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D E Cane

Publications and source records attributed to D E Cane.

At least 55 records · Page 3Linked to original sources
PubMed1996

Erythromycin biosynthesis: exploiting the catalytic versatility of the modular polyketide synthase.

DEBS 1 + TE is a recombinant modular polyketide synthase (PKS) in which the first two biosynthetic modules of the 6-deoxyerythronolide B synthase are linked to the thioesterase domain normally found at the C-terminus of DEBS 3. Incubation of DEBS 1 + TE with propionyl-CoA, methylamalonyl-CoA, and NADPH gives the triketide lactone (2R,3S,4S,5R)-2,4-dimethyl-3, 5-dihydroxy-n-heptanoic acid delta-lactone (2), the cyclized form of the normal triketide chain elongation product of DEBS 1. In order to probe the molecular recognition features of the PKS and to explore its synthetic versatility, [2,3-13C2]-(2S,3R)-2-methyl-3-hydroxypentanoyl-NAC thioester (3), an analogue of the normal diketide chain elongation intermediate, and (2RS)-methyl-malonyl-CoA were incubated with DEBS 1 + TE, leading to the formation of the predicted labeled triketide ketolactone [4,5-13C2]-8, as established by 13C NMR analysis and comparison with spectra of synthetic 8. This stereoselective conversion illustrates the potential of using modular PKSs as multifunctional catalysts for the enzymatic synthesis of novel polyketides.

Erythromycin↗
PubMed1996

A functional chimeric modular polyketide synthase generated via domain replacement.

BACKGROUND: Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzymes that catalyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized into 'modules', such that each module catalyzes the stereospecific addition of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate. The core of each module is made up of a 'reductive segment', which includes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organization of reductive segments within modules suggests that they might be able to function in unnatural contexts to generate novel organic molecules. RESULTS: To investigate domain substitution as a method for altering PKS function, a chimeric enzyme was engineered. Using a bimodular derivative of DEBS (DEBS1+TE), the reductive segment of module 2, which includes a functional KR, was replaced with its homolog from module 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predicted ketolactone with a yield (35 %) comparable to that of a control strain in which the KR2 domain was retained but mutationally inactivated. CONCLUSIONS: These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.

Multienzyme Complexes↗
PubMed1995

Cell-free synthesis of polyketides by recombinant erythromycin polyketide synthases.

Modular polyketide synthases (PKSs) are complex multi-enzyme proteins that catalyse the bacterial biosynthesis of many pharmaceutically useful polyketides. The PKSs are organized into a series of modules, each containing the active catalytic sites required for one step in the synthesis process. Here we report a method for cell-free enzymatic synthesis of 6-deoxyerythronolide B (6-dEB), the parent molecule of the antibiotic erythromycin A, using recombinant 6-deoxyerythronolide B synthase (DEBS), a modular PKS with at least 28 distinct active sites. We have also synthesized in vitro a triketide lactone by using a truncated mutant of DEBS. The availability of such cell-free synthetic routes will allow direct investigation of the structural and mechanistic basis for the unusual combination of high substrate specificity and tolerance to genetic reprogramming found in this enzyme family.

Amino Acid Sequence↗
PubMed1995

Trichodiene synthase. Substrate specificity and inhibition.

The substrate specificity of the sesquiterpene synthase trichodiene synthase was examined by determining the Vmax and Km parameters for the natural substrate, trans,trans-farnesyl diphosphate (1), its stereoisomer, cis,trans-farnesyl diphosphate, and the tertiary allylic isomer, (3R)-nerolidyl diphosphate (3), using both the native fungal and recombinant enzymes. A series of farnesyl diphosphate analogs, 15, 16, 20, 7, 8, and 9, was also tested as inhibitors of trichodiene synthase. 10-Fluorofarnesyl diphosphate (15) was the most effective competitive inhibitor, with a K1 of 16 nM compared to the Km for 1 of 87 nM, while the ether analog of farnesyl diphosphate, 8, an extremely potent inhibitor of squalene synthase, showed only modest inhibition of trichodiene synthase, with a K1/Km of 70.

Binding, Competitive↗
PubMed1995

Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Overproduction and characterization of the erythromycin C-12 hydroxylase, EryK.

Hydroxylation of C-12 is one of the final steps in the biosynthesis of erythromycin A (ErA). A point of uncertainty in the erythromycin pathway has been whether the C-12 hydroxylase operates on each of two possible substrates, erythromycin B (ErB) and erythromycin D (ErD). Stassi et al. have cloned the gene, designated eryK, which encodes the P-450 monooxygenase responsible for erythromycin C-12 hydroxylation in Saccharopolyspora erythraea [Stassi, D., Donadio, S., Staver, M. J., & Katz, L. (1993) J. Bacteriol. 175, 182-189]. We report the overproduction of EryK in Escherichia coli as insoluble inclusion bodies; the solubilization, refolding, and reconstitution of active holo-EryK; and kinetic confirmation of a 1200-1900-fold preference of the enzyme for ErD over the alternative C-12 hydroxylase substrate ErB. Our results indicate that ErB is a shunt metabolite in the erythromycin biosynthetic pathway.

Amino Acid Sequence↗
PubMed1995

Crystallization and preliminary X-ray diffraction analysis of recombinant pentalenene synthase.

Recombinant pentalenene synthase, a 42.5-kDa sesquiterpene cyclase originally isolated from Streptomyces UC5319 and cloned in Escherichia coli, has been crystallized in space group P6(3) with unit cell dimensions a = b = 183.5 A and c = 56.5 A. Hexagonal prismatic crystals, approximately 0.2 x 0.2 x 0.3 mm, diffract to approximately 2.9 A resolution using monochromatic synchrotron radiation. From the universal (and achiral) building block, farnesyl pyrophosphate, pentalenene synthase catalyzes the formation of four stereocenters in the construction of the three fused five-membered rings of pentalenene; this novel sesquiterpene is a precursor to the pentalenolactone family of antibiotics.

Crystallization↗
PubMed1995

Erythromycin biosynthesis. Highly efficient incorporation of polyketide chain elongation intermediates into 6-deoxyerythronolide B in an engineered Streptomyces host.

Feeding of (2S,3R)-[2,3-13C2]-2-methyl-3-hydroxypentanoyl NAC thioester (1a) to the recombinant organism Streptomyces coelicolor CH999/pCK7 harboring the complete set of eryA genes from Saccharopolyspora erythraea encoding the 6-deoxyerythronolide B synthase (DEBS) resulted in the formation of 6-deoxyerythronolide B (2a) labeled with 13C at C-12 and C-13, as evidenced by the appearance of a pair of enhanced and coupled doublets in the 13C NMR spectrum. The level of 13C enrichment was 15-20 atom% 13C, as much as 100 times higher than the usually observed efficiency of incorporation of NAC thioesters into polyketide metabolites. Similar incorporation of (2S,3R)-[3-2H,3-13C]-2-methyl-3-hydroxypentanoyl NAC thioester (1b) gave 6-deoxyerythronolide B (2b) labeled with both 13C and deuterium at C-13. The intact incorporation of both precursors confirms the normal functioning of the recombinant DEBS proteins in the heterologous host.

DNA, Recombinant↗
PubMed1994

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by pentalenolactone. 2. Identification of the site of alkylation by tetrahydropentalenolactone.

Incubation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the antibiotic pentalenolactone (3) results in time-dependent, irreversible inhibition of GAPDH by modification of a single Cys residue in each subunit of the homotetrameric enzyme. Reduction of pentalenolactone with tritium gas gave [2,3,7,8-3H4]tetrahydropentalenolactone (7), which also exhibited time-dependent, irreversible inactivation of GAPDH. The site of covalent attachment of 7 was determined. Tryptic digestion of inactivated GAPDH and purification of the resultant products by reverse-phase HPLC gave a single labeled peptide. Amino acid sequence analysis of the radioactive peptide gave Ile-Val-Ser-Asn-Ala-Ser-X-Thr-Thr-Asn-(...). This sequence is identical to the highly conserved region from Ile-143 to Asn-152 in pig muscle GAPDH, except for the active site Cys-149 to which the tetrahydropentalenolactone was covalently bound. Molecular modeling was used to compare both pentalenolactone (3) and heptelidic acid (4), a mechanistically related inactivator of GAPDH, with the normal substrate, glyceraldehyde 3-phosphate (1). Finally, pentalenolactone was shown by reaction with model thiols to undergo epoxide ring opening exclusively by nucleophilic attack at the primary carbon, C-10.

Alkylation↗
PubMed1994

Pentalenene synthase. Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319.

Pentalenene synthase, which catalyzes the cyclization of farnesyl diphosphate (1) to the tricyclic sesquiterpene hydrocarbon pentalenene (2), was purified from Streptomyces UC5319. A 450-bp hybridization probe, generated by PCR amplification of genomic DNA using primers based on N-terminal and internal tryptic peptide sequence data for pentalenene synthase, was used to screen both plasmid and phage DNA libraries of Streptomyces genomic DNA, resulting in the isolation and sequencing of the complete pentalenene synthase gene. PCR was used to insert the pentalenene synthase gene into the T7 expression vector pLM1. Cloning of the resulting construct in the expression host Escherichia coli BL21 (DE3) gave transformants that expressed pentalenene synthase as greater than 10% of soluble protein. The recombinant enzyme has been purified, and initial physical and kinetic characterization has been performed. The recombinant enzyme appears to be identical in every respect with the native Streptomyces synthase and exhibits the following steady-state kinetic parameters: Km = 0.31 +/- 0.05 microM, kcat = 0.32 +/- s-1, KI(PPi) = 3.2 +/- 0.6 microM. Both enzymes have an absolute requirement of Mg2+ for catalysis and an optimum pH of 8.2-8.4. Both proteins have M(r) values of 41-42 kDa, as determined by SDS-PAGE.

Amino Acid Sequence↗
PubMed1993

Overexpression in Escherichia coli of soluble aristolochene synthase from Penicillium roqueforti.

Aristolochene synthase, a fungal cyclase which has been isolated from Aspergillus terreus and Penicillium roqueforti, catalyzes the cyclization of farnesyl diphosphate to the sesquiterpene hydrocarbon aristolochene. The aristolochene synthase gene (Ari1) of P. roqueforti has previously been cloned and expressed at low levels as a protein A-aristolochene synthase fusion protein in Escherichia coli. We have now used the polymerase chain reaction to amplify the aristolochene synthase coding sequence using engineered primers which produced dsDNA carrying an EcoRI restriction site and the T7 gene 10 ribosome binding site and translational spacer element immediately upstream of the ATG start codon and a BamHI site adjacent to the TAA stop codon. The PCR product was digested with EcoRI and BamHI and inserted into the multiple cloning site of the expression vector pLM1 which carried the promoter and translational leader sequence from T7 gene 10 and the E. coli rrnBT1T2 tandem transcription terminator. Cloning of the resulting construct into E. coli XL1-Blue and subcloning into the expression host E. coli BL21 (DE3) gave transformants which expressed aristolochene synthase at levels up to 40% of soluble protein when induced with isopropyl beta-D-thiogalactoside. Purification of the recombinant protein by ammonium sulfate precipitation, ion-exchange chromatography on Q Sepharose, and affinity dye chromatography on Reactive Blue 4-agarose gave homogenous aristolochene synthase which had the expected N-terminal sequence, ATSTE, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and steady-state kinetic parameters when compared to native fungal protein.

Base Sequence↗
PubMed1993

Overproduction of soluble trichodiene synthase from Fusarium sporotrichioides in Escherichia coli.

Trichodiene synthase is a sesquiterpene cyclase isolated from various fungal species which catalyzes the cyclization of farnesyl diphosphate (FPP) to trichodiene. The trichodiene synthase gene (Tox5) of Fusarium sporotrichioides has previously been cloned and expressed as 0.05-0.1% of total cell protein in Escherichia coli. We have used polymerase chain reaction to amplify the trichodiene coding sequence carried on the plasmid pTS56-1. The resulting DNA, carrying a BamHI restriction site and the T7 gene 10 ribosome binding site and translational spacer element immediately upstream of the ATG start codon as well as a HindIII site adjacent to the translational stop codon, was inserted into the corresponding sites of the expression vector pLM1. The latter vector carried the promoter and translational leader sequence from T7 gene 10 and the E. coli rmBT1T2 tandem transcription terminator. This construct was cloned into E. coli BL21 (DE3). The resulting transformants, when induced with isopropyl beta-D-thiogalactoside, produced trichodiene synthase as 20-30% of total soluble protein. The recombinant synthase, which could be purified five-fold to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography on Q Sepharose, and gel filtration on Superose 12, was identical to native protein in steady-state kinetic parameters and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the expected MENFP N-terminal sequence.

Base Sequence↗
PubMed1993

Irreversible inactivation of monoterpene cyclases by a mechanism-based inhibitor.

Monoterpene synthases (cyclases) catalyze the divalent metal ion-dependent transformation of geranyl pyrophosphate to representative of the various monocyclic and bicyclic skeletal types by an electrophilic reaction mechanism involving coupled isomerization and cyclization steps. An analogue of the geranyl substrate, in which the terminal gem-dimethyl groups were joined to form a cyclopropyl function (6-cyclopropylidene-3E-methyl-hex-2-en-l-yl pyrophosphate) was shown to be a potent inhibitor of (-)-4S-limonene synthase from Mentha spicata and of several other monoterpene cyclases from diverse plant species. Inhibition was concentration and time dependent (pseudo-first-order kinetics), as well as absolutely contingent on the presence of the divalent metal ion cofactor. A double reciprocal plot of kinactivation versus inhibitor concentration gave an apparent Ki of approximately 0.3 microM and a maximum rate of inactivation of about 0.3 min-1 with limonene synthase. As expected for an active-site-directed process, the natural substrate, geranyl pyrophosphate, afforded protection against inactivation by the cyclopropylidene analogue. Selectivity of the inhibition was demonstrated with [1-3H]6-cyclopropylidene-3E-methyl-hex-2-en-1-yl pyrophosphate by specific labeling of limonene synthase in crude enzyme extracts as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radio-fluorography, and immunoblotting. The radioactive cyclase-inactivator complex was formed with 1:1 stoichiometry and was stable to extended dialysis and boiling in 2% sodium dodecyl sulfate, suggesting irreversible covalent modification of the enzyme involving a chemical reaction between cyclase and inhibitor. Thermally denatured limonene synthase and synthase that had been inactivated with the histidine-directed reagent diethylpyrocarbonate or the cysteine-directed reagent p-hydroxymercuribenzoate (two reagents known to modify the active site of the enzyme and inhibit catalysis) were not labeled when treated with the [1-3H]-analogue, indicating that the functional enzyme was necessary to effect complex formation. All of the evidence is consistent with the analogue serving as a mechanism-based inactivator that must undergo both ionization-dependent isomerization and cyclization steps to reveal an allylic cation which alkylates the protein. In addition to furnishing supporting evidence for the electrophilic reaction sequence, this mechanism-based inactivator provides a powerful new approach for the examination of cyclase active sites.

Intramolecular Lyases↗
PubMed1992

Terpenoid cyclases: design and function of electrophilic catalysts.

Terpenoid cyclases catalyse the cyclization of the universal acyclic precursors geranyl and farnesyl diphosphate to monoterpenes and sesquiterpenes, respectively. All such cyclases investigated to date are operationally soluble, moderately lipophilic proteins of relative molecular weight 40,000-100,000, requiring no cofactors other than a divalent metal, usually Mg2+ and occasionally Mn2+. The focus of most work has been on the mechanisms of the cyclization reactions themselves. It is currently proposed that the cyclase binds the acyclic substrate in a suitable conformation and initiates the cyclization by ionization of the labile allylic diphosphate moiety. The use of stereospecifically labelled substrates and analysis of the sites of labelling in the derived cyclization products has allowed the proposal of detailed cyclization mechanisms. Further insight into the architecture and function of the cyclase active site has come from the study of substrate and intermediate analogues designed to act as potential inhibitors or anomalous substrates of the normal cyclization reaction. Progress has also been made on the cloning of the relevant structural genes for sesquiterpene cyclases. This has led to new insights into the basic requirements for cyclase catalysis and specificity.

Amino Acid Sequence↗
PubMed1991

Terpenoid biosynthesis and the stereochemistry of enzyme-catalysed allylic addition-elimination reactions.

Allylic addition-elimination reactions are widely used in the enzyme-catalysed formation of terpenoid metabolites. It has earlier been shown that the isoprenoid chain elongation reaction catalysed by farnesyl pyrophosphate synthase involving successive condensations of dimethylallyl pyrophosphate (DMAPP) and geranyl pyrophosphate (GPP) with isopentenyl pyrophosphate (IPP) corresponds to such an SE' reaction with net syn stereochemistry for the sequential electrophilic addition and proton elimination steps. Studies of the enzymic cyclization of farnesyl pyrophosphate (FPP) to pentalenene have now established the stereochemical course of two additional biological SE' reactions. Incubation of both (9R)- and (9S)-[9-3H,4,8-14]FPP with pentalenene synthase and analysis of the resulting labelled pentalenene has revealed that H-9re of FPP becomes H-8 of pentalenene, while H-9si undergoes net intramolecular transfer to the adjacent carbon, becoming H-1re (H-1 alpha) of pentalenene, as confirmed by subsequent experiments with [10-2H, 11-13C]FPP. These results correspond to net anti-stereochemistry in the intramolecular allylic addition-elimination reaction. The stereochemical course of a second SE' reaction has now been examined by analogous incubations of (4S,8S)-[4,8-3H,4,8-14C]FPP and (4R,8R)-[4,8-3H, 4.8-14C]FPP with pentalenene synthase. Determination of the distribution of label in the derived pentalenenes showed stereospecific loss of the original H-8si proton. Analysis of the plausible conformation of the presumed reaction intermediates revealed that the stereochemical course of the latter reaction cannot properly be described as either syn or anti, since cyclization and subsequent double bond formation require significant internal motions to allow proper overlap of the scissile C-H bond with the developing carbocation.

Carbon Radioisotopes↗