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Structure–Function Analysis of the Steroid-Hydroxylating Cytochrome P450 109 (CYP109) Enzyme Family

Siphesihle M. Msweli, Tiara Padayachee Orcid Logo, Thembeka Khumalo, David R. Nelson Orcid Logo, David Lamb Orcid Logo, Khajamohiddin Syed Orcid Logo

International Journal of Molecular Sciences, Volume: 26, Issue: 13, Start page: 6219

Swansea University Author: David Lamb Orcid Logo

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DOI (Published version): 10.3390/ijms26136219

Abstract

Steroids are found in bacteria and eukaryotes, and genes potentially encoding steroid metabolic enzymes have also been identified in giant viruses. For decades, hydroxylated steroids have been utilized in medicine to treat various human diseases. The hydroxylation of steroids can be achieved using m...

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Published in: International Journal of Molecular Sciences
ISSN: 1422-0067
Published: MDPI 2025
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URI: https://cronfa.swan.ac.uk/Record/cronfa69940
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spelling 2025-07-10T13:33:14.3930059 v2 69940 2025-07-10 Structure–Function Analysis of the Steroid-Hydroxylating Cytochrome P450 109 (CYP109) Enzyme Family 1dc64e55c2c28d107ef7c3db984cccd2 0000-0001-5446-2997 David Lamb David Lamb true false 2025-07-10 MEDS Steroids are found in bacteria and eukaryotes, and genes potentially encoding steroid metabolic enzymes have also been identified in giant viruses. For decades, hydroxylated steroids have been utilized in medicine to treat various human diseases. The hydroxylation of steroids can be achieved using microbial enzymes, especially cytochrome P450 monooxygenases (CYPs/P450s) and is well documented. Understanding the structural determinants that govern the regio- and stereoselectivity of steroid hydroxylation by P450s is essential in order to fully exploit their potential. Herein, we present a comprehensive analysis of the steroid-hydroxylating CYP109 family across the domains of life and delineate the structural determinants that govern steroid hydroxylation. Data mining, annotation, and phylogenetic analysis revealed that CYP109 family members are highly populated in bacteria, and indeed, these members passed from bacteria to archaea by horizontal gene transfer, leading to the evolution of P450s in archaea. Analysis of twelve CYP109 crystal structures revealed large, flexible, and dynamic active site cavities that can accommodate multiple ligands. The correct positioning and orientation of the steroid in the active site cavity and the nature of the C17 substituent on the steroid molecule influence catalysis. In an analogous fashion to the CYP107 family, the amino acid residues within the CYP109 binding pocket involve hydrophilic and hydrophobic interactions, influencing substrate orientations and anchoring and determining the site of hydroxylation and catalytic activity. A handful of amino acids, such as Val84, Val292, and Ser387 in CYP109B4, have been found to play a role in determining the catalytic regiospecificity, and a single amino acid, such as Arg74 in CYP109A2, has been found to be essential for the enzymatic activity. This work serves as a reference for the precise understanding of CYP109 structure–function relationships and for P450 enzymes in general. The findings will guide the genetic engineering of CYP109 enzymes to produce valuable steroid molecules of medicinal and biotechnological importance. Journal Article International Journal of Molecular Sciences 26 13 6219 MDPI 1422-0067 cytochrome P450; CYP109; steroid; oxidation reactions; active site; crystal structure 27 6 2025 2025-06-27 10.3390/ijms26136219 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University Another institution paid the OA fee National Research Foundation (NRF), South Africa Grant: PMDS230527110616 Grant: MND210504599108; National Research Foundation (NRF), South Africa Grant: RA22102865602 2025-07-10T13:33:14.3930059 2025-07-10T13:25:50.6263376 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science Siphesihle M. Msweli 1 Tiara Padayachee 0000-0001-6667-9982 2 Thembeka Khumalo 3 David R. Nelson 0000-0003-0583-5421 4 David Lamb 0000-0001-5446-2997 5 Khajamohiddin Syed 0000-0002-1497-3570 6 69940__34732__5c6429399d784edf8f035acdd804c3a5.pdf ijms-26-06219-v3.pdf 2025-07-10T13:25:50.5951548 Output 5484366 application/pdf Version of Record true © 2025 by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. true eng https://creativecommons.org/licenses/by/4.0/
title Structure–Function Analysis of the Steroid-Hydroxylating Cytochrome P450 109 (CYP109) Enzyme Family
spellingShingle Structure–Function Analysis of the Steroid-Hydroxylating Cytochrome P450 109 (CYP109) Enzyme Family
David Lamb
title_short Structure–Function Analysis of the Steroid-Hydroxylating Cytochrome P450 109 (CYP109) Enzyme Family
title_full Structure–Function Analysis of the Steroid-Hydroxylating Cytochrome P450 109 (CYP109) Enzyme Family
title_fullStr Structure–Function Analysis of the Steroid-Hydroxylating Cytochrome P450 109 (CYP109) Enzyme Family
title_full_unstemmed Structure–Function Analysis of the Steroid-Hydroxylating Cytochrome P450 109 (CYP109) Enzyme Family
title_sort Structure–Function Analysis of the Steroid-Hydroxylating Cytochrome P450 109 (CYP109) Enzyme Family
author_id_str_mv 1dc64e55c2c28d107ef7c3db984cccd2
author_id_fullname_str_mv 1dc64e55c2c28d107ef7c3db984cccd2_***_David Lamb
author David Lamb
author2 Siphesihle M. Msweli
Tiara Padayachee
Thembeka Khumalo
David R. Nelson
David Lamb
Khajamohiddin Syed
format Journal article
container_title International Journal of Molecular Sciences
container_volume 26
container_issue 13
container_start_page 6219
publishDate 2025
institution Swansea University
issn 1422-0067
doi_str_mv 10.3390/ijms26136219
publisher MDPI
college_str Faculty of Medicine, Health and Life Sciences
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hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
document_store_str 1
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description Steroids are found in bacteria and eukaryotes, and genes potentially encoding steroid metabolic enzymes have also been identified in giant viruses. For decades, hydroxylated steroids have been utilized in medicine to treat various human diseases. The hydroxylation of steroids can be achieved using microbial enzymes, especially cytochrome P450 monooxygenases (CYPs/P450s) and is well documented. Understanding the structural determinants that govern the regio- and stereoselectivity of steroid hydroxylation by P450s is essential in order to fully exploit their potential. Herein, we present a comprehensive analysis of the steroid-hydroxylating CYP109 family across the domains of life and delineate the structural determinants that govern steroid hydroxylation. Data mining, annotation, and phylogenetic analysis revealed that CYP109 family members are highly populated in bacteria, and indeed, these members passed from bacteria to archaea by horizontal gene transfer, leading to the evolution of P450s in archaea. Analysis of twelve CYP109 crystal structures revealed large, flexible, and dynamic active site cavities that can accommodate multiple ligands. The correct positioning and orientation of the steroid in the active site cavity and the nature of the C17 substituent on the steroid molecule influence catalysis. In an analogous fashion to the CYP107 family, the amino acid residues within the CYP109 binding pocket involve hydrophilic and hydrophobic interactions, influencing substrate orientations and anchoring and determining the site of hydroxylation and catalytic activity. A handful of amino acids, such as Val84, Val292, and Ser387 in CYP109B4, have been found to play a role in determining the catalytic regiospecificity, and a single amino acid, such as Arg74 in CYP109A2, has been found to be essential for the enzymatic activity. This work serves as a reference for the precise understanding of CYP109 structure–function relationships and for P450 enzymes in general. The findings will guide the genetic engineering of CYP109 enzymes to produce valuable steroid molecules of medicinal and biotechnological importance.
published_date 2025-06-27T05:29:31Z
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