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.

Mutagenesis identifies a conserved tyrosine residue important for the activity of uroporphyrinogen III synthase from Anacystis nidulans.

Uroporphyrinogen III synthase from the cyanobacterium Anacystis nidulans was overproduced in Escherichia coli and analyzed by site specific mutagenesis. Of the nine conserved amino acids altered, only a single tyrosine mutant (Y166F) showed any significant decrease in activity suggesting this residue is critical for proper substrate binding and/or catalysis.

Sterol carrier protein-2: structure reveals function.

The multiple actions of sterol carrier protein-2 (SCP-2) in intracellular lipid circulation and metabolism originate from its gene and protein structure. The SCP-x/pro-SCP-2 gene is a fusion gene with separate initiation sites coding for 15-kDa pro-SCP-2 (no enzyme activity) and 58-kDa SCP-x (a 3-ketoacyl CoA thiolase). Both proteins share identical cDNA and amino acid sequences for 13-kDa SCP-2 at their C-termini. Cellular 13-kDa SCP-2 derives from complete, posttranslational cleavage of the 15-kDa pro-SCP-2 and from partial posttranslational cleavage of 58-kDa SCP-x. Putative physiological functions of SCP-2 have been proposed on the basis of enhancement of intermembrane lipid transfer (e.g., cholesterol, phospholipid) and activation of enzymes involved in fatty acyl CoA transacylation (cholesterol esters, phosphatidic acid) in vitro, in transfected cells, and in genetically manipulated animals. At least four important SCP-2 structural domains have been identified and related to specific functions. First, the 46-kDa N-terminal presequence present in 58-kDa SCP-x is a 3-ketoacyl-CoA thiolase specific for branched-chain acyl CoAs. Second, the N-terminal 20 amino acid presequence in 15-kDa pro-SCP-2 dramatically modulates the secondary and tertiary structure of SCP-2 as well as potentiating its intracellular targeting coded by the C-terminal peroxisomal targeting sequence. Third, the N-terminal 32 amino acids form an amphipathic a-helical region, one face of which represents a membrane-binding domain. Positively charged amino acid residues in one face of the amphipathic helices allow SCP-2 to bind to membrane surfaces containing anionic phospholipids. Fourth, the hydrophobic faces of the N-terminal amphipathic a helices along with beta strands 4, 5, and helix D form a ligand-binding cavity able to accommodate multiple types of lipids (e. g., fatty acids, fatty acyl CoAs, cholesterol, phospholipids, isoprenoids). Two-dimensional 1H-15N heteronuclear single quantum coherence spectra of both apo-SCP-2 and of the 1:1 oleate-SCP-2 complex, obtained at pH 6.7, demonstrated the homogenous formation of holo-SCP-2. While comparison of the apo- and holoprotein amide fingerprints revealed about 60% of the resonances remaining essentially unchanged, 12 assigned amide residues underwent significant chemical-shift changes upon oleic acid binding. These residues were localized in three regions: the juncture of helices A and B, the mid-section of the beta sheet, and the interface formed by the region of beta strands 4, 5, and helix D. Circular dichroism also showed that these chemical-shift changes, upon oleic acid binding, did not alter the secondary structure of SCP-2. The nuclear magnetic resonance chemical shift difference data, along with mapping of the nearby hydrophobic residues, showed the oleic acid-binding site to be comprised of a pocket created by the face of the beta sheet, helices A and B on one end, and residues associated with beta strands 4, 5, and helix D at the other end of the binding cavity. Furthermore, the hydrophobic nature of the previously ill-defined C-terminus suggested that these 20 amino acids may form a 'hydrophobic cap' which closes around the oleic acid upon binding. Thus, understanding the structural domains of the SCP-x/pro-SCP-2 gene and its respective posttranslationally processed proteins has provided new insights into their functions in intracellular targeting and metabolism of lipids.

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.

Crystal structure of precorrin-8x methyl mutase.

BACKGROUND: The crystal structure of precorrin-8x methyl mutase (CobH), an enzyme of the aerobic pathway to vitamin B12, provides evidence that the mechanism for methyl migration can plausibly be regarded as an allowed [1,5]-sigmatropic shift of a methyl group from C-11 to C-12 at the C ring of precorrin-8x to afford hydrogenobyrinic acid. RESULTS: The dimeric structure of CobH creates a set of shared active sites that readily discriminate between different tautomers of precorrin-8x and select a discrete tautomer for sigmatropic rearrangement. The active site contains a strictly conserved histidine residue close to the site of methyl migration in ring C of the substrate. CONCLUSION: Analysis of the structure with bound product suggests that the [1,5]-sigmatropic shift proceeds by protonation of the ring C nitrogen, leading to subsequent methyl migration.

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.

Multiple biosynthetic pathways for vitamin B12: variations on a central theme.

The manner in which vitamin B12 is synthesized is detailed with emphasis on the different mechanisms for ring contraction encountered in aerobic and anaerobic organisms. The aerobic process utilizes two enzymes and is dependent on molecular oxygen, in stark contrast to the anaerobic mechanism which is controlled by cobalt and requires only one enzyme.

Reflections on the discovery of nature's pathways to vitamin B12.

The chronology of the discoveries along the pathway of vitamin B12 biosynthesis is reviewed from a personal perspective, including discussion of the most recent finding that two pathways to B12 exist--one aerobic and one anaerobic--which differ mainly in the ring contraction mechanisms which convert porphyrin to corrin.

The effect of repeated bilateral electroconvulsive therapy on seizure threshold.

Seizure threshold was measured by empirical titration in 28 patients referred for bilateral electroconvulsive therapy to treat depressive illness at the outset of treatment and after another six treatments. No patient was given antiepileptic drug treatment, and anesthetic technique and concomitant psychotropic drug treatment were fixed. The average (+/- SD) initial seizure threshold measured by set charge was 79.5 mC (+/- 33.4 mC), and this increased to 95.5 mC (+/- 37.9 mC). The average percentage increase was 22.8% (95% confidence interval, 13.7% to 31.8%). The seizure threshold measured by set charge did not change in 15 patients (54%), and there was no significant relation between change in seizure threshold and patient sex, change in seizure duration measured by cuff technique, or global clinical improvement during the course of treatment.

Variation in rates of electroconvulsive therapy use among consultant teams in Edinburgh (1993-1996).

BACKGROUND: Critics of electroconvulsive therapy (ECT) have expressed concern about variations in ECT use among consultant teams within the same hospital. The aim was to establish whether or not there was a significant variation in rates of ECT use among consultant teams in the same hospital when in-patient workload was taken into account. METHODS: A computerised database was used to calculate annual and aggregate rates of ECT use by consultant team, expressed as the number of individual in-patients treated per 100 in-patients discharged between 1993 and 1996. RESULTS: The variation in aggregate rates of ECT use varied approximately 18-fold among the 11 general adult psychiatric teams (P<0.001), and twofold among the three sector old-age psychiatric teams (P<0.05). CONCLUSIONS: Substantial variation in the rates of ECT use was confirmed, but only among general adult psychiatric teams. LIMITATIONS: The extent to which findings from one teaching hospital can be generalised was unknown. Possible explanations of the variations were not assessed.

Evidence-based psychiatry--do psychiatrists want it and can they do it?

OBJECTIVE: To examine (a) psychiatrists' attitudes to evidence-based psychiatry, (b) whether psychiatrists have identifiable clinical information needs, (c) if such information is practically obtainable, and (d) how psychiatrists respond to the information obtained. DESIGN: We surveyed senior psychiatrists to enquire about their attitudes and to request up to three clinical questions they would like answered. We attempted to find evidence to answer the five most frequently asked questions using recommended strategies and timed how long it took us. We fed our answers back to those who had asked one of these questions and asked if they found the answers useful. SETTING: Specialist registrars, senior registrars and consultants in south-east Scotland. RESULTS: Ninety three (76%) of those surveyed returned usable questionnaires. Respondents thought that only 40% of their practice is evidence-based. They reported that 'insufficient time' was the biggest barrier to implementing evidence-based psychiatry. The most frequently asked questions concerned the treatment of major psychiatric disorders. It took us--three experienced researchers, trained in critical appraisal, with excellent local facilities--between 15-60 minutes to answer each question. Most of those who had asked the questions (15/22) thought they would not have been able to answer them and stated that such a 'question answering service' as piloted here would be valuable. CONCLUSIONS: An evidence-based psychiatry appears to be desirable and possible, but impractical for the individual clinician. There is a need for short accessible evidence-based summaries of optimal treatment in psychiatry.

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.

A survey of methohexitone use by anesthetists in the clinical practice of ECT in Edinburgh.

The dosage of methohexitone (methohexital) administered by anesthetic staff was surveyed in a consecutive series of 52 patients referred for electroconvulsive therapy (ECT) in routine clinical practice in Edinburgh. Patients were weighed before the first treatment, and the ratio of administered dose to weight in kilograms calculated. Anesthesia was administered by three consultant staff and six nonconsultant staff. In only one patient (2%) was the administered dose within the range recommended by the Royal College of Psychiatrists (0.75-0.9 mg/kg), and in only four patients (8%) were the doses within the range recommended by the American Psychiatric Association (0.75-1.0 mg/kg). In all other patients the dose exceeded these recommended ranges; the average dose was 1.5 (+/- 0.3) mg/kg. The possible implications of these findings are discussed.

Holo-sterol carrier protein-2. (13)C NMR investigation of cholesterol and fatty acid binding sites.

Although sterol carrier protein-2 (SCP-2) stimulates sterol transfer in vitro, almost nothing is known regarding the identity of the putative cholesterol binding site. Furthermore, the interrelationship(s) between this SCP-2 ligand binding site and the recently reported SCP-2 long chain fatty acid (LCFA) and long chain fatty acyl-CoA (LCFA-CoA) binding site(s) remains to be established. In the present work, two SCP-2 ligand binding sites were identified. First, both [4-(13)C]cholesterol and 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol) binding assays were consistent with a single cholesterol binding site in SCP-2. This ligand binding site had high affinity for NBD-cholesterol, K(d) = 4.15 +/- 0.71 nM. (13)C NMR-labeled ligand competition studies demonstrated that the SCP-2 high affinity cholesterol binding site also bound LCFA or LCFA-CoA. However, only the LCFA-CoA was able to effectively displace the SCP-2-bound [4-(13)C]cholesterol. Thus, the ligand affinities at this SCP-2 binding site were in the relative order cholesterol = LCFA-CoA > LCFA. Second, (13)C NMR studies demonstrated the presence of another ligand binding site on SCP-2 that bound either LCFA or LCFA-CoA but not cholesterol. Photon correlation spectroscopy was consistent with SCP-2 being monomeric in both liganded and unliganded states. In summary, both (13)C NMR and fluorescence techniques demonstrated for the first time that SCP-2 had a single high affinity binding site that bound cholesterol, LCFA, or LCFA-CoA. Furthermore, results with (13)C NMR supported the presence of a second SCP-2 ligand binding site that bound either LCFA or LCFA-CoA but not cholesterol. These data contribute to our understanding of a role for SCP-2 in both cellular cholesterol and LCFA metabolism.

Genetic engineering of Escherichia coli for the production of precorrin-3 in vivo and in vitro.

The construction of a new recombinant strain of Escherichia coli in which two vitamin B12 biosynthetic genes, cobA and cobI, from Pseudomonas denitrificans are simultaneously overexpressed has resulted in the in vivo synthesis and accumulation of Factor III, an isobacteriochlorin not normally synthesized in E. coli. A lysate of the new strain can take the place of two lysates normally required to provide uroporphyrinogen III methyltransferase (cobA) and precorrin-2 methyltransferase (cobI) in an anaerobic five-enzyme synthesis of the early B12 intermediate, precorrin-3 (the reduced form of Factor III) from delta-aminolevulinic acid.

Overexpression, purification, and characterization of the thermostable mevalonate kinase from Methanococcus jannaschii.

We report here the first overexpression and characterization of a thermostable mevalonate kinase from an archae, Methanococcus jannaschii, a strict anaerobe, which produces methane and grows at pressure of 200 atm and an optimum temperature near 85 degrees C. PCR-derived DNA fragments containing the structural gene for mevalonate kinase were cloned into an expression vector, pET28a, to form pETMVK. The mevalonate kinase was overexpressed from Escherichia coli pETMVK/BL21(DE3) (15-20% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside. The protein was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Talon metal-affinity resin column. The purified protein had a dimeric structure composed of identical subunits, and the M(r) of the enzyme determined by gel chromatography was 68K. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit M(r) was 36, 000. The pI for mevalonate kinase was 7.8. The Michaelis constant (K(m)) for (RS)-mevalonate was 68.5 microM and was 92 microM for ATP. The V(max) was 387 units mg(-1). The optimal temperature for mevalonate kinase activity was 70-75 degrees C.

Chemoenzymatic synthesis of an unnatural tetramethyl cobalt corphinoid.

The chemoenzymatic synthesis and structural characterization by 13C NMR of a tetramethyl cobalt-corphinoid produced by methylation of cobalt-precorrin-3 using CbiF are described.