Kinetics of cholesterol oxidation by cholesterol oxidase

1996 ◽  
Vol 60 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Palligarnai T. Vasudevan ◽  
Tao Zhou
Keyword(s):  
Cholesterol Oxidase ◽  
Cholesterol Oxidation ◽  
Kinetics Of

scholarly journals Localization of cholesterol in sphingomyelinase-treated fibroblasts

1995 ◽  
Vol 308 (1) ◽  
pp. 269-274 ◽  
Author(s):  
M I Pörn ◽  
J P Slotte
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Staining Pattern ◽  
Cholesterol Oxidase ◽  
Cholesterol Oxidation ◽  
Cellular Cholesterol ◽  
Hypotonic Buffer ◽  
Induced Changes ◽  
Kinetics Of

The distribution of cellular unesterified cholesterol was studied in fibroblasts, which had been depleted of plasma membrane sphingomyelin by exposure to exogenous sphingomyelinase. This treatment has previously been shown to induce an increase in cholesterol esterification, a decrease in the biosynthesis of cholesterol, and a decreased susceptibility of cell cholesterol to oxidation with cholesterol oxidase. When the cellular localization of cholesterol was studied with fluorescent filipin staining, sphingomyelin depletion did not cause any visible changes in the filipin-cholesterol staining pattern, suggesting that the major part of cellular cholesterol was retained in the plasma membrane after sphingomyelinase treatment. After the oxidation of cell-surface cholesterol with cholesterol oxidase, the plasma membrane was no longer stained by filipin, but the plasma membrane cholesterol of sphingomyelin-depleted cells appeared to be resistant to oxidation with cholesterol oxidase when sphingomyelinase was used as an oxidation-promoting agent. However, the use of hypotonic buffer or phosphatidylcholine-specific phospholipase C together with cholesterol oxidase resulted in a complete oxidation of the cell-surface cholesterol in sphingomyelin-depleted cells, as evidenced by the filipin-cholesterol staining pattern. Similar results were obtained when [3H]cholesterol-labelled fibroblasts were used for determination of the susceptibility to cholesterol oxidation. The kinetics of [3H]cholesterol oxidation in sphingomyelin-depleted cells with cholesterol oxidase in hypotonic buffer indicated that approximately 85% of the cellular cholesterol still resided in the plasma membrane after sphingomyelin depletion. These results are contradictory to earlier reports on sphingomyelinase-induced changes in cellular cholesterol distribution and suggest that minor changes in the kinetics of cholesterol transport from the plasma membrane to the endoplasmic reticulum may be responsible for the sphingomyelinase-induced changes in the rates of cholesterol metabolism. Whereas the use of phospholipases to promote the oxidation of cholesterol in some instances might lead to misinterpretations, the use of hypotonic buffer together with cholesterol oxidase proved to be a more reliable method for the determination of cellular cholesterol distribution.

The kinetics of cholesterol oxidase synthesis byNocardia rhodocrous

1976 ◽  
Vol 18 (5) ◽  
pp. 601-621 ◽  
Author(s):  
B. C. Buckland ◽  
M. D. Lilly ◽  
P. Dunnill
Keyword(s):  
Cholesterol Oxidase ◽  
Kinetics Of

scholarly journals Steroid Metabolism in Thermophilic Actinobacterium Saccharopolyspora hirsuta VKM Ac-666T

2021 ◽  
Vol 9 (12) ◽  
pp. 2554
Author(s):  
Tatyana Lobastova ◽  
Victoria Fokina ◽  
Sergey Tarlachkov ◽  
Andrey Shutov ◽  
Eugeny Bragin ◽  
...  
Keyword(s):  
Thermophilic Bacteria ◽  
Cholesterol Oxidase ◽  
Cascade Reactions ◽  
Cholesterol Oxidation ◽  
P450 Monooxygenase ◽  
Thermophilic Microorganisms ◽  
Mycobacterium Tuberculosis H37rv ◽  
Rhodococcus Jostii Rha1 ◽  
Saccharopolyspora Hirsuta ◽  
Steroid Catabolism

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3β-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.

scholarly journals Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis

2021 ◽  
Vol 11 ◽  
Author(s):  
Thabatta L. S. A. Rosa ◽  
Maria Angela M. Marques ◽  
Zachary DeBoard ◽  
Kelly Hutchins ◽  
Carlos Adriano A. Silva ◽  
...  
Keyword(s):  
Ldl Cholesterol ◽  
Cholesterol Oxidase ◽  
Close Association ◽  
Atp Synthesis ◽  
Mycobacterium Leprae ◽  
Cholesterol Oxidation ◽  
Bacterial Survival ◽  
Catabolic Pathway ◽  
Oxidation Activity ◽  
Microscopy Analysis

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3β-hydroxysteroid dehydrogenase (3β-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3β-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3β-HSD activity with the 17β-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3β-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

scholarly journals Some enzymatic properties of cholesterol oxidase produced by Brevibacterium sp

1999 ◽  
Vol 30 (4) ◽  
pp. 315-323 ◽  
Author(s):  
Terezinha J.G. Salva ◽  
Alcina M. Liserre ◽  
Aloísia L. Moretto ◽  
Marco A.T. Zullo ◽  
Gisleine Ventrucci ◽  
...  
Keyword(s):  
Enzyme Stability ◽  
Degradation Products ◽  
Cholesterol Oxidase ◽  
Cholesterol Oxidation ◽  
Optimum Temperature ◽  
Buffer Solution ◽  
First Order ◽  
Membrane Bound ◽  
Triton X ◽  
Reaction Media

In this study we determined some properties of the cholesterol oxidase from a Brevibacterium strain isolated from buffalo's milk and identified the cholesterol degradation products by the bacterial cell. A small fraction of the enzyme synthesized by cells cultured in liquid medium for 7days was released into the medium whereas a larger fraction remained bound to the cell membrane. The extraction of this fraction was efficiently accomplished in 1 mM phosphate buffer, pH 7.0, containing 0.7% Triton X-100. The enzyme stability under freezing and at 45oC was improved by addition of 20% glycerol. The optimum temperature and pH for the enzyme activity were 53°C and 7.5, respectively. The only steroidal product from cholesterol oxidation by the microbial cell and by the crude extract of the membrane-bound enzyme was 4-colesten-3-one. Chromatographic analysis showed that minor no steroidal compounds as well as 4-colesten-3-one found in the reaction media arose during fermentation process and were extracted together with the enzyme in the buffer solution. Cholesterol oxidation by the membrane-bound enzyme was a first order reaction type.

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