A comparison of steroid and lipid binding cytochrome P450s from Mycobacterium marinum and Mycobacterium tuberculosis

Child, Stella A.; Ghith, Amna; Bruning, John B.; Bell, Stephen

doi:10.1016/j.jinorgbio.2020.111116

Cited by 14 publications

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“…When cloned, produced, and purified, MulcCYP142A3 displayed a characteristic low spin resting state similar to those observed with MmarCYP142A3 and CYP142A1 from M. tuberculosis . ,, The extinction coefficient was calculated to be ε 416 = 139 ± 10 mM –1 cm –1 . When the substrates cholesterol and cholest-4-en-3-one were added to the MulcCYP142A3 enzyme, both induced a shift to the high spin state (55% and 90%, respectively, see Figure , Table ).…”

Section: Resultsmentioning

confidence: 64%

“…This is in agreement with the results obtained with the other CYP142 enzymes (Figure S2, Figure S3). ,,, The addition of cholesteryl sulfate to each CYP142 enzyme resulted in a larger shift to the high spin ferric state compared to cholesterol (Figure and Figure S4). The proportion of the high spin ferric enzyme was similar to that observed with cholest-4-en-3-one.…”

Section: Resultsmentioning

confidence: 97%

“…These steroid metabolizing CYP enzymes are also present across diverse Actinobacteria including Rhodococcus and other Mycobacterium species. ,,,, M. ulcerans contains genes encoding individual CYP125 and CYP142 enzymes. Recent work on M. smegmatis and M. marinum identified that each contain two CYP125 encoding genes (CYP125A3 and CYP125A4, and CYP125A6 and CYP125A7, respectively). , These mycobacterial species also have a gene encoding a CYP142 family member. This results in there being at least three potential cholesterol metabolizing enzymes in M. smegmatis and M. marinum .…”

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confidence: 99%

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The Structures of the Steroid Binding CYP142 Cytochrome P450 Enzymes from Mycobacterium ulcerans and Mycobacterium marinum

et al. 2022

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The steroid binding CYP142 cytochrome P450 enzymes of Mycobacterium species are involved in the metabolism of cholesterol and its derivatives. The equivalent enzyme from Mycobacterium ulcerans was studied to compare the degree of functional conservation between members of this CYP family. We compared substrate binding of the CYP142A3 enzymes of M. ulcerans and M. marinum and CYP142A1 from M. tuberculosis using UV–vis spectroscopy. The catalytic oxidation of cholesterol derivatives by all three enzymes was undertaken. Both CYP142A3 enzymes were structurally characterized by X-ray crystallography. The amino acid sequences of the CYP142A3 enzymes are more similar to CYP142A1 from M. tuberculosis than CYP142A2 from Mycolicibacterium smegmatis. Both CYP142A3 enzymes have substrate binding properties, which are more resemblant to CYP142A1 than CYP142A2. The cholest-4-en-3-one-bound X-ray crystal structure of both CYP142A3 enzymes were determined at a resolution of <1.8 Å, revealing the substrate binding mode at a high level of detail. The structures of the cholest-4-en-3-one binding CYP142 enzymes from M. ulcerans and M. marinum demonstrate how the steroid binds in the active site of these enzymes. They provide an explanation for the high selectivity of the enzyme for terminal methyl C–H bond oxidation to form 26-hydroxy derivatives. These enzymes in pathogenic Mycobacterium species are candidates for inhibition. The work here demonstrates that similar drug molecules could target these CYP142 enzymes from different species in order to combat Buruli ulcer or tuberculosis.

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Section: Resultsmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 97%

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confidence: 99%

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The Structures of the Steroid Binding CYP142 Cytochrome P450 Enzymes from Mycobacterium ulcerans and Mycobacterium marinum

et al. 2022

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“…The most closely related CYP enzymes to CYP168A1 that have been thoroughly characterized are from the pathogen M. tuberculosis (23,27,66,69). Previous studies have demonstrated that, in the case of this obligate pathogen, CYP121A1 (70,71), CYP125A1 (45,72), and CYP142A1 (73) are essential for bacterial survival, making them attractive drug targets (74,75). CYP125A1 and CYP142A1 from M. tuberculosis have been identified as cholesterol hydroxylases, suggesting that at least some CYP enzymes from pathogens have evolved functions as steroid or fatty acid hydroxylases, which are common roles for a number of microbial CYP enzymes (76,77).…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa cytochrome P450 CYP168A1 is a fatty acid hydroxylase that metabolizes arachidonic acid to the vasodilator 19-HETE

Tooker¹,

Kandel²,

Work

et al. 2022

Journal of Biological Chemistry

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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“…The most closely related CYP enzymes to CYP168A1 that have been thoroughly characterized are from the pathogen M. tuberculosis (23,27,67,70). Previous studies have demonstrated that, in the case of this obligate pathogen, CYP121A1 (71, 72), CYP125A1 (44, 73), and CYP142A1(74) are essential for bacterial survival, making them attractive drug targets (58, 75). CYP125A1 and CYP142A1 from M. tuberculosis have been identified as cholesterol hydroxylases, suggesting that at least some CYP enzymes from pathogens have evolved functions as steroid or fatty acid hydroxylases, which are common functions for a number of microbial CYP enzymes (76, 77).…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosacytochrome P450 CYP168A1 is a fatty acid hydroxylase that metabolizes arachidonic acid to the vasodilator 19-HETE

Tooker

Kandel

Work

et al. 2021

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Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen that is highly prevalent in individuals with cystic fibrosis (CF). A major problem in treating CF patients infected with P. aeruginosa is the development of antibiotic resistance. Therefore, the identification of novel P. aeruginosa antibiotic drug targets is of the upmost urgency. The genome of P. aeruginosa contains four putative cytochrome P450 enzymes (CYPs) of unknown function that have never before been characterized. Analogous to some of the CYPs from M. tuberculosis, the P. aeruginosa CYPs may be important for growth and colonization of the CF patient’s lung. In this study, we cloned, expressed, and characterized CYP168A1 from P. aeruginosa and identified it as a sub-terminal fatty acid hydroxylase. Spectral binding data and computational modeling of substrates and inhibitors suggest that CYP168A1 has a large, expansive active site preferring long chain fatty acids and large hydrophobic inhibitors. Furthermore, metabolism experiments confirm that the enzyme is capable of hydroxylating arachidonic acid, an important inflammatory signaling molecule present in abundance in the CF lung, to 19-hydroxyeicosatetraenoic acid (19-HETE; Km = 41.1 µM, Vmax = 222 pmol/min/nmol P450), a potent vasoconstrictor which may play a role in the pathogen’s ability to colonize the mammalian lung. Metabolism of arachidonic acid is subject to substrate inhibition and is also inhibited by the presence of ketoconazole. This study points to the discovery of a new potential drug target that may be of utility in treating drug resistant P. aeruginosa.

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A comparison of steroid and lipid binding cytochrome P450s from Mycobacterium marinum and Mycobacterium tuberculosis

Cited by 14 publications

References 51 publications

The Structures of the Steroid Binding CYP142 Cytochrome P450 Enzymes from Mycobacterium ulcerans and Mycobacterium marinum

The Structures of the Steroid Binding CYP142 Cytochrome P450 Enzymes from Mycobacterium ulcerans and Mycobacterium marinum

Pseudomonas aeruginosa cytochrome P450 CYP168A1 is a fatty acid hydroxylase that metabolizes arachidonic acid to the vasodilator 19-HETE

Pseudomonas aeruginosacytochrome P450 CYP168A1 is a fatty acid hydroxylase that metabolizes arachidonic acid to the vasodilator 19-HETE

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