Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are giant multienzymes that biosynthesize a number of clinically important natural products. The modular nature of PKSs suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides, but the efficacy of such a strategy depends critically on gaining fundamental insight into PKS structure and function, most directly through experiments with purified PKS proteins. Several recent investigations into important aspects of the activity of these enzymes have used only partially purified proteins (often 3-4% of total protein), reflecting how difficult it is to purify these multienzymes in amounts adequate for kinetic and structural analysis. We report here the steady-state kinetic analysis of a typical bimodular PKS, 6-deoxyerythronolide B synthase 1-thioesterase (DEBS 1-TE), purified from recombinant Saccharopolyspora erythraea JCB101 by a new, high-yielding procedure consisting of three steps: ammonium sulfate precipitation, hydrophobic interaction chromatography and size-exclusion chromatography. The method provides 13-fold purification with a recovery of 11% of the applied PKS activity. The essentially homogeneous synthase exhibits an intrinsic methylmalonyl-CoA hydrolase activity, which competes with polyketide chain extension. The most reliable value for the kcat for synthesis of (3S,5R)-dihydroxy-(2R,4R)-dimethyl-n-heptanoic acid-delta-lactone is 0.84 min-1, and the apparent Km for (2RS)-methylmalonyl-CoA is 17 microM. This kcat is approximately 10-fold lower than the value reported previously for a differently engineered version of the truncated PKS, DEBS 1+TE. The difference likely reflects the fact that the DEBS 1-TE contains a hybrid acyl carrier protein (ACP) domain in its second module, which lowers its catalytic efficiency.

Frightening dreams and spells: a case of ventricular asystole from Lyme disease.

We present a case of a 20-year-old woman who presented with a febrile illness, frightening dreams and repeated short episodes of apparent seizure activity. Third degree heart block and ventricular asystole were noted on the monitor when the patient experienced a spell during conscious sedation for a lumbar puncture. The combination of heart block and a predominantly lymphocytic cerebrospinal fluid led to the diagnosis of Lyme disease. Lyme titres were strongly positive and subsequently confirmed by Western Blot analysis. Cardiac aetiologies and specifically heart block associated with Lyme disease should be considered in patients from endemic areas presenting with fever and unexplained spells or seizure-like activity.

Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase.

BACKGROUND: Modular polyketide synthases (PKSs) catalyse the biosynthesis of complex polyketides using a different set of enzymes for each successive cycle of chain extension. Directed biosynthesis starting from synthetic diketides is a potentially valuable route to novel polyketides. We have used a purified bimodular derivative of the erythromycin-producing polyketide synthase (DEBS 1-TE) to study chain extension starting from a variety of diketide analogues and, in some cases, from the alternative acyl-CoA thioester substrates. RESULTS: Chain initiation in vitro by DEBS 1-TE module 2 using a synthetic diketide analogue as a substrate was tolerant of significant structural variation in the starter unit of the synthetic diketide, but other changes completely abolished activity. Interestingly, a racemic beta-keto diketide was found to be reduced in situ on the PKS and utilised in place of its more complex hydroxy analogue as a substrate for chain extension. The presence of a diketide analogue strongly inhibited chain initiation via the loading module. Significantly higher concentrations of diketide N-acetylcysteamine analogues than their corresponding acyl-CoA thioesters are required to achieve comparable yields of triketide lactones. CONCLUSIONS: Although a broad range of variation in the starter residue is acceptable, the substrate specificity of module 2 of a typical modular PKS in vitro is relatively intolerant of changes at C-2 and C-3. This will restrict the usefulness of approaches to synthesise novel erythromycins using synthetic diketides in vivo. The use of synthetic beta-keto diketides in vivo deserves to be explored.

Origin of starter units for erythromycin biosynthesis.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are multifunctional proteins that govern the synthesis of a number of clinically important natural products. The modular arrangement of active sites within these enzymes suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides. The efficacy of combinatorial strategies toward altering the starter unit specificity of polyketide synthases critically depends on controlling the supply of competing endogenous starter acids. Using DEBS 1-TE, a bimodular derivative of DEBS, we aimed to determine whether the beta-ketosynthase (KS) domain responsible for condensation in the first module also has the ability to prime its own biosynthesis by catalyzing the decarboxylation of methylmalonyl-CoA to produce propionyl-CoA. In contrast to earlier reports with a closely similar mini-PKS DEBS 1+TE, we have found that rigorously purified DEBS 1-TE does not catalyze the decarboxylation of methylmalonyl-CoA.

Brief motivational interviewing in a hospital setting for adolescent smoking: a preliminary study.

This study tested the feasibility and efficacy of a brief smoking intervention for adolescents in a hospital setting. Forty adolescent patients were randomized to receive either brief advice or a motivational interview, a nonconfrontational therapeutic intervention. Feasibility of brief smoking interventions with teen patients was supported by high rates of recruitment, retention, and quit attempts, and long periods of continuous abstinence. Although between-groups differences on smoking measures were not significant at 3-month follow-up, an effect size of h = .28 was noted. The sample showed significant decreases in smoking dependence and number of days smoked. Baseline stage of change, smoking rate, and depression were significant prospective predictors of smoking outcome. Implications for smoking intervention research with adolescents are discussed.

The molecular basis of Celmer's rules: the stereochemistry of the condensation step in chain extension on the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), for example, the 6-deoxyerythronolide B synthase (DEBS) responsible for synthesis of the aglycone core of the macrolide antibiotic erythromycin, generate an impressive diversity of asymmetric centers in their polyketide products. However, as noted by Celmer, macrolides have the same absolute configuration at all comparable stereocenters. Understanding how the stereochemistry of chain extension in controlled is therefore crucial to determining the common mechanism of action of these enzymes. We aimed to elucidate the molecular basis of Celmer's rules through in vitro studies with DEBS 1-TE, a bimodular derivative of DEBS from Saccharopolyspora erythraea, which uses (2S)-methylmalonyl-coenzyme. A to produce both D- and L-methyl centers in its triketide lactone product. We show here that condensation of (2S)-methylmalonyl-CoA in module 2 proceeds with decarboxylative inversion without cleavage of the C-H bond adjacent to the methyl group; in contrast, in module 1 the chain extension process involves loss of the hydrogen attached to C-2 of the methylmalonyl-CoA precursor. The production of the D-methyl center in module 2 without loss of hydrogen from the asymmetric center of the (2S)-methylmalonyl-CoA establishes that condensation takes place with inversion of configuration as in fatty acid biosynthesis. The loss of the key hydrogen from the (2S)-methylmalonyl-CoA to produce the L-methyl center generated in module 1 implies that an additional obligatory epimerization step takes place in that module. The nature and timing of the epimerization remain to be established.

Hospitalizing the suicidal adolescent: decision-making criteria of psychiatric residents.

OBJECTIVE: The primary purpose of this research is to investigate the criteria used by general psychiatric residents in determining the appropriateness of hospitalization. METHOD: A questionnaire containing 64 vignettes describing adolescent suicide attempts was completed by a sample of 33 residents from a general psychiatry training program. Six variables known to relate to lethality of attempt were systematically varied within the vignettes: gender, depression, conduct disorder/substance abuse, previous attempts, suicidal relative, and family supports. Respondents were asked to judge the appropriateness of hospitalization for each vignette. RESULTS: Hospitalization preference was significantly predicted by all risk factors except for gender, with the presence of depression emerging as the most important predictor of hospitalization. Residents recommended hospitalization more frequently than did experienced child and adolescent clinicians. In comparison with experienced clinicians, residents placed more importance on depression, and less importance on conduct disorder/substance abuse, in making decisions to hospitalize. CONCLUSIONS: Although psychiatric residents use known risk factors for adolescent suicide in assessing need for hospitalization, there was clear support for further training initiatives for psychiatric residents concerning the assessment of suicidal adolescents.

Chest wall movements prior to phonation.

Movements of the chest wall during the interval between an acoustic stimulus and the subject's vocal response were examined and timed in eight normal males. The reaction-time interval was divisible into two phases, a latency period with duration independent on chest wall status at the time of stimulus and an adjustment period during which the rib cage and abdomen usually moved oppositionally to achieve a prephonatory postural set. The time required for this adjustment varied significantly with lung volume, but was independent of the ventilatory phaze previously in progress.