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Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity

David Lamb Orcid Logo, Jared V. Goldstone, Bin Zhao Orcid Logo, Li Lei, Jonathan Mullins Orcid Logo, Michael J. Allen Orcid Logo, Steven Kelly Orcid Logo, John J. Stegeman

Biomolecules, Volume: 12, Issue: 8, Start page: 1107

Swansea University Authors: David Lamb Orcid Logo, Jonathan Mullins Orcid Logo, Steven Kelly Orcid Logo

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DOI (Published version): 10.3390/biom12081107

Abstract

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus...

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Published in: Biomolecules
ISSN: 2218-273X
Published: MDPI AG 2022
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URI: https://cronfa.swan.ac.uk/Record/cronfa62081
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The sequenced genome of 0305&#x3C6;8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305&#x3C6;8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305&#x3C6;8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305&#x3C6;8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305&#x3C6;8-36 flavodoxin is predicted to mediate the 2-electron reduction of the &#x3B2; subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.</abstract><type>Journal Article</type><journal>Biomolecules</journal><volume>12</volume><journalNumber>8</journalNumber><paginationStart>1107</paginationStart><paginationEnd/><publisher>MDPI AG</publisher><placeOfPublication/><isbnPrint/><isbnElectronic/><issnPrint/><issnElectronic>2218-273X</issnElectronic><keywords>flavodoxin, virus/phage, cytochrome P450, evolution, Bacteria</keywords><publishedDay>11</publishedDay><publishedMonth>8</publishedMonth><publishedYear>2022</publishedYear><publishedDate>2022-08-11</publishedDate><doi>10.3390/biom12081107</doi><url/><notes/><college>COLLEGE NANME</college><department>Biomedical Sciences</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>BMS</DepartmentCode><institution>Swansea University</institution><apcterm>Not Required</apcterm><funders>The study was supported by the National Institutes of Health grant 5U41HG003345 (J.V.G.), by the Woods Hole Center for Oceans and Human Health, NIH P01 ES021923 and NSF OCE-1314642 (J.J.S.), and by a Fulbright Scholarship (to D.C.L.). 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spelling 2022-12-07T15:54:14.7496169 v2 62081 2022-11-29 Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity 1dc64e55c2c28d107ef7c3db984cccd2 0000-0001-5446-2997 David Lamb David Lamb true false 4cf2dddedbe1dacb506ec925fdbd5b40 0000-0003-0144-2962 Jonathan Mullins Jonathan Mullins true false b17cebaf09b4d737b9378a3581e3de93 0000-0001-7991-5040 Steven Kelly Steven Kelly true false 2022-11-29 BMS Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the β subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships. Journal Article Biomolecules 12 8 1107 MDPI AG 2218-273X flavodoxin, virus/phage, cytochrome P450, evolution, Bacteria 11 8 2022 2022-08-11 10.3390/biom12081107 COLLEGE NANME Biomedical Sciences COLLEGE CODE BMS Swansea University Not Required The study was supported by the National Institutes of Health grant 5U41HG003345 (J.V.G.), by the Woods Hole Center for Oceans and Human Health, NIH P01 ES021923 and NSF OCE-1314642 (J.J.S.), and by a Fulbright Scholarship (to D.C.L.). Funding at Swansea University supported by the European Regional Development Fund/Welsh European Funding Office via the BEACON project (S.L.K). 2022-12-07T15:54:14.7496169 2022-11-29T10:26:01.6818423 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine David Lamb 0000-0001-5446-2997 1 Jared V. Goldstone 2 Bin Zhao 0000-0003-0104-7508 3 Li Lei 4 Jonathan Mullins 0000-0003-0144-2962 5 Michael J. Allen 0000-0001-8504-7171 6 Steven Kelly 0000-0001-7991-5040 7 John J. Stegeman 8 62081__26030__f79a912a039444e69fbdc923d91af87f.pdf 62081.pdf 2022-12-07T15:52:55.2814509 Output 2626550 application/pdf Version of Record true © 2022 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 Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity
spellingShingle Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity
David Lamb
Jonathan Mullins
Steven Kelly
title_short Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity
title_full Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity
title_fullStr Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity
title_full_unstemmed Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity
title_sort Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity
author_id_str_mv 1dc64e55c2c28d107ef7c3db984cccd2
4cf2dddedbe1dacb506ec925fdbd5b40
b17cebaf09b4d737b9378a3581e3de93
author_id_fullname_str_mv 1dc64e55c2c28d107ef7c3db984cccd2_***_David Lamb
4cf2dddedbe1dacb506ec925fdbd5b40_***_Jonathan Mullins
b17cebaf09b4d737b9378a3581e3de93_***_Steven Kelly
author David Lamb
Jonathan Mullins
Steven Kelly
author2 David Lamb
Jared V. Goldstone
Bin Zhao
Li Lei
Jonathan Mullins
Michael J. Allen
Steven Kelly
John J. Stegeman
format Journal article
container_title Biomolecules
container_volume 12
container_issue 8
container_start_page 1107
publishDate 2022
institution Swansea University
issn 2218-273X
doi_str_mv 10.3390/biom12081107
publisher MDPI AG
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
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 - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
document_store_str 1
active_str 0
description Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the β subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.
published_date 2022-08-11T04:21:25Z
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