Structural and Biochemical Characterization of Aminoglycoside Nucleotidyltransferase(6)‐Ib From Campylobacter fetus subsp. fetus
ABSTRACT Aminoglycoside antibiotics have played a critical role in the treatment of both Gram‐negative and Gram‐positive bacterial infections. However, antibiotic resistance has severely compromised the efficacy of aminoglycosides. A leading cause of aminoglycoside resistance is mediated by bacteria...
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| Veröffentlicht in: | Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2025-02, Vol.93 (2), p.413-419 |
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| container_title | Proteins, structure, function, and bioinformatics |
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| creator | Nalam, Pranav Cook, Paul D. Smith, Brian A. |
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Aminoglycoside antibiotics have played a critical role in the treatment of both Gram‐negative and Gram‐positive bacterial infections. However, antibiotic resistance has severely compromised the efficacy of aminoglycosides. A leading cause of aminoglycoside resistance is mediated by bacterial enzymes that inactivate these drugs via chemical modification. Aminoglycoside nucleotidyltransferase‐6 (ANT(6)) enzymes inactivate streptomycin by transferring an adenyl group from ATP to position 6 on the antibiotic. Despite the clinical significance of this activity, ANT(6) enzymes remain relatively uncharacterized. Here, we report the first high resolution x‐ray crystallographic structure of ANT(6)‐Ib from Campylobacter fetus subsp. fetus bound with streptomycin. Structural modeling and gel filtration chromatography experiments suggest that the enzyme exists as a dimer in which both subunits contribute to the active site. Moreover, superposition of the ANT(6)‐Ib structure with the structurally related enzyme lincosamide nucleotidyltransferase B (LinB) permitted the identification of a putative nucleotide binding site. These data also suggest that residues D44 and D46 coordinate essential divalent metal ions and D102 functions as the catalytic base. |
| doi_str_mv | 10.1002/prot.26745 |
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Aminoglycoside antibiotics have played a critical role in the treatment of both Gram‐negative and Gram‐positive bacterial infections. However, antibiotic resistance has severely compromised the efficacy of aminoglycosides. A leading cause of aminoglycoside resistance is mediated by bacterial enzymes that inactivate these drugs via chemical modification. Aminoglycoside nucleotidyltransferase‐6 (ANT(6)) enzymes inactivate streptomycin by transferring an adenyl group from ATP to position 6 on the antibiotic. Despite the clinical significance of this activity, ANT(6) enzymes remain relatively uncharacterized. Here, we report the first high resolution x‐ray crystallographic structure of ANT(6)‐Ib from Campylobacter fetus subsp. fetus bound with streptomycin. Structural modeling and gel filtration chromatography experiments suggest that the enzyme exists as a dimer in which both subunits contribute to the active site. Moreover, superposition of the ANT(6)‐Ib structure with the structurally related enzyme lincosamide nucleotidyltransferase B (LinB) permitted the identification of a putative nucleotide binding site. These data also suggest that residues D44 and D46 coordinate essential divalent metal ions and D102 functions as the catalytic base.</description><identifier>ISSN: 0887-3585</identifier><identifier>ISSN: 1097-0134</identifier><identifier>EISSN: 1097-0134</identifier><identifier>DOI: 10.1002/prot.26745</identifier><identifier>PMID: 39246239</identifier><language>eng</language><publisher>Hoboken, USA: John Wiley & Sons, Inc</publisher><subject>Adenosine triphosphate ; Amino Acid Sequence ; aminoglycoside ; Aminoglycoside antibiotics ; aminoglycoside modifying enzyme ; aminoglycoside nucleotidyltransferase‐6 ; Aminoglycosides ; Anti-Bacterial Agents - chemistry ; Anti-Bacterial Agents - metabolism ; Anti-Bacterial Agents - pharmacology ; Antibiotic resistance ; Antibiotics ; Bacterial diseases ; Bacterial Proteins - chemistry ; Bacterial Proteins - metabolism ; Binding Sites ; Campylobacter ; Campylobacter fetus ; Campylobacter fetus - chemistry ; Campylobacter fetus - enzymology ; Campylobacter fetus subsp. fetus ; Catalytic Domain ; Chemical modification ; Crystal structure ; Crystallography ; Crystallography, X-Ray ; Deactivation ; Drug resistance ; Enzymes ; Fetuses ; Gel chromatography ; Gel filtration ; Metal ions ; Models, Molecular ; Molecular structure ; Nucleotides ; Nucleotidyltransferases - chemistry ; Nucleotidyltransferases - metabolism ; Protein Binding ; Protein Multimerization ; Streptomycin ; Streptomycin - chemistry ; Streptomycin - metabolism ; Streptomycin - pharmacology ; Structural analysis</subject><ispartof>Proteins, structure, function, and bioinformatics, 2025-02, Vol.93 (2), p.413-419</ispartof><rights>2024 Wiley Periodicals LLC.</rights><rights>2025 Wiley Periodicals LLC.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c2465-4c4c9eb423c4ffaf07e74a9bef33fbdd7a141e8aa68403839d753063d91e9eb23</cites><orcidid>0009-0002-1743-5273 ; 0000-0002-7286-2860</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fprot.26745$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fprot.26745$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1416,27915,27916,45565,45566</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/39246239$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nalam, Pranav</creatorcontrib><creatorcontrib>Cook, Paul D.</creatorcontrib><creatorcontrib>Smith, Brian A.</creatorcontrib><title>Structural and Biochemical Characterization of Aminoglycoside Nucleotidyltransferase(6)‐Ib From Campylobacter fetus subsp. fetus</title><title>Proteins, structure, function, and bioinformatics</title><addtitle>Proteins</addtitle><description>ABSTRACT
Aminoglycoside antibiotics have played a critical role in the treatment of both Gram‐negative and Gram‐positive bacterial infections. However, antibiotic resistance has severely compromised the efficacy of aminoglycosides. A leading cause of aminoglycoside resistance is mediated by bacterial enzymes that inactivate these drugs via chemical modification. Aminoglycoside nucleotidyltransferase‐6 (ANT(6)) enzymes inactivate streptomycin by transferring an adenyl group from ATP to position 6 on the antibiotic. Despite the clinical significance of this activity, ANT(6) enzymes remain relatively uncharacterized. Here, we report the first high resolution x‐ray crystallographic structure of ANT(6)‐Ib from Campylobacter fetus subsp. fetus bound with streptomycin. Structural modeling and gel filtration chromatography experiments suggest that the enzyme exists as a dimer in which both subunits contribute to the active site. Moreover, superposition of the ANT(6)‐Ib structure with the structurally related enzyme lincosamide nucleotidyltransferase B (LinB) permitted the identification of a putative nucleotide binding site. These data also suggest that residues D44 and D46 coordinate essential divalent metal ions and D102 functions as the catalytic base.</description><subject>Adenosine triphosphate</subject><subject>Amino Acid Sequence</subject><subject>aminoglycoside</subject><subject>Aminoglycoside antibiotics</subject><subject>aminoglycoside modifying enzyme</subject><subject>aminoglycoside nucleotidyltransferase‐6</subject><subject>Aminoglycosides</subject><subject>Anti-Bacterial Agents - chemistry</subject><subject>Anti-Bacterial Agents - metabolism</subject><subject>Anti-Bacterial Agents - pharmacology</subject><subject>Antibiotic resistance</subject><subject>Antibiotics</subject><subject>Bacterial diseases</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - metabolism</subject><subject>Binding Sites</subject><subject>Campylobacter</subject><subject>Campylobacter fetus</subject><subject>Campylobacter fetus - chemistry</subject><subject>Campylobacter fetus - enzymology</subject><subject>Campylobacter fetus subsp. fetus</subject><subject>Catalytic Domain</subject><subject>Chemical modification</subject><subject>Crystal structure</subject><subject>Crystallography</subject><subject>Crystallography, X-Ray</subject><subject>Deactivation</subject><subject>Drug resistance</subject><subject>Enzymes</subject><subject>Fetuses</subject><subject>Gel chromatography</subject><subject>Gel filtration</subject><subject>Metal ions</subject><subject>Models, Molecular</subject><subject>Molecular structure</subject><subject>Nucleotides</subject><subject>Nucleotidyltransferases - chemistry</subject><subject>Nucleotidyltransferases - metabolism</subject><subject>Protein Binding</subject><subject>Protein Multimerization</subject><subject>Streptomycin</subject><subject>Streptomycin - chemistry</subject><subject>Streptomycin - metabolism</subject><subject>Streptomycin - pharmacology</subject><subject>Structural analysis</subject><issn>0887-3585</issn><issn>1097-0134</issn><issn>1097-0134</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2025</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp90c1qFTEUB_Agir1WNz6ABNzUwlyTSeYjy3ppa6FY0boeMpkTm5KZ3OYDma7EJ-gz9knM7VQXLlyFA7_z55A_Qq8pWVNCyvdb7-K6rBtePUErSkRTEMr4U7QibdsUrGqrPfQihGtCSC1Y_RztMVHyumRihX59jT6pmLy0WE4D_mCcuoLRqDxvrqSXKoI3tzIaN2Gn8dFoJvfdzsoFMwD-lJQFF80w2-jlFDR4GeCgfnf_8-6sxyfejXgjx-1sXf8QhTXEFHBIfdiul-EleqalDfDq8d1H306OLzcfi_OL07PN0Xmh8rFVwRVXAnpeMsW1lpo00HApetCM6X4YGkk5hVbKuuWEtUwMTcVIzQZBIe-VbB8dLLn5v24ShNiNJiiwVk7gUugYJSVpSs5Epm__odcu-Slfl1W1Q4S0WR0uSnkXggfdbb0ZpZ87SrpdM92ume6hmYzfPEamfoThL_1TRQZ0AT-Mhfk_Ud3nLxeXS-hvdqycbA</recordid><startdate>202502</startdate><enddate>202502</enddate><creator>Nalam, Pranav</creator><creator>Cook, Paul D.</creator><creator>Smith, Brian A.</creator><general>John Wiley & Sons, Inc</general><general>Wiley Subscription Services, Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><orcidid>https://orcid.org/0009-0002-1743-5273</orcidid><orcidid>https://orcid.org/0000-0002-7286-2860</orcidid></search><sort><creationdate>202502</creationdate><title>Structural and Biochemical Characterization of Aminoglycoside Nucleotidyltransferase(6)‐Ib From Campylobacter fetus subsp. fetus</title><author>Nalam, Pranav ; Cook, Paul D. ; Smith, Brian A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c2465-4c4c9eb423c4ffaf07e74a9bef33fbdd7a141e8aa68403839d753063d91e9eb23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2025</creationdate><topic>Adenosine triphosphate</topic><topic>Amino Acid Sequence</topic><topic>aminoglycoside</topic><topic>Aminoglycoside antibiotics</topic><topic>aminoglycoside modifying enzyme</topic><topic>aminoglycoside nucleotidyltransferase‐6</topic><topic>Aminoglycosides</topic><topic>Anti-Bacterial Agents - chemistry</topic><topic>Anti-Bacterial Agents - metabolism</topic><topic>Anti-Bacterial Agents - pharmacology</topic><topic>Antibiotic resistance</topic><topic>Antibiotics</topic><topic>Bacterial diseases</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - metabolism</topic><topic>Binding Sites</topic><topic>Campylobacter</topic><topic>Campylobacter fetus</topic><topic>Campylobacter fetus - chemistry</topic><topic>Campylobacter fetus - enzymology</topic><topic>Campylobacter fetus subsp. fetus</topic><topic>Catalytic Domain</topic><topic>Chemical modification</topic><topic>Crystal structure</topic><topic>Crystallography</topic><topic>Crystallography, X-Ray</topic><topic>Deactivation</topic><topic>Drug resistance</topic><topic>Enzymes</topic><topic>Fetuses</topic><topic>Gel chromatography</topic><topic>Gel filtration</topic><topic>Metal ions</topic><topic>Models, Molecular</topic><topic>Molecular structure</topic><topic>Nucleotides</topic><topic>Nucleotidyltransferases - chemistry</topic><topic>Nucleotidyltransferases - metabolism</topic><topic>Protein Binding</topic><topic>Protein Multimerization</topic><topic>Streptomycin</topic><topic>Streptomycin - chemistry</topic><topic>Streptomycin - metabolism</topic><topic>Streptomycin - pharmacology</topic><topic>Structural analysis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nalam, Pranav</creatorcontrib><creatorcontrib>Cook, Paul D.</creatorcontrib><creatorcontrib>Smith, Brian A.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Proteins, structure, function, and bioinformatics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nalam, Pranav</au><au>Cook, Paul D.</au><au>Smith, Brian A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structural and Biochemical Characterization of Aminoglycoside Nucleotidyltransferase(6)‐Ib From Campylobacter fetus subsp. fetus</atitle><jtitle>Proteins, structure, function, and bioinformatics</jtitle><addtitle>Proteins</addtitle><date>2025-02</date><risdate>2025</risdate><volume>93</volume><issue>2</issue><spage>413</spage><epage>419</epage><pages>413-419</pages><issn>0887-3585</issn><issn>1097-0134</issn><eissn>1097-0134</eissn><abstract>ABSTRACT
Aminoglycoside antibiotics have played a critical role in the treatment of both Gram‐negative and Gram‐positive bacterial infections. However, antibiotic resistance has severely compromised the efficacy of aminoglycosides. A leading cause of aminoglycoside resistance is mediated by bacterial enzymes that inactivate these drugs via chemical modification. Aminoglycoside nucleotidyltransferase‐6 (ANT(6)) enzymes inactivate streptomycin by transferring an adenyl group from ATP to position 6 on the antibiotic. Despite the clinical significance of this activity, ANT(6) enzymes remain relatively uncharacterized. Here, we report the first high resolution x‐ray crystallographic structure of ANT(6)‐Ib from Campylobacter fetus subsp. fetus bound with streptomycin. Structural modeling and gel filtration chromatography experiments suggest that the enzyme exists as a dimer in which both subunits contribute to the active site. Moreover, superposition of the ANT(6)‐Ib structure with the structurally related enzyme lincosamide nucleotidyltransferase B (LinB) permitted the identification of a putative nucleotide binding site. These data also suggest that residues D44 and D46 coordinate essential divalent metal ions and D102 functions as the catalytic base.</abstract><cop>Hoboken, USA</cop><pub>John Wiley & Sons, Inc</pub><pmid>39246239</pmid><doi>10.1002/prot.26745</doi><tpages>7</tpages><orcidid>https://orcid.org/0009-0002-1743-5273</orcidid><orcidid>https://orcid.org/0000-0002-7286-2860</orcidid></addata></record> |
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| subjects | Adenosine triphosphate Amino Acid Sequence aminoglycoside Aminoglycoside antibiotics aminoglycoside modifying enzyme aminoglycoside nucleotidyltransferase‐6 Aminoglycosides Anti-Bacterial Agents - chemistry Anti-Bacterial Agents - metabolism Anti-Bacterial Agents - pharmacology Antibiotic resistance Antibiotics Bacterial diseases Bacterial Proteins - chemistry Bacterial Proteins - metabolism Binding Sites Campylobacter Campylobacter fetus Campylobacter fetus - chemistry Campylobacter fetus - enzymology Campylobacter fetus subsp. fetus Catalytic Domain Chemical modification Crystal structure Crystallography Crystallography, X-Ray Deactivation Drug resistance Enzymes Fetuses Gel chromatography Gel filtration Metal ions Models, Molecular Molecular structure Nucleotides Nucleotidyltransferases - chemistry Nucleotidyltransferases - metabolism Protein Binding Protein Multimerization Streptomycin Streptomycin - chemistry Streptomycin - metabolism Streptomycin - pharmacology Structural analysis |
| title | Structural and Biochemical Characterization of Aminoglycoside Nucleotidyltransferase(6)‐Ib From Campylobacter fetus subsp. fetus |
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