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At least 199 records · Page 11Linked to original sources
PubMed1983

Novel transformations of i-cholesterol and 6 beta-methoxy-i-cholesterol by Moraxella sp.

A soil microorganism was isolated by the enrichment culture technique using cholesterol as the sole source of carbon. The organism has been identified as belonging to the genus Moraxella. With this organism two novel biotransformations of sterols were observed viz. (1) isomerization of 3 alpha,5 alpha-cyclocholestan-6 beta-ol (i-cholesterol) to cholesterol, (2) demethylation of 6 beta-methoxy-3 alpha,5 alpha-cyclocholestane (6 beta-methoxy-i-cholesterol) to i-cholesterol with subsequent isomerization to cholesterol. The enzymes responsible for these transformations were shown to be inducible. The pH optimum of the partially purified i-cholesterol isomerase was found to be 8.4. The apparent Km value for i-cholesterol was 1.43 microM. A plausible mechanism for the i-cholesterol isomerization has been discussed.

Biotransformation↗
PubMedDate not supplied

Drug design based on biosynthetic studies: synthesis, biological activity, and kinetics of new inhibitors of 2,3-oxidosqualene cyclase and squalene epoxidase.

Various classes of inhibitor of 2,3-oxido squalene cyclase have been synthesized and tested on rat liver and Saccharomyces cerevisiae microsomes, 3T3 fibroblast cultures, and various bacteria, fungi, and yeasts. The compounds include azasqualenes, azasqualanes, bis-azasqualenes, bis-azasqualanes, and N-oxide and ammonium derivatives of squalene. In order to better mimic the transition state involved in the SN2-like opening of 2,3-oxidosqualene, we synthesized squalene N-methyloxaziridine. Other derivatives tested were N-methylimine, aminalic hydroperoxide, and N-methylamide. We also attempted to produce new "suicide" inhibitors of SO cyclase, such as a squalenoid epoxide vinyl ether. Many of the products described inhibited the various cyclases, the best having an IC50 of 0.3 microM on plants and 1.5 microM on rat liver microsomes, and good antibacterial and antifungal activity. In a search for inhibitors of squalene epoxidase, a series of mono- and bifunctional squalenoid acetylenes and allenes were synthesized. Some of them proved to be inhibitors of squalene epoxidase.

Alkynes↗
PubMed1993

Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MAT alpha 2 repressor.

Attachment of ubiquitin to proteins is catalyzed by a family of ubiquitin-conjugating (UBC) enzymes. Although these enzymes are essential for many cellular processes; their molecular functions remain unclear because no physiological target has been identified for any of them. Here we show that four UBC proteins (UBC4, UBC5, UBC6, and UBC7) target the yeast MAT alpha 2 transcriptional regulator for intracellular degradation by two distinct ubiquitination pathways. UBC6 and UBC7 define one of the pathways and can physically associate. The UBC6/UBC7-containing complex targets the Deg1 degradation signal of alpha 2, a conclusion underscored by the finding that UBC6 is encoded by DOA2, a gene previously implicated in Deg1-mediated degradation. These data reveal an unexpected overlap in substrate specificity among diverse UBC enzymes and suggest a combinatorial mechanism of substrate selection in which UBC enzymes partition into multiple ubiquitination complexes.

Epistasis, Genetic↗
PubMed1990

Oxygen derepresses deacetoxycephalosporin C synthase and increases the conversion of penicillin N to cephamycin C in Streptomyces clavuligerus.

When dissolved oxygen (DO) was maintained at saturation level during batch fermentations of Streptomyces clavuligerus (NRRL 3585), the accumulation of the intermediate penicillin N was lowered while formation of the end product cephamycin C was increased relative to fermentations without DO control. The specific activity of the penicillin ring-expansion enzyme deacetoxycephalosporin C synthase (DAOCS) was increased 2.3-fold under oxygen saturated conditions, whereas the penicillin ring-cyclizing enzyme isopenicillin N synthase (IPNS) showed only a 1.3-fold increase. Thus oxygen derepression of DAOCS appears to be an important regulatory mechanism in the conversion of penicillin N to cephamycin C in S. clavuligerus. IPNS, an early acting enzyme in cephamycin C biosynthesis, and DAOCS, which acts late in the pathway, both disappeared from cell extracts at 60 h, just prior to cessation of cephamycin production.

Cephamycins↗
PubMed1992

Molecular design and catalytic function of a B12-enzyme mimetic.

Developing artificial holoenzymes requires attention to the molecular design, not only of the active site, but also of its microenvironment, which in the naturally occurring enzyme is provided by the apoprotein. Microenvironmental effects play a major role in determining the catalytic performance of an enzyme. Using the development of a B12-dependent holoenzyme mimetic as an example, the important factors and concepts underlying the approaches to developing an artificial holoenzyme are discussed.

Amino Acid Isomerases↗
PubMed1985

Differences in the inhibitory effects of N-(1-n-dodecyl)-heterocycles on the 2,3-oxidosqualene lanosterol-cyclase of rat liver and yeast.

The ability of thirteen N-(1-n-dodecyl)-heterocyclic compounds and one N-(1-n-fluoroalkyl)-heterocyclic compound to inhibit the 2,3-oxidosqualene lanosterol-cyclase of rat liver and yeast has been tested. Eight of the N-(1-n-dodecyl)-heterocycles inhibited the rat liver enzyme well but none inhibited the yeast enzyme at 100 microM concentrations. The N-(1-fluoroalkyl)-heterocycle inhibited the rat liver enzyme mildly but did not inhibit the yeast enzyme at 100 microM concentration. It is evident that the rat liver and yeast cyclases are different; the possible nature of these differences is discussed.

Animals↗
PubMed1989

delta-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA.

Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of delta-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis. The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.

Alanine↗
PubMed1996

A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene.

A new gene (menF) encoding an isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis has been cloned and sequenced. Overexpression of the encoded polypeptide under the influence of a T7 promoter showed an increase in specific activity of 2200-fold. Treatment with protamine sulfate resulted in another 3.5-fold increase in specific activity (7700-fold compared to the parent strain). The relative molecular mass of the overexpressed protein was M(r) 49 000, which is in full agreement with the DNA sequence predicted molecular mass of 48 777 Da. Purified enzyme converted chorismate to isochorismate with the product of the reaction shown to be isochorismate by its thermal conversion to salicylic acid. The fluorescence spectrum generated by the formed salicylic acid was identical to that of authentic salicylic acid. The 5' end of the flanking menD gene has also be redefined.

Amino Acid Sequence↗
PubMed1989

An electrophoretic molecular karyotype for an industrial strain of Cephalosporium acremonium.

An electrophoretic molecular karyotype has been established for an industrial strain of Cephalosporium acremonium using transverse alternating field electrophoresis. Eight chromosome bands were detected in gently prepared DNA samples. The size of the chromosomes ranged from approx. 1700 kb up to greater than 4000 kb. The total genomic content for this strain of C. acremonium is at least 22,500 kb. Hybridization analyses revealed that two key genes involved in cephalosporin C biosynthesis are not physically linked to one another. The isopenicillin N synthetase gene (pcbC) resides on chromosome (chr.) VI while the deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthetase gene (cefEF) resides on chr. II. The ribosomal RNA genes were located on chr. VII, while the beta-isopropylmalate dehydrogenase gene (LEU2) was found to be linked to the pcbC gene on chr. VI.

3-Isopropylmalate Dehydrogenase↗
PubMed1990

Cloning and characterization of the 2,3-oxidosqualene cyclase-coding gene of Candida albicans.

2,3-Oxidosqualene (OS) cyclase (OSC) catalyzes the conversion of OS to lanosterol, an essential step in the biosynthesis of sterols. The Candida albicans gene (ERG7) encoding OSC was cloned by complementation of a Saccharomyces cerevisiae OSC mutant (erg7). Two different Erg+ clones were isolated that contain a common overlapping region. The minimum region required for complementation was determined to be approx. 3.2 kb and a single 2.7-kb ERG7 transcript was detected. The cloned Candida ERG7 DNA complemented an additional nonconditional erg7 allele and a temperature-sensitive erg7 mutation. OSC activity was restored in the mutants as determined by [14C]acetate incorporation in vivo as well as incorporation in vitro in cell-free extracts using either [14C]isopentenyl pyrophosphate or [3H]OS as substrate. The level of OSC produced from expression of a single copy of the Candida ERG7 sequence was sufficient to allow growth of the S. cerevisiae erg7 mutants in the absence of exogenous ergosterol. These data support the contention that the Candida ERG7 sequence is the structural gene for OSC.

Alleles↗
PubMed1992

Cloning and characterization of genes involved in the biosynthesis of delta-aminolevulinic acid in Escherichia coli.

Several mutants of Escherichia coli that had lost their ability to synthesize delta-aminolevulinic acid (ALA) via the C5 pathway were isolated. Their defective loci were classified into two groups, AlaA- and AlaB-. The genes that complemented these mutations were cloned. Nucleotide sequencing indicated that the gene that complemented AlaA- was identical to hemL which is located at 4 min on the E. coli chromosome and encodes glutamate 1-semialdehyde aminotransferase. The gene complementing AlaB- contained an open reading frame (ORF) encoding a polypeptide of 207 amino acids that was found to be a new gene involved in the synthesis of ALA via the C5 pathway. Thus, we designated the gene hemM. The hemM gene was adjacent to hemA that is located at 27 min and previously thought to encode glutamyl-tRNA dehydrogenase. However, we found that hemA complemented both the AlaA- (hemL) and AlaB- (hemM) mutants defective in the C5 pathway although the transformants showed small colonies on the selective medium without ALA. These results suggest that hemA is not involved in the C5 pathway, but controls a second, minor pathway for the synthesis of ALA.

Amino Acid Sequence↗
PubMed1993

Sequence of the Candida albicans erg7 gene.

The nucleotide sequence of the Candida albicans erg7 gene, which complements erg7 mutants of Saccharomyces cerevisiae and restores oxidosqualene cyclase activity, was determined. The gene encodes a 728-aa protein that displays homology with squalene-hopene cyclase, providing further evidence that erg7 is the gene encoding 2,3-oxidosqualene cyclase.

Amino Acid Sequence↗
PubMed1995

Structural organization of a Bacillus subtilis operon encoding menaquinone biosynthetic enzymes.

Menaquinone (MK) is a non-protein component of the Bacillus subtilis (Bs) electron transport chain synthesized from chorismate through a series of MK-specific reactions. The genes encoding biosynthesis of the naphthoquinone ring of MK are clustered at 273 degrees on the Bs chromosome. A 3.9-kb region capable of rescuing men mutants blocked in the early stages of MK biosynthesis was sequenced and found to contain three major open reading frames (ORFs). The first ORF (menF) has a predicted size of 51.8 kDa and 34% amino-acid identity with the isochorismate synthases of Escherichia coli (EntC) and Aeromonas hydrophila (AmoA), ORF2 (menD) a predicted size of 60.2 kDa and 21% identity with MenD of E. coli. ORF3 has a predicted size of 21.4 kDa and 29% identity to triacylglycerol lipase of Psychrobacter immobilis. No sequence corresponding to menC was identified. Plasmid integrational studies of the men gene cluster had suggested the presence of promoters secondary to the previously identified p1 men promoter. Sequence analysis revealed a putative promoter region upstream from ORF3.

Bacillus subtilis↗
PubMed1994

Highlights in haem biosynthesis.

The haem biosynthesis pathway continues to provide surprises, from the first enzyme, 5-aminolaevulinic acid synthase, the mRNA of which contains an iron-responsive element, to the last, ferrochelatase, that contains an iron sulphur cluster. 5-Aminolaevulinate dehydratase from animals are zinc-dependent enzymes while those from plants require magnesium. The first X-ray structure of a haem synthesis enzyme, porphobilinogen deaminase, has not only yielded clues about the mechanism of tetrapyrrole assembly but has also provided insight into the molecular basis of the human disease acute intermittent porphyria. Evidence is growing to suggest that a previously unsuspected alternative haem pathway may exist.

5-Aminolevulinate Synthetase↗
PubMed1994

Cobalamin-dependent methionine synthase: the structure of a methylcobalamin-binding fragment and implications for other B12-dependent enzymes.

Cobalamin-dependent methionine synthase is a large enzyme composed of structurally and functionally distinct regions. Recent studies have begun to define the roles of several regions of the protein. In particular, the structure of a 27 kDa cobalamin-binding fragment of the enzyme from Escherichia coli has been determined by X-ray crystallography, and has revealed the motifs and interactions responsible for recognition of the cofactor. The amino acid sequences of several adenosylcobalamin-dependent enzymes, the methylmalonyl coenzyme A mutases and glutamate mutases, show homology with the cobalamin-binding region of methionine synthase and retain conserved residues that are determinants for the binding of the prosthetic group, suggesting that these mutases and methionine synthase share common three-dimensional structures.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1992

Glutamyl-transfer RNA: a precursor of heme and chlorophyll biosynthesis.

In green plants, archaebacteria and many eubacteria, the porphyrin ring that is common to both chlorophyll and heme is synthesized from 5-aminolevulinic acid (ALA) via an interesting pathway. This two-step process involves the unusual enzymes glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase. Interest in this pathway has increased since it was discovered that a tRNA cofactor was required for the formation of ALA. This tRNA(Glu) is common to the biosyntheses of both porphyrins and proteins.

Aldehyde Oxidoreductases↗