{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T17:03:30Z","timestamp":1789751010541,"version":"4.0.1"},"reference-count":25,"publisher":"American Chemical Society (ACS)","issue":"37","license":[{"start":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T00:00:00Z","timestamp":1788307200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T00:00:00Z","timestamp":1788307200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T00:00:00Z","timestamp":1788307200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100017170","name":"Thailand Science Research and Innovation","doi-asserted-by":"publisher","award":["215520"],"award-info":[{"award-number":["215520"]}],"id":[{"id":"10.13039\/501100017170","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004352","name":"Suranaree University of Technology","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004352","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22477049"],"award-info":[{"award-number":["22477049"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22567020"],"award-info":[{"award-number":["22567020"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["U22A20451"],"award-info":[{"award-number":["U22A20451"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"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>Nucleoside analogues are important therapeutic scaffolds, but their synthesis is often protecting-group-intensive. Purine nucleoside phosphorylases offer a mild enzymatic alternative, yet broad substrate accommodation does not necessarily lead to productive glycosyl transfer. Here, we repurposed HpnG, a hopanoid-associated purine nucleoside phosphorylase, through second-shell engineering. Structure-guided substitution of D124 with glycine increased 2-fluoroadenosine formation from 11% to 91% yield and accelerated inosine phosphorolysis by more than 100-fold with little change in the apparent Km for inosine. HpnG-D124G also accepted diverse purine nucleobases and ribose, 2\u2032-deoxyribose, and arabinose donors, enabling access to drug-relevant nucleosides. Molecular dynamics simulations and electronic-structure calculations suggest that D124G enriches donor\u2013acceptor conformations that are properly aligned for glycosyl transfer. These findings establish productive active-site preorganization as a practical strategy for engineering broad-scope nucleoside biocatalysts.<\/jats:p>","DOI":"10.1021\/acs.orglett.6c03331","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T16:49:16Z","timestamp":1788454156000},"page":"11673-11677","source":"Crossref","is-referenced-by-count":0,"title":["A Second-Shell\nMutation Preorganizes HpnG for Broad-Scope\nNucleoside Transglycosylation"],"prefix":"10.1021","volume":"28","author":[{"given":"Xuxue","family":"Liu","sequence":"first","affiliation":[{"name":"Jiangxi Normal University , , No. 99 Ziyang Avenue , ,","place":["Nanchang, China, 330022"]},{"name":"Fudan University , , No. 2005 Songhu Road , , ,","place":["Shanghai, China, 200433"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deshuai","family":"Yang","sequence":"additional","affiliation":[{"name":"Jiangxi Normal University , , No. 99 Ziyang Avenue , ,","place":["Nanchang, China, 330022"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2076-0969","authenticated-orcid":true,"given":"Rung-Yi","family":"Lai","sequence":"additional","affiliation":[{"name":"Suranaree University of Technology , , ,","place":["Nakhon Ratchasima, Thailand, 30000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8135-2221","authenticated-orcid":true,"given":"Qi","family":"Zhang","sequence":"additional","affiliation":[{"name":"Jiangxi Normal University , , No. 99 Ziyang Avenue , ,","place":["Nanchang, China, 330022"]},{"name":"Fudan University , , No. 2005 Songhu Road , , ,","place":["Shanghai, China, 200433"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,2]]},"reference":[{"issue":"3","key":"2026091809411274500_cit1","doi-asserted-by":"publisher","DOI":"10.1002\/cpz1.376","article-title":"An Overview of Marketed Nucleoside\nand Nucleotide Analogs","volume":"2","author":"De Jonghe","year":"2022","journal-title":"Curr. Protoc."},{"key":"2026091809411274500_cit2","doi-asserted-by":"publisher","first-page":"66","DOI":"10.1016\/j.antiviral.2018.04.004","article-title":"The evolution of\nnucleoside analogue antivirals: A\nreview for chemists and non-chemists. Part 1: Early structural modifications\nto the nucleoside scaffold","volume":"154","author":"Seley-Radtke","year":"2018","journal-title":"Antiviral Res."},{"issue":"50","key":"2026091809411274500_cit3","doi-asserted-by":"publisher","first-page":"6522","DOI":"10.1038\/onc.2008.316","article-title":"Nucleoside\nanalogs: molecular mechanisms\nsignaling cell death","volume":"27","author":"Ewald","year":"2008","journal-title":"Oncogene"},{"issue":"23","key":"2026091809411274500_cit4","doi-asserted-by":"publisher","first-page":"14379","DOI":"10.1021\/acs.chemrev.6b00209","article-title":"Metabolism, Biochemical Actions, and Chemical Synthesis\nof Anticancer Nucleosides, Nucleotides, and Base Analogs","volume":"116","author":"Shelton","year":"2016","journal-title":"Chem. Rev."},{"issue":"26","key":"2026091809411274500_cit5","doi-asserted-by":"publisher","first-page":"11700","DOI":"10.1039\/D5SC03026A","article-title":"Preparation of nucleoside analogues:\nopportunities for innovation at the interface of synthetic chemistry\nand biocatalysis","volume":"16","author":"Salihovic","year":"2025","journal-title":"Chem. Sci."},{"issue":"11","key":"2026091809411274500_cit6","doi-asserted-by":"publisher","first-page":"1419","DOI":"10.1021\/op4002005","article-title":"A Stereoselective Process for the Manufacture\nof a 2\u2032-Deoxy-\u03b2-d-Ribonucleoside\nUsing the Vorbru\u0308ggen Glycosylation","volume":"17","author":"Henschke","year":"2013","journal-title":"Org.\nProcess Res. Dev."},{"issue":"9","key":"2026091809411274500_cit7","doi-asserted-by":"publisher","first-page":"2050","DOI":"10.1021\/acs.oprd.9b00304","article-title":"Follow the Silyl Cation: Insights\ninto the Vorbru\u0308ggen\nReaction","volume":"23","author":"Beutner","year":"2019","journal-title":"Org. Process Res. Dev."},{"key":"2026091809411274500_cit8","doi-asserted-by":"publisher","first-page":"102829","DOI":"10.1016\/j.copbio.2022.102829","article-title":"Industrial potential\nof the enzymatic synthesis of nucleoside analogs: existing challenges\nand perspectives","volume":"78","author":"Westarp","year":"2022","journal-title":"Curr. Opin. Biotechnol."},{"key":"2026091809411274500_cit9","doi-asserted-by":"publisher","first-page":"102873","DOI":"10.1016\/j.copbio.2022.102873","article-title":"Biocatalytic cascade\ntransformations for the synthesis\nof C-nucleosides and N-nucleoside analogs","volume":"79","author":"Pfeiffer","year":"2023","journal-title":"Curr.\nOpin. Biotechnol."},{"issue":"10","key":"2026091809411274500_cit10","doi-asserted-by":"publisher","first-page":"1428","DOI":"10.1002\/cbic.201900740","article-title":"General Principles for Yield Optimization of Nucleoside\nPhosphorylase-Catalyzed Transglycosylations","volume":"21","author":"Kaspar","year":"2020","journal-title":"ChemBioChem."},{"issue":"2","key":"2026091809411274500_cit11","doi-asserted-by":"publisher","first-page":"454","DOI":"10.1021\/jacsau.3c00581","article-title":"Engineering Substrate Promiscuity of Nucleoside Phosphorylase\nVia an Insertions\u2013Deletions Strategy","volume":"4","author":"Liu","year":"2024","journal-title":"JACS Au"},{"issue":"1","key":"2026091809411274500_cit12","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1042\/bj3610001","article-title":"Structural analyses reveal two distinct families of\nnucleoside phosphorylases","volume":"361","author":"Pugmire","year":"2002","journal-title":"Biochem. J."},{"issue":"6470","key":"2026091809411274500_cit13","doi-asserted-by":"publisher","first-page":"1255","DOI":"10.1126\/science.aay8484","article-title":"Design of an in vitro\nbiocatalytic cascade for the manufacture of islatravir","volume":"366","author":"Huffman","year":"2019","journal-title":"Science"},{"issue":"2","key":"2026091809411274500_cit14","doi-asserted-by":"publisher","first-page":"127","DOI":"10.1021\/ar960131y","article-title":"Ground\nState and Transition State Contributions to\nthe Rates of Intramolecular and Enzymatic Reactions","volume":"32","author":"Bruice","year":"1999","journal-title":"Acc. Chem. Res."},{"issue":"20","key":"2026091809411274500_cit15","doi-asserted-by":"publisher","first-page":"11333","DOI":"10.1021\/jacs.3c02423","article-title":"Second-Shell Residues\nContribute to Catalysis by Predominately Preorganizing\nthe Apo State in PafA","volume":"145","author":"Deng","year":"2023","journal-title":"J. Am. Chem. Soc."},{"issue":"10","key":"2026091809411274500_cit16","doi-asserted-by":"publisher","first-page":"4078","DOI":"10.1529\/biophysj.107.121913","article-title":"Remote Mutations\nand Active Site Dynamics Correlate with Catalytic Properties of Purine\nNucleoside Phosphorylase","volume":"94","author":"Saen-Oon","year":"2008","journal-title":"Biophys. J."},{"issue":"6","key":"2026091809411274500_cit17","doi-asserted-by":"publisher","first-page":"431","DOI":"10.1038\/nchembio.1503","article-title":"The role of distant mutations and allosteric regulation\non LovD active site dynamics","volume":"10","author":"Jime\u0301nez-Ose\u0301s","year":"2014","journal-title":"Nat. Chem. Biol."},{"issue":"1","key":"2026091809411274500_cit18","doi-asserted-by":"publisher","first-page":"3900","DOI":"10.1038\/s41467-018-06305-y","article-title":"The evolution of multiple\nactive site configurations in a designed enzyme","volume":"9","author":"Hong","year":"2018","journal-title":"Nat. Commun."},{"issue":"17","key":"2026091809411274500_cit19","doi-asserted-by":"publisher","first-page":"10830","DOI":"10.1021\/acscatal.1c02589","article-title":"Diversification of 4\u2032-Methylated\nNucleosides by Nucleoside Phosphorylases","volume":"11","author":"Kaspar","year":"2021","journal-title":"ACS\nCatal.s"},{"issue":"33","key":"2026091809411274500_cit20","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202405040","article-title":"Biocatalytic Nucleobase Diversification of 4\u2032-Thionucleosides\nand Application of Derived 5-Ethynyl-4\u2032-thiouridine for RNA\nSynthesis Detection","volume":"63","author":"Westarp","year":"2024","journal-title":"Angew. Chem., Int. Ed."},{"issue":"19","key":"2026091809411274500_cit21","doi-asserted-by":"publisher","first-page":"2351","DOI":"10.1002\/cjoc.202400233","article-title":"Characterization of HpnG as a Purine\nNucleoside Phosphorylase in Bacteriohopanepolyol Biosynthesis","volume":"42","author":"Li","year":"2024","journal-title":"Chin. J. Chem."},{"issue":"3","key":"2026091809411274500_cit22","doi-asserted-by":"publisher","first-page":"1652","DOI":"10.1021\/acs.jctc.8b01176","article-title":"GFN2-xTB\u2500An\nAccurate and Broadly\nParametrized Self-Consistent Tight-Binding Quantum Chemical Method\nwith Multipole Electrostatics and Density-Dependent Dispersion Contributions","volume":"15","author":"Bannwarth","year":"2019","journal-title":"J. Chem. Theory Comput."},{"issue":"7","key":"2026091809411274500_cit23","doi-asserted-by":"publisher","first-page":"4250","DOI":"10.1021\/acs.jctc.1c00471","article-title":"Robust and Efficient\nImplicit Solvation Model for Fast Semiempirical Methods","volume":"17","author":"Ehlert","year":"2021","journal-title":"J. Chem. Theory Comput."},{"issue":"6","key":"2026091809411274500_cit24","doi-asserted-by":"publisher","first-page":"064104","DOI":"10.1063\/1.5012601","article-title":"B97\u20133c: A revised\nlow-cost variant of the B97-D density functional method","volume":"148","author":"Brandenburg","year":"2018","journal-title":"J. Chem. Phys."},{"issue":"6","key":"2026091809411274500_cit25","doi-asserted-by":"publisher","first-page":"064103","DOI":"10.1063\/5.0040021","article-title":"r2SCAN-3c: A \u201cSwiss\narmy knife\u201d composite electronic-structure method","volume":"154","author":"Grimme","year":"2021","journal-title":"J. Chem. Phys."}],"container-title":["Organic Letters"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.orglett.6c03331","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/orlef7\/article-pdf\/28\/37\/11673\/68224707\/acs.orglett.6c03331.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/orlef7\/article-pdf\/28\/37\/11673\/68224707\/acs.orglett.6c03331.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T16:04:04Z","timestamp":1789747444000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/orlef7\/article\/28\/37\/11673\/5406932\/A-Second-Shell-Mutation-Preorganizes-HpnG-for"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,2]]},"references-count":25,"journal-issue":{"issue":"37","published-online":{"date-parts":[[2026,9,2]]},"published-print":{"date-parts":[[2026,9,18]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.orglett.6c03331","relation":{},"ISSN":["1523-7060","1523-7052"],"issn-type":[{"value":"1523-7060","type":"print"},{"value":"1523-7052","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,2]]}}}