Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b 5 (b 5 ). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k on ϳ 10 6 M ؊1 s ؊1 ), and the k off rates range from 5.7 to 36 s ؊1 (at 23°C). Reduction of ferric P450 2A6 is rapid (7.5 s ؊1 ) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O 2 is rapid (k > 10 6 M ؊1 s ؊1 ), and the putative Fe 2؉ ⅐O 2 complex decayed at a rate of ϳ0.3 s ؊1 at 23°C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b 5 . Kinetic analyses showed that ϳ 1 ⁄3 of the reduced b 5 was rapidly oxidized in the presence of the Fe 2؉ ⅐O 2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6 -10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s. P4501 enzymes are involved in the oxygenation of a variety of natural products and xenobiotic chemicals in microbial systems (3, 4). Much is known about the structure, function, and catalytic features of some of the P450s, particularly the more extensively studied of the bacterial P450s (4, 5). In mammalian systems P450s oxidize many drugs, steroids, carcinogens, fatty acids and eicosanoids, fat-soluble vitamins, and other endobiotic and xenobiotic chemicals (6). Less information is available about the biochemical details of most of the 57 human P450s (7). In particular, the basis of the inherently lower catalytic activities of these and other mammalian P450s relative to some of the microbial forms is not clear.P450 2A6 is a low-to-medium abundance P450 in human liver (7-9) and is also expressed in some extrahepatic tissues (10). The history of this gene/protein goes back to Phillips et al. (11), who identified a human P450 cDNA as a relative of rat P450 2B1. The 7-hydroxylation of coumarin has long been used as an assay of P450 activity in animal and human liver microsomes (12, 13), and Yamano et al. (14) isolated a P450 2A6 cDNA (then termed 2A3) and first showed that the protein derived from heterologous expression had coumarin 7-hydroxylation activity. Miles et al. (15) also provided similar evidence for this particular sequence being associated with coumarin 7-hydroxylation. Our group purified a pr...

Study Design:Narrative review of available literature.Objective:To summarize current trends in pathogenesis and management of spinal epidural lipomatosis (SEL) and suggest areas where more research would be of benefit.Methods:The available literature relevant to SEL was reviewed. PubMed, Medline, OVID, EMBASE, Cochrane, and Google Scholar were used to review the literature. Institutional review board approval is not applicable for this study.Results:This article clearly summarizes current trends in the pathogenesis and management of SEL.Conclusions:Possible etiologies of SEL include exogenous steroid use, endogenous steroid hormonal disease, obesity, surgery induced, and idiopathic disease. Comorbidities such as acquired immunodeficiency syndrome and Scheuermann’s disease have also been implicated in the pathogenesis of SEL. Steroid-induced SEL seems to have a proclivity for the thoracic region of the spine and has a higher incidence of paraplegia when compared with other forms. Several treatment modalities exist for SEL and are dictated by the underlying cause of the disorder. These include weight reduction, cessation of steroid medications, treatment of underlying endocrine abnormalities, and surgical decompression. Conservative treatments generally aim to decrease the thickness of adipose tissue in the epidural space, but the majority of patients tend to undergo surgical decompression to relieve neurologic symptoms. Surgical decompression provides a statistically significant reduction in symptoms, but postoperative mortality is high, influenced primarily by the patient’s preoperative comorbidities. Physicians should consider the underlying cause of SEL in a given patient before pursuing specific treatment modalities, but alarm symptoms, such as the development of acute cauda equina syndrome, should likely be treated with urgent surgical decompression.

An extreme diversity of substrates and catalytic reactions of cytochrome P450 (P450) enzymes is considered to be the consequence of evolutionary adaptation driven by different metabolic or environmental demands. Here we report the presence of numerous natural variants of P450 BM3 (CYP102A1) within a species of Bacillus megaterium. Extensive amino acid substitutions (up to 5% of the total 1049 amino acid residues) were identified from the variants. Phylogenetic analyses suggest that this P450 gene evolve more rapidly than the rRNA gene locus. It was found that key catalytic residues in the substrate channel and active site are retained. Although there were no apparent variations in hydroxylation activity towards myristic acid (C14) and palmitic acid (C16), the hydroxylation rates of lauric acid (C12) by the variants varied in the range of >25-fold. Interestingly, catalytic activities of the variants are promiscuous towards non-natural substrates including human P450 substrates. It can be suggested that CYP102A1 variants can acquire new catalytic activities through site-specific mutations distal to the active site.

Human cytochrome P450 (CYP) 3A4, a membrane anchoring protein, is the major CYP enzyme present in both liver and small intestine. The enzyme plays a major role in the metabolism of many drugs and procarcinogens. The roles of individual phospholipids and membrane properties in the catalytic activity, membrane binding, and insertion into the membrane of CYP3A4 are poorly understood. Here we report that the catalytic activity of testosterone 6beta-hydroxylation, membrane binding, and membrane insertion of CYP3A4 increase as a function of anionic phospholipid concentration in the order phosphatidic acid (PA) > phosphatidylserine (PS) in a binary system of phosphatidylcholine (PC)/anionic phospholipid and as a function of phosphatidylethanolamine (PE) content in ternary systems of PC/PE/PA or PC/PE/PS having a fixed concentration of anionic phospholipids. These results suggest that PA and PE might help the binding of CYP3A4 to the membrane and the interaction with NPR. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4 in all tested phospholipids vesicles with various compositions. Phospholipid-dependent changes of the CYP3A4 conformation were also revealed by altered Trp fluorescence and CD spectra. We also found that PE induced the formation of anionic phospholipid-enriched domains in ternary systems using extrinsic fluorescent probes incorporated into lipid bilayers. Taken together, it can be suggested that the chemical and physical properties of membranes induced by anionic phospholipids and PE are critical for the membrane binding and catalytic activity of CYP3A4.

ABSTRACT:Recently, wild-type and mutant forms of bacterial cytochrome P450 BM3 (CYP102A1) have been found to metabolize various drugs through reactions similar to those catalyzed by human cytochromes P450 (P450s). Therefore, it has been suggested that CYP102A1 may be used to produce large quantities of the metabolites of human P450-catalyzed reactions. In this report, we show that the oxidation of 7-ethoxycoumarin, a typical human P450 substrate, is catalyzed by both wild-type and mutant forms of CYP102A1. Two major products were produced as a result of O-deethylation and 3-hydroxylation reactions. These results demonstrate that CYP102A1 mutants catalyze the same reactions as human P450s. High noncompetitive intermolecular kinetic deuterium isotope effects were observed for 7-ethoxycoumarin O-deethylation in the CYP102A1 system. These results suggest that there is a common mechanism for the oxidation reactions catalyzed by both the bacterial CYP102A1 and human P450 enzymes.

No abstract

No abstract

Extracellular DNA (exDNA) pool in aquatic environments is a valuable source for biomonitoring and bioassessment. However, degradation under particular environmental conditions can hamper exDNA detectability over time. In this study, we analyzed how different biotic and abiotic factors affect the degradation rate of extracellular environmental DNA using 16S rDNA sequences extracted from the sediment of a eutrophic lake and Anabaena variabilis cultured in the laboratory. We exposed the extracted exDNA to different levels of temperature, light, pH, and bacterial activity, and quantitatively analyzed the concentration of exDNA during 4 days. The solution containing bacteria for microbial activity treatment was obtained from the lake sediment using four consecutive steps of filtration; two mesh filters (100 μm and 60 μm mesh) and two glass fiber filters (2.7 μm and 1.2 μm pore-sized). We found that temperature individually and in combination with bacterial abundance had significant positive effects on the degradation of exDNA. The highest degradation rate was observed in samples exposed to high microbial activity, where exDNA was completely degraded within 1 day at a rate of 3.27 day−1. Light intensity and pH had no significant effects on degradation rate of exDNA. Our results indicate that degradation of exDNA in freshwater ecosystems is driven by the combination of both biotic and abiotic factors and it may occur very fast under particular conditions.