Biosynthesis of cobalamin (vitamin B(12)).

The biosynthesis of vitamin B(12) is summarized, emphasizing the differences observed between the aerobic and anaerobic pathways. The biosynthetic route to adenosylcobalamin from its five-carbon precursor, 5-aminolaevulinic acid, can be divided into three sections: (1) the biosynthesis of uroporphyrinogen III from 5-aminolaevulinic acid, which is common to both pathways; (2) the conversion of uroporphyrinogen III into the ring-contracted, deacylated intermediate precorrin 6 or cobalt-precorrin 6, which includes the primary differences between the two pathways; and (3) the transformation of this intermediate to form adenosylcobalamin.

Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol.

Taxadiene, the key intermediate of paclitaxel (Taxol) biosynthesis, has been prepared enzymatically from isopentenyl diphosphate in cell-free extracts of Escherichia coli by overexpressing genes encoding isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase and taxadiene synthase. In addition, by the expression of three genes encoding four enzymes on the terpene biosynthetic pathway in a single strain of E. coli, taxadiene can be conveniently synthesized in vivo, at the unoptimized yield of 1.3mg per liter of cell culture. The success of both in vitro and in vivo synthesis of taxadiene bodes well for the future production of taxoids by non-paclitaxel producing organisms through pathway engineering.

An improved synthesis of the dinucleotides pdCpA and pdCpdA.

An improved route was developed for the preparation of the dinucleotide hybrid 5'-O-phosphoryl-2'-deoxycytidylyl-(3'--> 5')adenosine (pdCpA) 7. This simple and concise synthesis involves the successive coupling of 2-cyanoethyl N, N, N', N'-tetra- isopropylphosphorodiamidite with 4-N-benzoyl-5'-O-(4, 4'-dimethoxytrityl)-2'-deoxy-cytidine 1 and 6-N,6-N,2'-O,3'-O-tetrabenzoyladenosine 2 as the key step. Some dinucleotide derivatives bearing different protecting groups were also synthesized and the selective deprotection conditions were studied in detail. The utility and efficiency of this approach has been further demonstrated by its application to the synthesis of total DNA dinucleotide pdCpdA 17 and total RNA dinucleotide 21.

Microvascular complications in cystic fibrosis-related diabetes mellitus: a case report.

CONTEXT, The prevalence of cystic fibrosis-related diabetes mellitus is increasing and is associated with increased survival from cystic fibrosis. CASE REPORT, This study describes a case of the premature onset of disabling and widespread microvascular complications resulting from cystic fibrosis-related diabetes mellitus. Previously asymptomatic retinopathy was diagnosed on recognition of diabetic nephropathy. CONCLUSIONS, The treatment of pulmonary exacerbations has become more complex due to the nephrotoxic potential of intravenous aminoglycoside drugs which are frequently used to control chronic Pseudomonas infection in cystic fibrosis.

Detection of nascent polyproline II helices in solution by NMR in synthetic insect kinin neuropeptide mimics containing the X-Pro-Pro-X motif.

The conformations of three synthetic peptide analogs containing the dPro-dPro-dXaa motif (dXaa = dThr, dGlu, dAsn) in aqueous solution were studied by a combination of NMR and molecular modeling simulations. The three compounds were identified from a random D-amino acid tripeptide library on the basis of their ability to either mimic or block the diuretic activity of neuropeptides of the insect kinin family. TOCSY and ROESY correlations, as well as abnormal secondary chemical shifts for protons on the D-proline residues were employed to obtain conformational ensembles consistent with the experimental NMR data for the three analogs using an in vacuo simulated annealing protocol. Similar secondary structures were found for the three molecules after refinement, in agreement with the similarities observed between their NMR spectra. Unrestrained molecular dynamics simulations with explicit water representation indicate that the structural motifs found in vacuo are stable in aqueous solution. The three analogs can be considered initiators of right-handed poly D-proline II helices, mirror images of the poly L-proline II left-handed helical motifs normally found in proline-rich proteins. The role of these secondary folds on binding of the analogs to the kinin receptors is discussed.

Synthesis, biological activity, and conformational studies of insect allatostatin neuropeptide analogues incorporating turn-promoting moieties.

Allatostatins are 6-18 amino acid peptides synthezed by insects to control production of juvenile hormones, which in turn regulate functions including metamorphosis and egg production. Four insect allatostatin neuropeptide analogues incorporating turn-promoting pseudopeptide moieties in the region responsible for biological activity were prepared by solid phase peptide synthetic methods. Bioassay indicated that activities approached those of the natural neuropeptides, and molecular models based on NMR data showed similar conformations and the presence of a beta-turn in the active core region for the four analogues. Differences in activity are believed to be due to differences in bulk and relative position of atoms in the unnatural portion of the analogues, and their differing degrees of conformational freedom. The studies support the feasibility of development of neuropeptide-based insect control agents resistant to peptidase deactivation.

Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway.

Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein. Taxadiene synthase cDNA was amplified by polymerase chain reaction and then subcloned into pET3d and pET32a(+) to form pET3dTX and pET32TX, respectively. The expressed taxadiene synthase from E. coli BL21(DE3)/pET3dTX was present completely as inclusion bodies. The transformant E. coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble protein) when induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C). The recombinant enzyme was purified by a single step with a His-binding metal affinity column. The maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(DE3)/pET32TX. The purified recombinant taxadiene synthase fusion protein was similar to native protein in steady-state kinetic parameters and mobility on sodium sulfate-polyacrylamide gel electrophoresis. The protein purified from E. coli BL21(DE3)/pET3dTX had the expected N-terminal (AQLSFNA) sequence.

Overexpression, single-step purification, and site-directed mutagenetic analysis of casbene synthase.

Casbene synthase is a diterpene cyclase isolated from castor bean (Ricinus communis L), which catalyzes the cyclization of geranylgeranyl diphosphate to form the phytoalexin casbene. We here report the overexpression of casbene synthase in Escherichia coli in soluble form using a thioredoxin fusion system. The amplified DNA by PCR carried on pCS7 was inserted into the expression vector pET32b(+) to form pCAS.2. The resulting transformants of pCAS. 2/BL21(DE3) produced a thioredoxin casbene synthase fusion protein (20-30% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside at 20 degrees C. Recombinant casbene synthase was purified to homogeneity in a single step with a His-binding metal-affinity column. Casbene synthase has a conserved aspartate-rich region [amino acids 355-359 (DDTID)], one cysteine, and three histidines with several prenyl transferases and terpene cyclases. Seven mutants were constructed by site-directed mutagenesis. The importance of Asp 355 and Asp 356 for catalysis was established by an increase in Km as well as a reduction in kcat in the corresponding glutamate mutants. These results indicate that the first and the second aspartate are involved in catalysis, while the third aspartate and the conserved cysteine and histidine residues selected for mutagenesis appear not to be involved in catalysis.

Conformational studies of paclitaxel analogs modified at the C-2' position in hydrophobic and hydrophilic solvent systems.

The conformations of two paclitaxel analogs modified at the C-2' position, 2'-deoxypaclitaxel and 2'-methoxypaclitaxel, were studied in hydrophobic and hydrophilic solvent systems by a combination of NMR spectroscopy, CD measurements, and molecular modeling. Both analogs have hydrophobic and hydrophilic conformations that resemble those of paclitaxel itself in the same media. Since the two have diminished biological activities in a number of bioactivity assays and the hydrogen-bonding capability of the 2'-hydroxyl group has been eliminated, we postulate that this group is involved in hydrogen bonding with tubulin and plays an important role in molecular recognition. The results of this study are in agreement with our earlier report on paclitaxel 2'-acetate, an analog in which the 2'-hydroxyl group hydrogen-bonding capacity has also been eliminated.

Enzymatic synthesis of S-adenosyl-L-methionine on the preparative scale.

The problems inherent in the enzymatic and chemical synthesis of S-adenosyl-L-methionine (SAM) led us to develop an efficient, simple method for the synthesis of large amounts of labeled SAM. Previously, we reported that the problem of product inhibition of E. coli SAM synthetase encoded by the metK gene was successfully overcome in the presence of sodium p-toluenesulfonate (pTsONa). This research has now been expanded to demonstrate that product inhibition of this enzyme can also be overcome by adding a high concentration of beta-mercaptoethanol (beta ME), acetonitrile, or urea. In addition a recombinant strain of E. coli has been constructed that expresses the yeast SAM synthetase encoded by the sam2 gene. The yeast enzyme does not have the problem of product inhibition seen with the E. coli enzyme. Complete conversion of 10 mM methionine to SAM was achieved in incubations with either the recombinant yeast enzyme and 1 molar potassium ion or the E. coli enzyme in the presence of additives such as beta ME, acetonitrile, urea, or pTsONa. The recombinant yeast SAM synthetase was used to generate SAM in situ for use in the multi-enzymatic synthesis of precorrin 2.

A simple algorithm for superimposing sets of NMR derived structures: its application to the conformational study of cephalomannine in lipophobic and lipophilic solution.

A simple iterative method for superimposing sets of NMR derived structures and calculation of the root mean square deviation (RMSD) of the sets is described. It was compared to the commonly used algorithm involving pairwise best fitting in the conformational study of the taxoid anticancer drug cephalomannine in lipophobic and lipophilic solvents. Lower RMSD values were obtained, indicating a better superposition of the structures in the sets. The conformations of cephalomannine in the two solvent systems reported are in good agreement with earlier conformational studies on other active taxoids.

NMR and molecular modeling study of the conformations of taxol 2'-acetate in chloroform and aqueous dimethyl sulfoxide solutions.

Taxol 2'-acetate, an analog of the antitumor drug taxol, displays no significant in vitro microtubule polymerization activity, thus underscoring the importance of a free 2'-OH group to the biological activity of taxol. Previous work had suggested that the inactivity of taxol 2'-acetate is not due to steric interference by the acetyl group. The present study examined the conformations of taxol 2'-acetate in deuteriochloroform and (2)H2O-deuteriodimethyl sulfoxide solutions and found them to be essentially the same as the respective conformations adopted by taxol itself. Thus, neither destabilization of an active taxol conformation by the acetyl group nor the formation of an important taxol conformation determining role for the 2'-OH group appears likely. The implication of these findings is that the taxol 2'-OH group interacts directly with a protein residue in the taxol-microtubule complex, perhaps as a hydrogen bond donor.

Evidence for conformational changes in Escherichia coli porphobilinogen deaminase during stepwise pyrrole chain elongation monitored by increased reactivity of cysteine-134 to alkylation by N-ethylmaleimide.

Porphobilinogen deaminase from Escherichia coli becomes progressively more susceptible to inactivation by the thiophilic reagent N-ethylmaleimide (NEM) as the catalytic cycle proceeds through the enzyme-intermediate complexes ES, ES2, ES3, and ES4. Site-directed mutagenesis of potentially reactive cysteines has been used to identify cysteine-134 as the key residue that becomes modified by the reagent and leads to inactivation. Since cysteine-134 is buried at the interface between domains 2 and 3 of the E. coli deaminase molecule, the observations suggest that a stepwise conformational change occurs between these domains during each stage of tetrapyrrole assembly. Interestingly, mutation of the invariant active-site cysteine-242 to serine leads to an enzyme with up to a third of the catalytic activity found in the wild-type enzyme. Electrospray mass spectrometry indicates that serine can substitute for cysteine as the dipyrromethane cofactor attachment site.

Genetically engineered synthesis of precorrin-6x and the complete corrinoid, hydrogenobyrinic acid, an advanced precursor of vitamin B12.

BACKGROUND: Genetically engineered synthesis, in which the gene products, cofactors, and substrates of a complete pathway are combined in vitro in a single flask to give the target, can be a viable alternative to conventional chemical construction of molecules of complex structure and stereochemistry. We chose to attempt to synthesize the metal-free corrinoid hydrogenobyrinic acid, an advanced precursor of vitamin B12. RESULTS: Cloning and overexpression of the genes necessary for the S-adenosyl methionine dependent conversion of 5-aminolevulinic acid (ALA) to precorrin-3 and those required for the synthesis of hydrogenobyrinic acid from precorrin-3 completed the repertoire of the 12 biosynthetic enzymes involved in corrin synthesis. Using these enzymes and the necessary cofactors, the multi-enzyme synthesis of hydrogenobyrinic acid from ALA can be achieved in 20% overall yield in a single reaction vessel, corresponding to an average of at least 90% conversion for each of the 17 steps involved. CONCLUSIONS: By replacing the cell wall with glass, and by mixing the soluble biosynthetic enzymes and necessary cofactors, the major segment of the physiological synthesis of vitamin B12 has been accomplished. Since only those enzymes necessary for the synthesis of hydrogenobyrinic acid from ALA are supplied, none of the intermediates is deflected from the direct pathway. This results in an efficiency which in fact surpasses that of nature.

Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein.

The C-terminus of the Escherichia coli CysG protein, consisting of amino acids 202-457, was expressed as a recombinant protein using gene dissection methodology. Analysis of the activity of this truncated protein, termed CysGA, revealed that it was able to methylate uroporphyrinogen III in the same S-adenosyl-L-methionine (SAM)-dependent manner as the complete CysG protein. However, this truncated protein was not able to complement E. coli cysG cells, thereby suggesting that the first 201 amino acids of the CysG protein had an enzymic activity associated with the conversion of dihydrosirohydrochlorin into sirohaem. Analysis of the N-terminus of the CysG protein revealed the presence of a putative pyridine dinucleotide binding site. When the purified CysG protein was incubated with NADP+, uroporphyrinogen III and SAM the enzyme was found to catalyse a coenzyme-mediated dehydrogenation to form sirohydrochlorin. The CysGA protein on the other hand showed no such coenzyme-dependent activity. Analysis of the porphyrinoid material isolated from strains harbouring plasmids containing the complete and truncated cysG genes suggested that the CysG protein was also involved in ferrochelation. The evidence presented in this paper suggests that the CysG protein is a multifunctional protein involved in SAM-dependent methylation, pyridine dinucleotide dependent dehydrogenation and ferrochelation.

Biosynthesis of vitamin B12. Discovery of the enzymes for oxidative ring contraction and insertion of the fourth methyl group.

In the vitamin B12 biosynthetic pathway the enzymes responsible for the conversion of precorrin-3 to precorrin-4 have been identified as the gene products of cobG and cobJ from Pseudomonas denitrificans. CobG catalyzes the oxidation of precorrin-3 to precorrin-3x (a hydroxy lactone) whereas CobJ is a SAM-dependent C-17 methyl transferase and is necessary for ring contraction. A mechanism for ring contraction is proposed.

Interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 17 beta-estradiol in MCF-7 human breast cancer cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are both tumor promoters which act through different mechanisms. In MCF-7 human breast cancer cells, both TCDD and TPA inhibited constitutive and 17 beta-estradiol-induced cell proliferation but showed no apparent interactive effects. TCDD also inhibited the 17 beta-estradiol-induced secretion of the 52-kDa protein (procathepsin D) and induced CYP1A1 gene expression whereas TPA alone was inactive for these responses. Moreover, TPA did not modulate the TCDD-mediated antiestrogenic or induction responses and did not decrease levels of the nuclear Ah receptor complex as determined in a gel mobility shift assay using a 32P-dioxin responsive element (DRE). The interactions of TPA and TCDD on the metabolism of [13C]glucose to [13C]lactate was also investigated using 13C-nuclear magnetic resonance spectroscopy. The rate of formation of [13C]lactate from [13C]glucose in MCF-7 cells treated with DMSO (control), 1 nM 17 beta-estradiol, 1 nM TCDD, 1 nM TCDD plus 1 nM 17 beta-estradiol, and 0.1 ng/ml TPA plus 1 nM 17 beta-estradiol was 28, 48, 20, 22 and 50 fmol lactate formed/cell/h, respectively. Thus, TCDD, but not TPA, inhibited this estrogen-induced response. However, a comparison of the rate of lactate formation in cells treated with TCDD plus 17 beta-estradiol (22 fmol/cell/h) or TCDD plus 17 beta-estradiol plus TPA (61 fmol/cell/h) showed that TPA significantly inhibited the TCDD-mediated antiestrogenic response. The results of these studies in MCF-7 cells demonstrate that the interactions of TCDD and TPA are highly response-specific and do not involve TPA-mediated downregulation of the nuclear Ah receptor complex.

Expression of 9 Salmonella typhimurium enzymes for cobinamide synthesis. Identification of the 11-methyl and 20-methyl transferases of corrin biosynthesis.

Nine of the cbi genes from the 17.5 kb cob operon of Salmonella typhimurium previously shown by genetic studies to be involved in the biosynthesis of cobinamide from precorrin-2, have been subcloned and expressed in Escherichia coli. Seven of the gene products were found in the soluble fraction of cell lysates and have been purified. The gene products corresponding to cbi E, F, H and L were shown by SAM binding and by homology with other SAM-binding proteins to be candidates for the methyltransferases of vitamin B12 biosynthesis. The enzymatic functions of the gene products of cbiL and cbiF are associated with C-methylation at C-20 of precorrin-2 and C-11 of precorrin-3.

Enzymatic synthesis and structure of precorrin-3, a trimethyldipyrrocorphin intermediate in vitamin B12 biosynthesis.

The trimethylated intermediate of vitamin B12 (corrin) biosynthesis, precorrin-3, was produced from various 13C-enriched isotopomers of 5-aminolevulinic acid (ALA), using a multiple-enzyme system containing ALA dehydratase, porphobilinogen deaminase, uro'gen III synthetase, and the S-adenosyl-L-methionine-(SAM)-dependent uro'gen III methyltransferase (M-1) and precorrin-2 methyltransferase (M-2) in the presence of [13C]SAM. Structural analysis of the resulting product, precorrin-3, reveals a close similarity to precorrin-2 but with several subtle differences in the conjugated array of C = C and C = N bonds which reflect the presence of the new C-methyl group at C20 and its influence on the electronic distribution in the dipyrrocorphin chromophore. The implications of this structure for corrin biosynthesis are discussed.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on 17 beta-estradiol-induced glucose metabolism in MCF-7 human breast cancer cells: 13C nuclear magnetic resonance spectroscopy studies.

The effects of 17 beta-estradiol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and their combination on the metabolism of [1-13C] glucose were determined in cell suspensions of wild-type MCF-7 human breast cancer cells, by 13C NMR spectroscopy. Preliminary studies showed that, during the 7-hr duration of the NMR experiment, the cells maintained their viability and their aryl hydrocarbon responsiveness. Lactate was the major glucose metabolite detected in these studies, and the rate of lactate formation in the untreated (control) and 17 beta-estradiol (10(-9) M)-treated cells was 60 and 86 fmol/cell/hr, respectively; this represented a 40% increase in lactate formation in the cells treated with 17 beta-estradiol; comparable results were observed for the percentage of glucose converted into lactate. In contrast, TCDD (10(-9) M) did not significantly alter the rate of glucose metabolism or lactate formation. Co-treatment of the cells with 17 beta-estradiol (10(-9) M) plus TCDD (10(-8) to 10(-10) M) showed that TCDD completely inhibited the 17 beta-estradiol-induced metabolism of [13C] glucose to lactate in MCF-7 cells. In contrast, 2,8-dichlorodibenzo-p-dioxin (10(-8) M), a weak aryl hydrocarbon receptor agonist, did not inhibit estrogen-induced glucose-to-lactate metabolism in MCF-7 cells. In addition, it was shown that TCDD caused a significant decrease in 17 beta-estradiol-induced lactate formation within 1 hr after treatment, whereas the induction of monooxygenase activity was not observed until 3 hr after exposure of the cells to TCDD. These data indicate that TCDD-induced 17 beta-estradiol metabolism is not related to the decrease in the rate of conversion of glucose to lactate. These results further define the antiestrogenic responses elicited by TCDD and show that 13C NMR spectroscopy provides a unique method for measuring, in real time, the effects of TCDD on specific metabolic pathways.