Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin: purification and characterization of the recombinant enzyme.

The oxidation of phenols to quinones is an important reaction in the oxidative tailoring of many aromatic polyketides from bacterial and fungal systems. Sequence similarity between ActVA-Orf6 protein from the actinorhodin biosynthetic cluster and the previously characterized TcmH protein that is involved in tetracenomycin biosynthesis suggested that ActVA-Orf6 might catalyze this transformation as a step in actinorhodin biosynthesis. To investigate the role of ActVA-Orf6 in this oxidation, we have expressed the actVA-Orf6 gene in Escherichia coli and purified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate TcmF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a similar intermediate in actinorhodin biosynthesis. The monooxygenase obeys simple reaction kinetics and has a Km of 4.8 +/- 0.9 microM, close to the figure reported for the homologous enzyme TcmH. The enzyme contains no prosthetic groups and requires only molecular oxygen to catalyze the oxidation. Site-directed mutagenesis was used to investigate the role of histidine residues thought to be important in the reaction; mutants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a general base during catalysis.     

Inhibition of choline transport into human erythrocytes by choline mustard aziridinium ion.

Acetylcholine mustard aziridinium ion inhibited the transport of [3H]choline into human erythrocytes. Treatment of the erythrocytes with 1 X 10(-4) M tetraethylpyrophosphate prevented the inhibition of [3H]choline transport by acetylcholine mustard aziridinium ion. Hydrolyzed acetylcholine mustard aziridinium ion inhibited choline transport both in the presence and absence of 1 X 10(-4) M tetraethylpyrophosphate. The product of hydrolysis was equipotent with acetylcholine mustard in its ability to inhibit choline transport; incubation of this product with sodium thiosulfate prevented inhibition of choline transport thereby indicating the presence of an aziridinium ion. The hydrolysis product is likely to be choline mustard aziridinium ion. Results on the efflux of [3H]choline from erythrocytes in the presence of the proposed choline mustard aziridinium ion showed that the mustard moiety was transported into the red cells on the choline carrier. The rate of efflux of [3H]choline produced by choline mustard aziridinium ion was 55% of that produced by the same concentration of choline. It is concluded that acetylcholinesterase (EC 3.1.1.7) of red cells rapidly hydrolyzes acetylcholine mustard aziridinium ion to acetate and choline mustard aziridinium and the latter compound can act as a potent inhibitor of choline transport. This finding would indicate that the hemicholinium-like toxicity of acetylcholine mustard in the mouse is due to the formation of choline mustard aziridinium ion.     

DnrD cyclase involved in the biosynthesis of doxorubicin: purification and characterization of the recombinant enzyme

Mutations in the Streptomyces peucetius dnrD gene block the ring cyclization leading from aklanonic acid methyl ester (AAME) to aklaviketone (AK), an intermediate in the biosynthetic pathway to daunorubicin (DNR) and doxorubicin. To investigate the role of DnrD in this transformation, its gene was overexpressed in Escherichia coli and the DnrD protein was purified to homogeneity and characterized. The enzyme was shown to catalyze the conversion of AAME to AK presumably via an intramolecular aldol condensation mechanism. In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin (TCM) C pathway in Streptomyces glaucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occurs after anthraquinone formation and requires activation of a carboxylic acid group by esterification of aklanonic acid, the AAME precursor. Also, the cyclization is not coupled with a subsequent dehydration step that would result in an aromatic ring. As the substrates for the DnrD and TcmI enzymes are among the earliest isolable intermediates of aromatic polyketide biosynthesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic parameters that are important for aromatic ring formation. In the future, these parameters may be able to be applied to some of the earlier polyketide cyclization processes that currently are difficult to study in vitro.     

Status report of lipid-lowering trials in diabetes

The prevention and treatment of coronary heart disease is a major challenge in the overall management of the patient with type 2 diabetes. Diabetic dyslipidaemia is an important risk factor and is open to therapeutic intervention. However, as yet there are no primary or secondary coronary heart disease prevention trials of lipid-lowering therapy reported in diabetic populations. In this review, on-going clinical trials of lipid-lowering therapy in specific diabetic populations will be described.     

The Practice of Cranial Neurosurgery and the Malpractice Liability Environment in the United States

Object

The potential imbalance between malpractice liability cost and quality of care has been an issue of debate. We investigated the association of malpractice liability with unfavorable outcomes and increased hospitalization charges in cranial neurosurgery.

Methods

We performed a retrospective cohort study involving patients who underwent cranial neurosurgical procedures from 2005-2010, and were registered in the National Inpatient Sample (NIS) database. We used data from the National Practitioner Data Bank (NPDB) from 2005 to 2010 to create measures of volume and size of malpractice claim payments. The association of the latter with the state-level mortality, length of stay (LOS), unfavorable discharge, and hospitalization charges for cranial neurosurgery was investigated.

Results

During the study period, there were 189,103 patients (mean age 46.4 years, with 48.3% females) who underwent cranial neurosurgical procedures, and were registered in NIS. In a multivariable regression, higher number of claims per physician in a state was associated with increased ln-transformed hospitalization charges (beta 0.18; 95% CI, 0.17 to 0.19). On the contrary, there was no association with mortality (OR 1.00; 95% CI, 0.94 to 1.06). We observed a small association with unfavorable discharge (OR 1.09; 95% CI, 1.06 to 1.13), and LOS (beta 0.01; 95% CI, 0.002 to 0.03). The size of the awarded claims demonstrated similar relationships. The average claims payment size (ln-transformed) (Pearson’s rho=0.435, P=0.01) demonstrated a positive correlation with the risk-adjusted hospitalization charges but did not demonstrate a correlation with mortality, unfavorable discharge, or LOS.

Conclusions

In the present national study, aggressive malpractice environment was not correlated with mortality but was associated with higher hospitalization charges after cranial neurosurgery. In view of the association of malpractice with the economics of healthcare, further research on its impact is necessary.     

An aromatic hydroxylation reaction catalyzed by a two-component FMN-dependent Monooxygenase. The ActVA-ActVB system from Streptomyces coelicolor

The ActVA-ActVB system from Streptomyces coelicolor isatwo-component flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction.