{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T13:02:24Z","timestamp":1791291744220,"version":"4.1.0"},"reference-count":34,"publisher":"American Chemical Society (ACS)","issue":"19","license":[{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R01 GM040541"],"award-info":[{"award-number":["R01 GM040541"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R35 GM148356"],"award-info":[{"award-number":["R35 GM148356"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R35 GM153203"],"award-info":[{"award-number":["R35 GM153203"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>AprD5 is a member of the NAD+-dependent short-chain dehydrogenase\/reductase (SDR) superfamily. It participates in the apramycin biosynthetic pathway by catalyzing 4\u2032-oxidation of UDP-\u03b2-d-glucose (UBG) to initiate the assembly of UDP-4\u2032-amino-4\u2032-deoxy-\u03b2-d-glucose in partnership with the aminotransferase AprL. Here, we report a 1.9 \u00c5 crystal structure of AprD5 in complex with NAD+ and its substrate UBG, which reveals a well-ordered ternary complex poised for catalysis. The cofactor and substrate are precisely aligned for hydride transfer by a conserved Ser\u2013Tyr\u2013Lys catalytic triad, while the nucleotide-sugar is constrained in a single reactive conformation through an extensive hydrogen-bonding network and a tight electrostatic clamp on the diphosphate group. Although 4\u2032-oxidation is a common initiating step in SDR-catalyzed 4\u2032-epimerization and 4\u2032,6\u2032-dehydration, the structural features that direct these closely related enzymes toward distinct reaction outcomes remain poorly understood. Comparative structural analysis reveals that AprD5 lacks the catalytic architecture required for dehydration while a rigid phosphate clamp and extensive substrate-anchoring network restrict the conformational rearrangements necessary for epimerization. Together, these features suggest how a conserved SDR scaffold is specialized for dedicated 4\u2032-dehydrogenation and highlight general structural principles that may govern catalytic divergence among nucleotidyl-sugar SDR enzymes.<\/jats:p>","DOI":"10.1021\/acs.biochem.6c00553","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T04:02:24Z","timestamp":1790222544000},"page":"3106-3115","source":"Crossref","is-referenced-by-count":0,"title":["Structural and\nMechanistic Insights into 4\u2032-Dehydrogenation\nCatalyzed by AprD5 in Apramycin Biosynthesis"],"prefix":"10.1021","volume":"65","author":[{"given":"Wantae","family":"Kim","sequence":"first","affiliation":[{"name":"University of Texas at Austin , , , ,","place":["Austin, Texas, United States, 78712"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Hong","sequence":"additional","affiliation":[{"name":"University of Texas at Austin , , , ,","place":["Austin, Texas, United States, 78712"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3518-8309","authenticated-orcid":true,"given":"Shusuke","family":"Sato","sequence":"additional","affiliation":[{"name":"University of Texas at Austin , , , ,","place":["Austin, Texas, United States, 78712"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8953-4794","authenticated-orcid":true,"given":"Hung-wen","family":"Liu","sequence":"additional","affiliation":[{"name":"University of Texas at Austin , , , ,","place":["Austin, Texas, United States, 78712"]},{"name":"University of Texas at Austin , , , ,","place":["Austin, Texas, United States, 78712"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9360-5388","authenticated-orcid":true,"given":"Y. Jessie","family":"Zhang","sequence":"additional","affiliation":[{"name":"University of Texas at Austin , , , ,","place":["Austin, Texas, United States, 78712"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,23]]},"reference":[{"issue":"1","key":"2026100608064065100_cit1","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1021\/cb3005116","article-title":"Aminoglycoside Antibiotics in the 21st Century","volume":"8","author":"Becker","year":"2013","journal-title":"ACS Chem. Biol."},{"issue":"6","key":"2026100608064065100_cit2","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a027029","article-title":"Aminoglycosides:\nAn Overview","volume":"6","author":"Krause","year":"2016","journal-title":"Cold Spring Harbor Perspect. Med."},{"issue":"39","key":"2026100608064065100_cit3","doi-asserted-by":"publisher","first-page":"21361","DOI":"10.1021\/jacs.3c06354","article-title":"Biosynthetic Origin\nof the Octose Core and Its Mechanism of Assembly during Apramycin\nBiosynthesis","volume":"145","author":"Fan","year":"2023","journal-title":"J. Am. Chem. Soc."},{"issue":"4","key":"2026100608064065100_cit4","doi-asserted-by":"publisher","first-page":"1365","DOI":"10.1021\/jacs.7b10501","article-title":"1,2-Diol Dehydration\nby the Radical SAM Enzyme AprD4: A Matter of Proton Circulation and\nSubstrate Flexibility","volume":"140","author":"Liu","year":"2018","journal-title":"J. Am. Chem. Soc."},{"issue":"18","key":"2026100608064065100_cit5","doi-asserted-by":"publisher","first-page":"16186","DOI":"10.1021\/acscatal.5c02753","article-title":"Apramycin Biosynthesis: Structure\nand Mechanism of\nAction of a New-Type Transaldolase AprG from Streptoalloteichus Tenebrarius","volume":"15","author":"Xie","year":"2025","journal-title":"ACS Catal."},{"issue":"13","key":"2026100608064065100_cit6","doi-asserted-by":"publisher","first-page":"5038","DOI":"10.1021\/jacs.1c00076","article-title":"Mechanistic\nInvestigation of 1,2-Diol\nDehydration of Paromamine Catalyzed by the Radical S-Adenosyl-l-Methionine\nEnzyme AprD4","volume":"143","author":"Yeh","year":"2021","journal-title":"J. Am. Chem. Soc."},{"issue":"14","key":"2026100608064065100_cit7","doi-asserted-by":"publisher","first-page":"10103","DOI":"10.1021\/jacs.4c01233","article-title":"Complete In Vitro Reconstitution\nof the Apramycin Biosynthetic Pathway Demonstrates the Unusual Incorporation\nof a \u03b2-d-Sugar Nucleotide in the Final Glycosylation Step","volume":"146","author":"Sato","year":"2024","journal-title":"J. Am. Chem. Soc."},{"issue":"24","key":"2026100608064065100_cit8","doi-asserted-by":"publisher","first-page":"3895","DOI":"10.1007\/s00018-008-8588-y","article-title":"Medium- and\nShort-Chain Dehydrogenase\/Reductase Gene and Protein Families","volume":"65","author":"Kavanagh","year":"2008","journal-title":"Cell. Mol. Life Sci."},{"issue":"10","key":"2026100608064065100_cit9","doi-asserted-by":"publisher","first-page":"1201","DOI":"10.1039\/C2NP20039B","article-title":"The Structural Biology of Enzymes\nInvolved in Natural Product Glycosylation","volume":"29","author":"Singh","year":"2012","journal-title":"Nat.\nProd. Rep."},{"issue":"12","key":"2026100608064065100_cit10","doi-asserted-by":"publisher","first-page":"4615","DOI":"10.1021\/cr9902998","article-title":"Novel Enzymatic Mechanisms in Carbohydrate\nMetabolism","volume":"100","author":"He","year":"2000","journal-title":"Chem. Rev."},{"key":"2026100608064065100_cit11","doi-asserted-by":"publisher","first-page":"701","DOI":"10.1146\/annurev.biochem.71.110601.135339","article-title":"Formation of Unusual\nSugars: Mechanistic Studies and\nBiosynthetic Applications","volume":"71","author":"He","year":"2002","journal-title":"Annu. Rev. Biochem."},{"issue":"20","key":"2026100608064065100_cit12","doi-asserted-by":"publisher","first-page":"7777","DOI":"10.1021\/ja00020a059","article-title":"Stereochemical and\nMechanistic Studies of CDP-D-Glucose\nOxidoreductase Isolated from Yersinia Pseudotuberculosis","volume":"113","author":"Russell","year":"1991","journal-title":"J. Am. Chem. Soc."},{"issue":"37","key":"2026100608064065100_cit13","doi-asserted-by":"publisher","first-page":"9698","DOI":"10.1021\/ja982198h","article-title":"CDP-6-Deoxy-6,6-Difluoro-d-Glucose:\nA Mechanism-Based Inhibitor for CDP-d-Glucose 4,6-Dehydratase","volume":"120","author":"Chang","year":"1998","journal-title":"J. Am. Chem. Soc."},{"issue":"1","key":"2026100608064065100_cit14","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1016\/S0969-2126(01)00694-3","article-title":"Toward\na Structural Understanding of the Dehydratase Mechanism","volume":"10","author":"Allard","year":"2002","journal-title":"Structure"},{"issue":"22","key":"2026100608064065100_cit15","doi-asserted-by":"publisher","first-page":"6598","DOI":"10.1021\/bi010441a","article-title":"Probing Catalysis\nby Escherichia coli dTDP-Glucose-4,6-Dehydratase:\nIdentification\nand Preliminary Characterization of Functional Amino Acid Residues\nat the Active Site","volume":"40","author":"Hegeman","year":"2001","journal-title":"Biochemistry"},{"issue":"16","key":"2026100608064065100_cit16","doi-asserted-by":"publisher","first-page":"5137","DOI":"10.1021\/bi9601114","article-title":"Molecular Structure of the NADH\/UDP-Glucose\nAbortive Complex of UDP-Galactose 4-Epimerase from Escherichia coli: Implications for the Catalytic\nMechanism","volume":"35","author":"Thoden","year":"1996","journal-title":"Biochemistry"},{"issue":"35","key":"2026100608064065100_cit17","doi-asserted-by":"publisher","first-page":"10675","DOI":"10.1021\/bi970430a","article-title":"Mechanistic Roles\nof Tyrosine 149 and Serine 124 in UDP-Galactose 4-Epimerase from Escherichia coli","volume":"36","author":"Liu","year":"1997","journal-title":"Biochemistry"},{"issue":"52","key":"2026100608064065100_cit18","doi-asserted-by":"publisher","first-page":"15975","DOI":"10.1016\/S0040-4020(98)01005-9","article-title":"Stereospecificity\nof Hydride Transfer for the Catalytically Recycled\nNAD+ in CDP-d-Glucose 4,6-Dehydratase","volume":"54","author":"Hallis","year":"1998","journal-title":"Tetrahedron"},{"issue":"1","key":"2026100608064065100_cit19","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1107\/S0021889809045701","article-title":"Xia2: An Expert System for Macromolecular Crystallography Data Reduction","volume":"43","author":"Winter","year":"2010","journal-title":"J. Appl. Crystallogr."},{"issue":"2","key":"2026100608064065100_cit20","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1107\/S0907444909052925","article-title":"PHENIX: A Comprehensive\nPython-Based System for Macromolecular Structure Solution","volume":"66","author":"Adams","year":"2010","journal-title":"Acta Crystallogr., Sect. D: Biol. Crystallogr."},{"issue":"7873","key":"2026100608064065100_cit21","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly Accurate Protein\nStructure Prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"issue":"4","key":"2026100608064065100_cit22","doi-asserted-by":"publisher","first-page":"486","DOI":"10.1107\/S0907444910007493","article-title":"Features and Development\nof Coot","volume":"66","author":"Emsley","year":"2010","journal-title":"Acta Crystallogr.,\nSect. D: Biol. Crystallogr."},{"issue":"1","key":"2026100608064065100_cit23","doi-asserted-by":"publisher","first-page":"293","DOI":"10.1002\/pro.3330","article-title":"MolProbity:\nMore and Better Reference Data for Improved\nAll-atom Structure Validation","volume":"27","author":"Williams","year":"2018","journal-title":"Protein Sci."},{"issue":"1","key":"2026100608064065100_cit24","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4519","article-title":"DALI Shines\na Light on Remote Homologs: One Hundred Discoveries","volume":"32","author":"Holm","year":"2023","journal-title":"Protein Sci."},{"issue":"4","key":"2026100608064065100_cit25","doi-asserted-by":"publisher","first-page":"1160","DOI":"10.1093\/bib\/bbx108","article-title":"MAFFT Online Service: Multiple Sequence\nAlignment, Interactive Sequence Choice and Visualization","volume":"20","author":"Katoh","year":"2019","journal-title":"Briefings Bioinf."},{"issue":"9","key":"2026100608064065100_cit26","doi-asserted-by":"publisher","first-page":"1189","DOI":"10.1093\/bioinformatics\/btp033","article-title":"Jalview\nVersion 2\u2500a Multiple Sequence Alignment Editor and Analysis\nWorkbench","volume":"25","author":"Waterhouse","year":"2009","journal-title":"Bioinformatics"},{"issue":"11","key":"2026100608064065100_cit27","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4792","article-title":"UCSF ChimeraX:\nTools for Structure\nBuilding and Analysis","volume":"32","author":"Meng","year":"2023","journal-title":"Protein Sci."},{"issue":"16","key":"2026100608064065100_cit28","doi-asserted-by":"publisher","first-page":"2785","DOI":"10.1002\/jcc.21256","article-title":"AutoDock4\nand AutoDockTools4: Automated\nDocking with Selective Receptor Flexibility","volume":"30","author":"Morris","year":"2009","journal-title":"J. Comput. Chem."},{"issue":"2","key":"2026100608064065100_cit29","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: Improving\nthe Speed and Accuracy of\nDocking with a New Scoring Function, Efficient Optimization, and Multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."},{"issue":"3","key":"2026100608064065100_cit30","doi-asserted-by":"publisher","first-page":"774","DOI":"10.1016\/j.jmb.2007.05.022","article-title":"Inference of Macromolecular\nAssemblies from Crystalline\nState","volume":"372","author":"Krissinel","year":"2007","journal-title":"J. Mol. Biol."},{"issue":"2","key":"2026100608064065100_cit31","doi-asserted-by":"publisher","first-page":"529","DOI":"10.1110\/ps.03393904","article-title":"Crystal Structure of a Tetrameric\nGDP-d-Mannose 4,6-Dehydratase\nfrom a Bacterial GDP-d-Rhamnose Biosynthetic Pathway","volume":"13","author":"Webb","year":"2004","journal-title":"Protein Sci."},{"issue":"34","key":"2026100608064065100_cit32","doi-asserted-by":"publisher","first-page":"24489","DOI":"10.1074\/jbc.M602393200","article-title":"Structural Studies\nof FlaA1 from Helicobacter pylori Reveal\nthe Mechanism for Inverting 4,6-Dehydratase Activity*","volume":"281","author":"Ishiyama","year":"2006","journal-title":"J. Biol. Chem."},{"issue":"13","key":"2026100608064065100_cit33","doi-asserted-by":"publisher","first-page":"1314","DOI":"10.1021\/acs.biochem.0c00096","article-title":"Structural Analysis\nof Cj1427, an Essential NAD-Dependent\nDehydrogenase for the Biosynthesis of the Heptose Residues in the\nCapsular Polysaccharides of Campylobacter Jejuni","volume":"59","author":"Huddleston","year":"2020","journal-title":"Biochemistry"},{"issue":"1","key":"2026100608064065100_cit34","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1016\/j.bbrc.2019.10.035","article-title":"Structure of an Antibiotic-Synthesizing\nUDP-Glucuronate 4-Epimerase MoeE5 in Complex with Substrate","volume":"521","author":"Sun","year":"2020","journal-title":"Biochem. Biophys. Res. Commun."}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.biochem.6c00553","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/65\/19\/3106\/68631211\/acs.biochem.6c00553.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/65\/19\/3106\/68631211\/acs.biochem.6c00553.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T12:49:34Z","timestamp":1791290974000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/65\/19\/3106\/5437229\/Structural-and-Mechanistic-Insights-into-4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,23]]},"references-count":34,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,9,23]]},"published-print":{"date-parts":[[2026,10,6]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.biochem.6c00553","relation":{},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,23]]}}}