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Biomedical subjects

A I Scott

Publications and source records attributed to A I Scott.

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

Biosynthesis of vitamin B12: concerning the identity of the two-carbon fragment eliminated during anaerobic formation of cobyrinic acid.

It has been proved that, during anaerobic biosynthesis of the corrin macrocycle, the two-carbon fragment excised from the precursor, precorrin-3, is acetaldehyde, which originates from C-20 and its attached methyl group. This apparently contradictory finding is rationalized in terms of the subsequent enzymatic oxidation of acetaldehyde to acetic acid, which was previously regarded as the volatile fragment released by the action of the biosynthetic enzymes of Propionibacterium shermanii. The observation that acetaldehyde (rather than acetic acid) is extruded during anaerobic B12 synthesis is in full accord with the structure of factor IV, a new intermediate on the pathway.

Anaerobiosis↗
PubMed1996

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.

Acetates↗
PubMed1996

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.

Escherichia coli↗
PubMed1996

Achieving natural product synthesis and diversity via catalytic networking ex vivo.

Recent studies on ex vivo synthesis of natural products reveal that even complex multistep pathways can be successfully reconstructed. Genetic engineering of such reconstituted pathways has already been used to generate 'unnatural' natural products related to the original compound. In the future, it may be possible to use these approaches to make natural products that are currently inaccessible to conventional synthesis.

Carbohydrates↗
PubMed1996

Genetically engineered synthesis of natural products: from alkaloids to corrins.

Because many natural products are of biological and medicinal importance, methods are continually being sought for studying their biosynthetic pathways, which may eventually result in increased production and the generation of novel compounds. Advances in genetic engineering have enabled the homologous or heterologous expression of many natural product biosynthetic genes from divergent sources, resulting in a supply of enzymes not readily available by isolation from the producing organism. Mixing and matching of these enzymes in cell-free reactions can provide information, not available by any other means, about enzyme mechanisms, pathway intermediates, and possible variations in the structure of the final product.

Alkaloids↗
PubMed1995

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.

Alkylation↗
PubMed1995

Overexpression in Escherichia coli of 12 vitamin B12 biosynthetic enzymes.

The first 12 enzymes involved in the biosynthesis of vitamin B12 from its five-carbon precursor, aminolevulinic acid, have been overexpressed in recombinant strains of Escherichia coli. The activity of each enzyme has been demonstrated by the biosynthesis of hydrogenobyrinic acid from aminolevulinic acid.

Aminolevulinic Acid↗
PubMed1995

Diversity in cyclic sesquiterpene production by Gossypium hirsutum.

Major sesquiterpene components of oil of Texas Race Stock 810 of Gossypium hirsutum were alpha- and beta-selinene. This is the seventh cyclic terpene type found to date in this genus. Both alpha- and beta-selinene, along with aromadendrene, were found but only as minor components of extracts of several domestic cultivars of G. hirsutum.

Gossypium↗
PubMed1995

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).

We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.

Amino Acid Sequence↗
PubMed1995

Frequent deliberate self-harm: repetition, suicide and cost after three or more years.

This is a follow-up study of 44 patients who were admitted to the Royal Infirmary of Edinburgh after frequent deliberate self-harm (defined as three or more episodes of deliberate self-harm within one week) at least once between 1982 and 1987. Forty-two patients (95%) were traced after a minimum of three years (range 3yr 1 mo 8yr 9mo). Thirty eight patients were known to have repeated self-harm an average of 13 times during the follow-up. Only two patients had died, one of these by probable suicide. The average cost solely attributable to the in-patient accommodation and psychiatric assessment of this group during follow-up was 34,036.60 pounds (1986-87 prices).

Adolescent↗
PubMed1995

ECT seizure threshold.

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Antidepressive Agents, Second-Generation↗
PubMed1995

The Edinburgh Primary Care Depression Study: personality disorder and outcome.

BACKGROUND: Little is known about the impact of personality pathology on the treatment outcome of major depressive illness in primary care in the UK. METHOD: Patients meeting criteria for DSM-III major depressive disorder were randomly allocated to one of four treatments each lasting 16 weeks, then followed up for 18 months. Assessments were made of depressive symptoms, personality and social functioning. Personality was assessed at maximum improvement or 16 weeks. RESULTS: The prevalence of personality disorder (PD) in the sample of 113 patients was 26%. Patients with a PD were significantly younger and rated more depressed at entry than patients with no personality disorder (NoPD). On completion of treatment patients with a PD were significantly more depressed and had poorer social functioning than the NoPD group. After 18 months there were no differences in ratings of depression or social functioning between the groups. CONCLUSIONS: There was substantial improvement in both the PD and NoPD groups. The presence of personality pathology delays recovery from major depressive illness.

Adolescent↗
PubMed1995

Fluorescence-based method for selection of recombinant plasmids.

A fluorescence-based method for the selection of recombinant plasmids has been developed. Escherichia coli strains bearing plasmids for the overexpression of the gene encoding uroporphyrinogen III methyltransferase (cobA or cysG gene) accumulate fluorescent porphyrinoid compounds. When illuminated with ultraviolet light, the cells fluoresce with a bright red color. Replacement or disruption of the gene with other fragments of DNA results in loss of enzymatic activity and nonfluorescent cells. The detection of the recombinant plasmids can be accomplished without special host strains or expensive chromogenic reagents required for the blue-white screening technique.

Aminolevulinic Acid↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

Recent studies of enzymically controlled steps in B12 biosynthesis.

The acquisition and sequencing of the genes encoding the enzymes for vitamin B12 biosynthesis in Salmonella typhimurium and Pseudomonas denitrificans has dramatically altered the direction of research on the pathway from uroporphyrinogen III to the corrinoids. Through a combination of molecular biology, organic chemistry and NMR spectroscopy, logical progression along the sequence is being made. Recent work from our laboratory is focused on the discovery and specificities of the methyltransferases connecting uroporphyrinogen III with cobyrinic acid, the temporal resolution of cobalt insertion and a comparison of the anaerobic pathway in S. typhimurium and the aerobic pathway in Ps. denitrificans. The implication of two parallel routes to corrins in these bacteria is discussed.

Chromosome Mapping↗