Structural and spectroscopic characterization of RufO indicates a new biological role in rufomycin biosynthesis
Rufomycins constitute a class of cyclic heptapeptides isolated from actinomycetes. They are secondary metabolites that show promising treatment against Mycobacterium tuberculosis infections by inhibiting a novel drug target. Several nonproteinogenic amino acids are integrated into rufomycins, includ...
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creator | Jordan, Stephanie Li, Bingnan Traore, Ephrahime Wu, Yifei Usai, Remigio Liu, Aimin Xie, Zhong-Ru Wang, Yifan |
description | Rufomycins constitute a class of cyclic heptapeptides isolated from actinomycetes. They are secondary metabolites that show promising treatment against Mycobacterium tuberculosis infections by inhibiting a novel drug target. Several nonproteinogenic amino acids are integrated into rufomycins, including a conserved 3-nitro-tyrosine. RufO, a cytochrome P450 (CYP)-like enzyme, was proposed to catalyze the formation of 3-nitro-tyrosine in the presence of O2 and NO. To define its biological function, the interaction between RufO and the proposed substrate tyrosine is investigated using various spectroscopic methods that are sensitive to the structural change of a heme center. However, a low-to high-spin state transition and a dramatic increase in the redox potential that are commonly found in CYPs upon ligand binding have not been observed. Furthermore, a 1.89-Å crystal structure of RufO shows that the enzyme has flexible surface regions and a wide-open substrate access tunnel, and the heme center is largely exposed to solvent. Comparison with a closely related nitrating CYP reveals a spacious and hydrophobic distal pocket in RufO, which is incapable of stabilizing a free amino acid. Molecular docking validates the experimental data and proposes a possible substrate. Collectively, our results disfavor tyrosine as the substrate of RufO and point to the possibility that the nitration occurs during or after the assembly of the peptides. This study indicates a new function of the unique nitrating enzyme and provides insights into the biosynthesis of nonribosomal peptides. |
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They are secondary metabolites that show promising treatment against Mycobacterium tuberculosis infections by inhibiting a novel drug target. Several nonproteinogenic amino acids are integrated into rufomycins, including a conserved 3-nitro-tyrosine. RufO, a cytochrome P450 (CYP)-like enzyme, was proposed to catalyze the formation of 3-nitro-tyrosine in the presence of O2 and NO. To define its biological function, the interaction between RufO and the proposed substrate tyrosine is investigated using various spectroscopic methods that are sensitive to the structural change of a heme center. However, a low-to high-spin state transition and a dramatic increase in the redox potential that are commonly found in CYPs upon ligand binding have not been observed. Furthermore, a 1.89-Å crystal structure of RufO shows that the enzyme has flexible surface regions and a wide-open substrate access tunnel, and the heme center is largely exposed to solvent. Comparison with a closely related nitrating CYP reveals a spacious and hydrophobic distal pocket in RufO, which is incapable of stabilizing a free amino acid. Molecular docking validates the experimental data and proposes a possible substrate. Collectively, our results disfavor tyrosine as the substrate of RufO and point to the possibility that the nitration occurs during or after the assembly of the peptides. This study indicates a new function of the unique nitrating enzyme and provides insights into the biosynthesis of nonribosomal peptides.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/j.jbc.2023.105049</identifier><identifier>PMID: 37451485</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>cytochrome P450 ; heme-dependent catalysis ; nonribosomal peptide synthesis ; rufomycin ; tyrosyl nitration</subject><ispartof>The Journal of biological chemistry, 2023-08, Vol.299 (8), p.105049-105049, Article 105049</ispartof><rights>2023 The Authors</rights><rights>Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.</rights><rights>2023 The Authors 2023</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c404t-75397e2e73ba3f57753a7bb7aa0c130ff3f70c2958767f8df7164ec60598e29e3</cites><orcidid>0000-0002-4182-8176 ; 0000-0003-0378-2469 ; 0009-0006-7658-5107 ; 0000-0002-3202-3368</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10424215/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10424215/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37451485$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jordan, Stephanie</creatorcontrib><creatorcontrib>Li, Bingnan</creatorcontrib><creatorcontrib>Traore, Ephrahime</creatorcontrib><creatorcontrib>Wu, Yifei</creatorcontrib><creatorcontrib>Usai, Remigio</creatorcontrib><creatorcontrib>Liu, Aimin</creatorcontrib><creatorcontrib>Xie, Zhong-Ru</creatorcontrib><creatorcontrib>Wang, Yifan</creatorcontrib><title>Structural and spectroscopic characterization of RufO indicates a new biological role in rufomycin biosynthesis</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Rufomycins constitute a class of cyclic heptapeptides isolated from actinomycetes. They are secondary metabolites that show promising treatment against Mycobacterium tuberculosis infections by inhibiting a novel drug target. Several nonproteinogenic amino acids are integrated into rufomycins, including a conserved 3-nitro-tyrosine. RufO, a cytochrome P450 (CYP)-like enzyme, was proposed to catalyze the formation of 3-nitro-tyrosine in the presence of O2 and NO. To define its biological function, the interaction between RufO and the proposed substrate tyrosine is investigated using various spectroscopic methods that are sensitive to the structural change of a heme center. However, a low-to high-spin state transition and a dramatic increase in the redox potential that are commonly found in CYPs upon ligand binding have not been observed. Furthermore, a 1.89-Å crystal structure of RufO shows that the enzyme has flexible surface regions and a wide-open substrate access tunnel, and the heme center is largely exposed to solvent. Comparison with a closely related nitrating CYP reveals a spacious and hydrophobic distal pocket in RufO, which is incapable of stabilizing a free amino acid. Molecular docking validates the experimental data and proposes a possible substrate. Collectively, our results disfavor tyrosine as the substrate of RufO and point to the possibility that the nitration occurs during or after the assembly of the peptides. This study indicates a new function of the unique nitrating enzyme and provides insights into the biosynthesis of nonribosomal peptides.</description><subject>cytochrome P450</subject><subject>heme-dependent catalysis</subject><subject>nonribosomal peptide synthesis</subject><subject>rufomycin</subject><subject>tyrosyl nitration</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><recordid>eNp9kU2LFDEQhoMo7rj6A7xIjl56zOekGw8ii1-wsOAHeAvp6spOhp5kTNIr4683y6yLXswlqdRbbyX1EPKcszVnfPNqt96NsBZMyBZrpoYHZMVZLzup-feHZMWY4N0gdH9GnpSyY22pgT8mZ9IozVWvVyR9qXmBumQ3UxcnWg4INacC6RCAwtZlBxVz-OVqSJEmTz8v_oqGOAVwFQt1NOJPOoY0p-t2NdOcZmx5mhef9kdop5Ysx1i3WEJ5Sh55Nxd8drefk2_v3329-NhdXn34dPH2sgPFVO2MloNBgUaOTnptWuzMOBrnGHDJvJfeMBCD7s3G-H7yhm8UwobpoUcxoDwnb06-h2Xc4wQYa_uiPeSwd_lokwv230wMW3udbixnSijBdXN4eeeQ048FS7X7UADn2UVMS7Gil73QkmvTpPwkhTa5ktHf9-HM3pKyO9tI2VtS9kSq1bz4-4H3FX_QNMHrkwDbmG4CZlsgYAScQm6M7JTCf-x_A6MNpuo</recordid><startdate>20230801</startdate><enddate>20230801</enddate><creator>Jordan, Stephanie</creator><creator>Li, Bingnan</creator><creator>Traore, Ephrahime</creator><creator>Wu, Yifei</creator><creator>Usai, Remigio</creator><creator>Liu, Aimin</creator><creator>Xie, Zhong-Ru</creator><creator>Wang, Yifan</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-4182-8176</orcidid><orcidid>https://orcid.org/0000-0003-0378-2469</orcidid><orcidid>https://orcid.org/0009-0006-7658-5107</orcidid><orcidid>https://orcid.org/0000-0002-3202-3368</orcidid></search><sort><creationdate>20230801</creationdate><title>Structural and spectroscopic characterization of RufO indicates a new biological role in rufomycin biosynthesis</title><author>Jordan, Stephanie ; Li, Bingnan ; Traore, Ephrahime ; Wu, Yifei ; Usai, Remigio ; Liu, Aimin ; Xie, Zhong-Ru ; Wang, Yifan</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c404t-75397e2e73ba3f57753a7bb7aa0c130ff3f70c2958767f8df7164ec60598e29e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>cytochrome P450</topic><topic>heme-dependent catalysis</topic><topic>nonribosomal peptide synthesis</topic><topic>rufomycin</topic><topic>tyrosyl nitration</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jordan, Stephanie</creatorcontrib><creatorcontrib>Li, Bingnan</creatorcontrib><creatorcontrib>Traore, Ephrahime</creatorcontrib><creatorcontrib>Wu, Yifei</creatorcontrib><creatorcontrib>Usai, Remigio</creatorcontrib><creatorcontrib>Liu, Aimin</creatorcontrib><creatorcontrib>Xie, Zhong-Ru</creatorcontrib><creatorcontrib>Wang, Yifan</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jordan, Stephanie</au><au>Li, Bingnan</au><au>Traore, Ephrahime</au><au>Wu, Yifei</au><au>Usai, Remigio</au><au>Liu, Aimin</au><au>Xie, Zhong-Ru</au><au>Wang, Yifan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structural and spectroscopic characterization of RufO indicates a new biological role in rufomycin biosynthesis</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2023-08-01</date><risdate>2023</risdate><volume>299</volume><issue>8</issue><spage>105049</spage><epage>105049</epage><pages>105049-105049</pages><artnum>105049</artnum><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Rufomycins constitute a class of cyclic heptapeptides isolated from actinomycetes. They are secondary metabolites that show promising treatment against Mycobacterium tuberculosis infections by inhibiting a novel drug target. Several nonproteinogenic amino acids are integrated into rufomycins, including a conserved 3-nitro-tyrosine. RufO, a cytochrome P450 (CYP)-like enzyme, was proposed to catalyze the formation of 3-nitro-tyrosine in the presence of O2 and NO. To define its biological function, the interaction between RufO and the proposed substrate tyrosine is investigated using various spectroscopic methods that are sensitive to the structural change of a heme center. However, a low-to high-spin state transition and a dramatic increase in the redox potential that are commonly found in CYPs upon ligand binding have not been observed. Furthermore, a 1.89-Å crystal structure of RufO shows that the enzyme has flexible surface regions and a wide-open substrate access tunnel, and the heme center is largely exposed to solvent. Comparison with a closely related nitrating CYP reveals a spacious and hydrophobic distal pocket in RufO, which is incapable of stabilizing a free amino acid. Molecular docking validates the experimental data and proposes a possible substrate. Collectively, our results disfavor tyrosine as the substrate of RufO and point to the possibility that the nitration occurs during or after the assembly of the peptides. This study indicates a new function of the unique nitrating enzyme and provides insights into the biosynthesis of nonribosomal peptides.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>37451485</pmid><doi>10.1016/j.jbc.2023.105049</doi><tpages>1</tpages><orcidid>https://orcid.org/0000-0002-4182-8176</orcidid><orcidid>https://orcid.org/0000-0003-0378-2469</orcidid><orcidid>https://orcid.org/0009-0006-7658-5107</orcidid><orcidid>https://orcid.org/0000-0002-3202-3368</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | cytochrome P450 heme-dependent catalysis nonribosomal peptide synthesis rufomycin tyrosyl nitration |
title | Structural and spectroscopic characterization of RufO indicates a new biological role in rufomycin biosynthesis |
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