in nocardioform actinomycetes.

The novel enzyme 4-methyl-2-enelactone methyl-isomerase was detected in, and purified to electrophoretic homogeneity from, p-toluate-grown cells of Rhodococcus rhodocrous N75, a nocardioform actinomycete. The enzyme was very thermostable and had a native Mr of 75,500; as the monomer had an Mr of 17,000, the enzyme is probably tetrameric. The new isomerase is highly specific with respect to its lactone substrate, only accepting (+)-(4S)-4-methylmuconolactone (4-carboxymethyl-4-methylbut-2-en-1,4-olide), and the putative isomerization reaction intermediate 1-methylbislactone ((-)-1-methyl-3,7-dioxo-2,6-dioxabicyclo-[3.3.0]octane) as substrates, and yielding (-)-(4S)-3-methylmuconolactone (4-carboxymethyl-3-methylbut-2-en-1,4-olide) as product. Some other lactone analogues acted as competitive inhibitors. Our data suggest that the isomerization does not involve actual methyl migration, but proceeds via the 1-methybislactone.     

Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein.

Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosylmethionine. Another V. fischeri gene, ainS, directs the synthesis of N-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the Vibrio harveyi LuxM protein. The luxM gene is required for the synthesis of N-(3-hydroxybutyryl)-L-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of N-octanoylhomoserine lactone from S-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes.     

The monoterpenes of Artemisia tridentata ssp. vaseyana, Artemisia cana ssp. viscidula and Artemisia tridentata ssp. spiciformis.

Monoterpenes from three different members of the Anthemideae family, Artemisia tridentata ssp. vaseyana, Artemisia cana ssp. viscidula and Artemisia tridentata ssp. spiciformis were isolated and their structures determined using spectroscopic techniques. A total of 26 irregular and regular monoterpenes were identified. Among these, 20 had previously been identified in the Anthemideae family. Of the remaining six, four were known, but previously unidentified in this family. 2,2-Dimethyl-6-isopropenyl-2H-pyran, 2,3-dimethyl-6-isopropyl-4H-pyran and 2-isopropenyl-5-methylhexa-trans-3,5-diene-1-ol were isolated from both A. tridentata ssp. vaseyana and A. cana ssp. viscidula. The irregular monoterpene 2,2-dimethyl-6-isopropenyl-2H-pyran has a carbon skeleton analogous to the biologically important triterpene squalene. Two additional irregular monoterpenes, artemisia triene and trans-chrysanthemal were isolated from A. cana ssp. viscidula and lavandulol was isolated from A. tridentata ssp. spiciformis. This is the first time a compound possessing a lavandulyl-skeletal type has been found in the Anthemideae family.     

Synthesis and biological evaluation of substrate-based inhibitors of 6-phosphogluconate dehydrogenase as potential drugs against African trypanosomiasis

The synthesis and biological evaluation of three series of 6-phosphogluconate (6PG) analogues is described. (2R)-2-Methyl-4,5-dideoxy, (2R)-2-methyl-4-deoxy and 2,4-dideoxy analogues of 6PG were tested as inhibitors of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver and also Trypanosoma brucei where the enzyme is a validated drug target. Among the three series of analogues, seven compounds were found to competitively inhibit 6PGDH from T. brucei and sheep liver enzymes at micromolar concentrations. Six inhibitors belong to the (2R)-2-methyl-4-deoxy series (6, 8, 10, 12, 21, 24) and one is a (2R)-2-methyl-4,5-dideoxy analogue (29b). The 2,4-dideoxy analogues of 6PG did not inhibit both enzymes. The trypanocidal effect of the compounds was also evaluated in vitro against T. brucei rhodesiense as well as other related trypanosomatid parasites (i.e., Trypanosoma cruzi and Leishmania donovani).     

Isolation and characterization of N-acylhomoserine lactonase from the thermophilic bacterium, Geobacillus caldoxylosilyticus YS-8

Geobacillus caldoxylosilyticus YS-8, which was isolated from volcanic soil in Indonesia, was found to degrade various N-acylhomoserine lactones (AHLs) with different lengths and acyl side-chain substitutions over a wide temperature range of 30-70 °C. The purified AHL-degrading enzyme showed a single band of 32 kDa, and its N-terminal amino acid sequence was determined to be ANVIKARPKLYVMDN, tentatively suggesting that the AHL-degrading enzyme was AHL lactonase. The AHL-degrading activity of the purified enzyme was maximized at pH 7.5 and 50 °C, and it retained about 50% of its activity even after a heat treatment at 60 °C for 3 h, exhibiting properties consistent with a thermostable enzyme. The mass spectrometric analysis demonstrated that the AHL-degrading enzyme catalyzed lactone ring opening of N-3-oxohexanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone by hydrolyzing the lactones and working as an AHL lactonase.     

Stereochemistry of catalysis by the ketoreductase activity in the first extension module of the erythromycin polyketide synthase

Multiple ketoreductase activities play a crucial role in establishing the stereochemistry of the products of modular polyketide synthases (PKSs), but there has been little systematic scrutiny of catalysis by individual ketoreductases. To allow this, a diketide synthase, consisting of the loading module, first extension module, and the chain-terminating thioesterase of the erythromycin-producing PKS of Saccharopolyspora erythraea, has been expressed and purified. The DNA encoding the ketoreductase-1 domain in this construct is flanked by unique restriction sites so that another ketoreductase domain can be readily substituted. The purified recombinant diketide synthase catalyzes, at a very low rate (k(cat) equals 2.5 x 10(-3) s(-1)), the specific production of the diketide (2S,3R)-2-methyl-3-hydroxypentanoic acid. The activity of the ketoreductase domain in this model synthase was analyzed using as a model substrate (+/-)-2-methyl-3-oxopentanoic acid N-acetylcysteaminyl (NAC) ester for which k(cat)/K(m) was 21.7 M(-1) s(-1). The NAC thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid was the major product and was strongly preferred over other stereoisomers as a substrate in the reverse reaction. The bicyclic ketone (9RS)-trans-1-decalone, a known substrate for ketoreductase in fatty acid synthase, was found also to be an effective substrate for the ketoreductase of the diketide synthase. Only the (9R)-trans-1-decalone was reduced, selectively and reversibly, to the (1S,9R)-trans-decalol. The stereochemical course of reduction and oxidation is exactly as found previously for the ketoreductase of animal fatty acid synthase, an additional indication of the close similarity of these enzymes.     

The desymmetrization of bicyclic beta -diketones by an enzymatic retro-Claisen reaction. A new reaction of the crotonase superfamily

The enzyme 6-oxocamphor hydrolase, which catalyzes the desymmetrization of 6-oxocamphor to yield (2R,4S)-alpha-campholinic acid, has been purified with a factor of 35.7 from a wild type strain of Rhodococcus sp. NCIMB 9784 grown on (1R)-(+)-camphor as the sole carbon source. The enzyme has a subunit molecular mass of 28,488 Da by electrospray mass spectrometry and a native molecular mass of approximately 83,000 Da indicating that the active protein is trimeric. The specific activity was determined to be 357.5 units mg(-)1, and the K(m) was determined to be 0.05 mm for the natural substrate. The N-terminal amino acid sequence was obtained from the purified protein, and using this information, the gene encoding the enzyme was cloned. The translation of the gene was found to bear significant homology to the crotonase superfamily of enzymes. The gene is closely associated with an open reading frame encoding a ferredoxin reductase that may be involved in the initial step in the biodegradation of camphor. A mechanism for 6-oxocamphor hydrolase based on sequence homology and the known mechanism of the crotonase enzymes is proposed.     

Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for disruption of quorum sensing

SsoPox, a bifunctional enzyme with organophosphate hydrolase and N-acyl homoserine lactonase activities from the hyperthermophilic archaeon Sulfolobus solfataricus, was overexpressed and purified from recombinant Pseudomonas putida KT2440 with a yield of 9.4 mg of protein per liter of culture. The enzyme has a preference for N-acyl homoserine lactones (AHLs) with acyl chain lengths of at least 8 carbon atoms, mainly due to lower K(m) values for these substrates. The highest specificity constant obtained was for N-3-oxo-decanoyl homoserine lactone (k(cat)/K(m) = 5.5 × 10(3) M(-1)·s(-1)), but SsoPox can also degrade N-butyryl homoserine lactone (C(4)-HSL) and N-oxo-dodecanoyl homoserine lactone (oxo-C(12)-HSL), which are important for quorum sensing in our Pseudomonas aeruginosa model system. When P. aeruginosa PAO1 cultures were grown in the presence of SsoPox-immobilized membranes, the production of C(4)-HSL- and oxo-C(12)-HSL-regulated virulence factors, elastase, protease, and pyocyanin were significantly reduced. This is the first demonstration that immobilized quorum-quenching enzymes can be used to attenuate the production of virulence factors controlled by quorum-sensing signals.     

Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates

Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase.     

Reactor operation and scale-up of whole cell Baeyer-Villiger catalyzed lactone synthesis

The recombinant whole cell biocatalyst Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871, was used in 1.5- and 55-L fed-batch processes to oxidize bicyclo[3.2.0]hept-2-en-6-one to its corresponding regioisomeric lactones, (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. By employing a bicyclo[3.2.0]hept-2-en-6-one feed rate below that of the theoretical volumetric biocatalyst activity (275 micromol x min(-1) x L(-1)), the reactant concentration in the bioreactor was successfully maintained below the inhibitory concentration of 0.2-0.4 g x L(-1). In this way approximately 3.5 g x L(-1) of the combined regioisomeric lactones was produced with a yield of product on reactant of 85-90%. The key limitation to the process was shown to be product inhibition. This process was scaled up to 55 L, producing over 200 g of combined lactone product. Using a simple downstream process (centrifugation, adsorption to activated charcoal, 5-fold concentration with ethyl acetate elution, and silica gel chromatography), we have shown that the two regioisomeric lactone products could be isolated and purified at this scale.     

Molecular cloning and characterization of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (CaHDR) from Camptotheca acuminata and its functional identification in Escherichia coli

Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 microM methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.     

Monoterpene coumarins from Ferula ferulago.

Two monoterpene coumarins, designated ferulagol A and B, as well as three known monoterpene coumarins and three sesquiterpene lactones were isolated from the chloroform extract of the roots of Ferula ferulago. The structures of ferulagol A and B were determined to be 7-[(E)-3'-hydroxy-3',7'-dimethyl-4',6'-octadienyloxy]coumarin and 7-[(3'Z,5'E)-7'-hydroxy-3',7'-dimethyl-3',5'-octadienyloxy]coumarin, respectively.     

Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [(14)C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M(r)s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.