{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T16:45:40Z","timestamp":1792082740748,"version":"4.4.0"},"reference-count":33,"publisher":"American Chemical Society (ACS)","issue":"9","license":[{"start":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T00:00:00Z","timestamp":1788739200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100012043","name":"Kindai University","doi-asserted-by":"publisher","award":["IP002"],"award-info":[{"award-number":["IP002"]}],"id":[{"id":"10.13039\/100012043","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007684","name":"Asahi Glass Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100007684","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002241","name":"Japan Science and Technology Agency","doi-asserted-by":"publisher","award":["JPMJTR25U3"],"award-info":[{"award-number":["JPMJTR25U3"]}],"id":[{"id":"10.13039\/501100002241","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Institute Fermentation for Science, Osaka"},{"name":"Anti-aging Project for Private Universities"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Nonribosomal peptide synthetases (NRPSs) are modular assembly-line enzymes that generate structurally diverse and biologically active natural products. Although adenylation (A) domain selectivity has been extensively characterized and engineered, the specificity of condensation (C) domains, which catalyze peptide bond formation and can constrain NRPS reprogramming, remains less well understood. Here, we report a systematic functional analysis of VibH, a stand-alone C domain VibH from the vibriobactin biosynthetic pathway. By integrating VibH with wild-type and engineered variants of the upstream aryl acid A domain EntE, we bypassed intrinsic A-domain constraints and independently interrogated donor- and acceptor-site substrate tolerance. The donor site of VibH exhibited stringent specificity, accepting only mono- and disubstituted benzoic acid derivatives closely resembling 2,3-dihydroxybenzoic acid (DHB), whereas bulkier aryl substrates were not processed. In contrast, the acceptor site displayed broad tolerance toward structurally diverse amines. Monoamines showed clear chain-length dependence, with productive turnover restricted to medium-chain substrates, whereas diamines were more broadly accepted. Systematic analysis further revealed distance-dependent steric tolerance, in which bulky substituents were accommodated when positioned distal to the reactive amine but were restricted beyond an upper steric threshold. This engineered reconstitution strategy enabled the synthesis of numerous non-native amide conjugates and uncovered an asymmetric substrate-recognition architecture characterized by a stringent donor site and a permissive yet spatially constrained acceptor site. Molecular docking analysis provided a structural rationale for these trends by suggesting distinct binding modes for native and non-native donor substrates and distance-dependent accommodation of bulky amine acceptors. These findings provide mechanistic insights into C-domain specificity and establish a framework for rational NRPS reprogramming.<\/jats:p>","DOI":"10.1021\/acschembio.6c00537","type":"journal-article","created":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T12:01:58Z","timestamp":1788782518000},"page":"2427-2440","source":"Crossref","is-referenced-by-count":0,"title":["Exploring the Substrate Selectivity of the Condensation Domain VibH through Engineered Adenylation Domains, Non-Native Amide Synthesis, and Substrate Recognition Analysis"],"prefix":"10.1021","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8681-9396","authenticated-orcid":true,"given":"Fumihiro","family":"Ishikawa","sequence":"first","affiliation":[{"name":"Kindai University , , 3-4-1 Kowakae , , ,","place":["Higashi-osaka, Osaka, Japan, 577-8502"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroyuki","family":"Izuno","sequence":"additional","affiliation":[{"name":"Kindai University , , 3-4-1 Kowakae , , ,","place":["Higashi-osaka, Osaka, Japan, 577-8502"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinya","family":"Nakamura","sequence":"additional","affiliation":[{"name":"Kindai University , , 3-4-1 Kowakae , , ,","place":["Higashi-osaka, Osaka, Japan, 577-8502"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryotaro","family":"Wakano","sequence":"additional","affiliation":[{"name":"Kindai University , , 3-4-1 Kowakae , , ,","place":["Higashi-osaka, Osaka, Japan, 577-8502"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinta","family":"Ijichi","sequence":"additional","affiliation":[{"name":"Gakushuin University , , 1-5-1 Mejiro , , ,","place":["Toshima, Tokyo, Japan, 171-8588"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7178-5410","authenticated-orcid":true,"given":"Shumpei","family":"Asamizu","sequence":"additional","affiliation":[{"name":"Kobe University , , 1-1 Rokkodai, Nada , ,","place":["Kobe, Japan, 657-8501"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nao","family":"Miyazaki","sequence":"additional","affiliation":[{"name":"Kindai University , , 3-4-1 Kowakae , , ,","place":["Higashi-osaka, Osaka, Japan, 577-8502"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saaya","family":"Kusuhara","sequence":"additional","affiliation":[{"name":"Kindai University , , 3-4-1 Kowakae , , ,","place":["Higashi-osaka, Osaka, Japan, 577-8502"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4536-1881","authenticated-orcid":true,"given":"Hiroyasu","family":"Onaka","sequence":"additional","affiliation":[{"name":"Gakushuin University , , 1-5-1 Mejiro , , ,","place":["Toshima, Tokyo, Japan, 171-8588"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isao","family":"Nakanishi","sequence":"additional","affiliation":[{"name":"Kindai University , , 3-4-1 Kowakae , , ,","place":["Higashi-osaka, Osaka, Japan, 577-8502"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7954-8874","authenticated-orcid":true,"given":"Genzoh","family":"Tanabe","sequence":"additional","affiliation":[{"name":"Kindai University , , 3-4-1 Kowakae , , ,","place":["Higashi-osaka, Osaka, Japan, 577-8502"]},{"name":"Kindai University , , 3-4-1 Kowakae , , ,","place":["Higashi-osaka, Osaka, Japan, 577-8502"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,7]]},"reference":[{"key":"2026101511072361900_cit1","doi-asserted-by":"publisher","first-page":"3770","DOI":"10.1002\/anie.201609079","article-title":"Nonribosomal peptide synthesis\u2013principles and prospects","volume":"56","author":"S\u00fcssmuth","year":"2017","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026101511072361900_cit2","doi-asserted-by":"publisher","first-page":"1550","DOI":"10.1039\/d3np00003f","article-title":"Structural advances toward understanding the catalytic activity and conformational dynamics of modular nonribosomal peptide synthetases","volume":"40","author":"Patel","year":"2023","journal-title":"Nat. Prod. Rep."},{"key":"2026101511072361900_cit3","doi-asserted-by":"publisher","DOI":"10.1002\/psc.3545","article-title":"Recent progress in the reprogramming of nonribosomal peptide synthetases","volume":"30","author":"Ishikawa","year":"2024","journal-title":"J. Pept. Sci."},{"key":"2026101511072361900_cit4","doi-asserted-by":"publisher","first-page":"1180","DOI":"10.1039\/d3np00064h","article-title":"Structural, biochemical and bioinformatic analyses of nonribosomal peptide synthetase adenylation domains","volume":"41","author":"Heard","year":"2024","journal-title":"Nat. Prod. Rep."},{"key":"2026101511072361900_cit5","doi-asserted-by":"publisher","first-page":"761","DOI":"10.1038\/s41589-023-01532-x","article-title":"High-throughput reprogramming of an NRPS condensation domain","volume":"20","author":"Folger","year":"2024","journal-title":"Nat. Chem. Biol."},{"key":"2026101511072361900_cit6","doi-asserted-by":"publisher","first-page":"599","DOI":"10.1021\/acschembio.3c00678","article-title":"Controlling substrate- and stereospecificity of condensation domains in nonribosomal peptide synthetases","volume":"19","author":"Peng","year":"2024","journal-title":"ACS Chem. Biol."},{"key":"2026101511072361900_cit7","doi-asserted-by":"publisher","first-page":"1587","DOI":"10.1016\/j.bbapap.2017.05.010","article-title":"Structural and functional aspects of the nonribosomal peptide synthetase condensation domain superfamily: discovery, dissection and diversity","volume":"1865","author":"Bloudoff","year":"2017","journal-title":"Biochim. Biophys. Acta, Proteins Proteomics"},{"key":"2026101511072361900_cit8","doi-asserted-by":"publisher","first-page":"522","DOI":"10.1038\/nsb810","article-title":"The structure of VibH represents nonribosomal peptide synthetase condensation, cyclization and epimerization domains","volume":"9","author":"Keating","year":"2002","journal-title":"Nat. Struct. Biol."},{"key":"2026101511072361900_cit9","doi-asserted-by":"publisher","first-page":"331","DOI":"10.1016\/j.chembiol.2016.02.012","article-title":"Chemical probes allow structural insight into the condensation reaction of nonribosomal peptide synthetases","volume":"23","author":"Bloudoff","year":"2016","journal-title":"Cell Chem. Biol."},{"key":"2026101511072361900_cit10","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaw4388","article-title":"Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility","volume":"366","author":"Reimer","year":"2019","journal-title":"Science"},{"key":"2026101511072361900_cit11","doi-asserted-by":"publisher","first-page":"440","DOI":"10.1016\/j.str.2024.01.010","article-title":"Modular catalytic activity of nonribosomal peptide synthetases depends on the dynamic interaction between adenylation and condensation domains","volume":"32","author":"Peng","year":"2024","journal-title":"Structure"},{"key":"2026101511072361900_cit12","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1038\/s41586-024-08306-y","article-title":"Crosslinking intermodular condensation in non-ribosomal peptide biosynthesis","volume":"638","author":"Heberlig","year":"2025","journal-title":"Nature"},{"key":"2026101511072361900_cit13","doi-asserted-by":"publisher","first-page":"270","DOI":"10.1038\/s41586-024-08417-6","article-title":"Structures and mechanism of condensation in non-ribosomal peptide synthesis","volume":"638","author":"Pistofidis","year":"2025","journal-title":"Nature"},{"key":"2026101511072361900_cit14","doi-asserted-by":"publisher","first-page":"4183","DOI":"10.1073\/pnas.0808880106","article-title":"A free-standing condensation enzyme catalyzing ester bond formation in C-1027 biosynthesis","volume":"106","author":"Lin","year":"2009","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026101511072361900_cit15","doi-asserted-by":"publisher","first-page":"383","DOI":"10.1038\/nature14100","article-title":"\u03b2-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis","volume":"520","author":"Gaudelli","year":"2015","journal-title":"Nature"},{"key":"2026101511072361900_cit16","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2026017118","article-title":"Evolutionary and functional analysis of an NRPS condensation domain integrates \u03b2-lactam, D-amino acid, and dehydroamino acid synthesis","volume":"118","author":"Wheadon","year":"2021","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026101511072361900_cit17","doi-asserted-by":"publisher","first-page":"3229","DOI":"10.1002\/anie.202012902","article-title":"Discovery and biosynthetic investigation of a new antibacterial dehydrated non-ribosomal tripeptide","volume":"60","author":"Wang","year":"2021","journal-title":"Angew. Chem., Int. 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J."},{"key":"2026101511072361900_cit23","doi-asserted-by":"publisher","first-page":"2039","DOI":"10.1021\/acs.biochem.4c00770","article-title":"Teicoplanin nonribosomal peptide synthetase is unable to incorporate alpha-ketoacid building blocks","volume":"64","author":"Ratnayake","year":"2025","journal-title":"Biochemistry"},{"key":"2026101511072361900_cit24","doi-asserted-by":"publisher","first-page":"486","DOI":"10.1126\/science.284.5413.486","article-title":"Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis","volume":"284","author":"Belshaw","year":"1999","journal-title":"Science"},{"key":"2026101511072361900_cit25","doi-asserted-by":"publisher","first-page":"765","DOI":"10.1016\/s1074-5521(00)00022-3","article-title":"Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases","volume":"7","author":"Ehmann","year":"2000","journal-title":"Chem. Biol."},{"key":"2026101511072361900_cit26","doi-asserted-by":"publisher","first-page":"2511","DOI":"10.1038\/s41467-021-22623-0","article-title":"Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity","volume":"12","author":"Izor\u00e9","year":"2021","journal-title":"Nat. Commun."},{"key":"2026101511072361900_cit27","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1016\/bs.mie.2024.06.010","article-title":"An in vitro assay to explore condensation domain specificity from non-ribosomal peptide synthesis","volume":"702","author":"Ratnayake","year":"2024","journal-title":"Methods Enzymol."},{"key":"2026101511072361900_cit28","doi-asserted-by":"publisher","first-page":"9466","DOI":"10.1039\/c9sc03678d","article-title":"A proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics","volume":"10","author":"Kaniusaite","year":"2019","journal-title":"Chem. Sci."},{"key":"2026101511072361900_cit29","doi-asserted-by":"publisher","first-page":"2648","DOI":"10.1021\/bi9726584","article-title":"Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF","volume":"37","author":"Gehring","year":"1998","journal-title":"Biochemistry"},{"key":"2026101511072361900_cit30","doi-asserted-by":"publisher","first-page":"15522","DOI":"10.1021\/bi0016523","article-title":"Reconstitution and Characterization of the Vibrio cholerae Vibriobactin Synthetase from VibB, VibE, VibF, and VibH","volume":"39","author":"Keating","year":"2000","journal-title":"Biochemistry"},{"key":"2026101511072361900_cit31","doi-asserted-by":"publisher","first-page":"7055","DOI":"10.1128\/jb.179.22.7055-7062.1997","article-title":"Cloning of a Vibrio cholerae vibriobactin gene cluster: identification of genes required for early steps in siderophore biosynthesis","volume":"179","author":"Wyckoff","year":"1997","journal-title":"J. Bacteriol."},{"key":"2026101511072361900_cit32","doi-asserted-by":"publisher","first-page":"15513","DOI":"10.1021\/bi001651a","article-title":"Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains","volume":"39","author":"Keating","year":"2000","journal-title":"Biochemistry"},{"key":"2026101511072361900_cit33","doi-asserted-by":"publisher","first-page":"14129","DOI":"10.1039\/c6cc07793e","article-title":"A chemical proteomic probe for detecting native carrier protein motifs in nonribosomal peptide synthetases","volume":"52","author":"Kasai","year":"2016","journal-title":"Chem. Commun."}],"container-title":["ACS Chemical Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acschembio.6c00537","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/21\/9\/2427\/68403488\/cb-2026-00537q.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acbcct\/article-pdf\/21\/9\/2427\/68403488\/cb-2026-00537q.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T15:49:26Z","timestamp":1792079366000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acbcct\/article\/21\/9\/2427\/5422384\/Exploring-the-Substrate-Selectivity-of-the"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,7]]},"references-count":33,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9,7]]},"published-print":{"date-parts":[[2026,9,18]]}},"URL":"https:\/\/doi.org\/10.1021\/acschembio.6c00537","relation":{},"ISSN":["1554-8929","1554-8937"],"issn-type":[{"value":"1554-8929","type":"print"},{"value":"1554-8937","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,7]]}}}