{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T13:45:13Z","timestamp":1792590313757,"version":"4.5.0"},"reference-count":41,"publisher":"American Chemical Society (ACS)","issue":"41","license":[{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32572499"],"award-info":[{"award-number":["32572499"]}],"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":["82274040"],"award-info":[{"award-number":["82274040"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004608","name":"Natural Science Foundation of Jiangsu Province","doi-asserted-by":"publisher","award":["BK20240849"],"award-info":[{"award-number":["BK20240849"]}],"id":[{"id":"10.13039\/501100004608","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,21]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>L-Xylulose, a potent \u03b1-glucosidase inhibitor and indispensable chiral precursor for L-nucleoside drugs, is highly valuable. However, chemical synthesis often suffers from poor atom economy and tedious protection and deprotection steps of functional groups. Here, a green biocatalytic route was explored by retrieving five XDH4 genes and selecting AYXDH4 as the best candidate. Through homology modeling and site-directed mutagenesis targeting potential synergy sites in the \u03b1-helix, the double mutant T171A\/S172A was generated, which exhibited 43.97% higher catalytic efficiency (kcat\/Km) than the wild type, alongside an increase in optimal temperature from 35 \u00b0C to 40 \u00b0C. Structural analysis revealed that flexibility modulation of an adjacent \u03b1-helix triggers hydrogen bond rearrangements, thereby enhancing substrate binding and hydride transfer. Coupling with NADH oxidase regenerates NAD+ in situ, eliminating cofactor addition and producing only water. The system produced 6.61 \u00b1 0.52 g\/L L-xylulose, 1.9-fold higher than the wild type, demonstrating a green, atom-economic platform for L-xylulose production.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c11391","type":"journal-article","created":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T19:01:41Z","timestamp":1791313301000},"page":"32562-32574","source":"Crossref","is-referenced-by-count":0,"title":["Structure-Guided\nEngineering of Xylitol 4-dehydrogenase\nUnlocks Efficient L-Xylulose Biosynthesis: Mechanistic Insights\ninto Catalytic Enhancement"],"prefix":"10.1021","volume":"74","author":[{"given":"Xiaolan","family":"Li","sequence":"first","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziye","family":"Wang","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia le","family":"Long","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhongyi","family":"Hua","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7875-1283","authenticated-orcid":true,"given":"Zhi-Hong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0246-9809","authenticated-orcid":true,"given":"Zhen","family":"Ouyang","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212013"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5817-1609","authenticated-orcid":true,"given":"Xianghui","family":"Qi","sequence":"additional","affiliation":[{"name":"Jiangsu University , , , ,","place":["Zhenjiang, Jiangsu, China, 212013"]},{"name":"Guangzhou University , , ,","place":["Guangzhou, China, 510006"]},{"name":"Guangzhou University , , ,","place":["Guangzhou, China, 510006"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,6]]},"reference":[{"key":"2026102109084027900_cit1","doi-asserted-by":"publisher","first-page":"119125","DOI":"10.1016\/j.foodres.2026.119125","article-title":"L-xylulose, a new versatile\nrare sugar: recent biosynthetic advances and challenges","volume":"235","author":"Li","year":"2026","journal-title":"Food Res. Int."},{"issue":"2","key":"2026102109084027900_cit2","doi-asserted-by":"publisher","first-page":"535","DOI":"10.1007\/s00253-015-7087-y","article-title":"Advances in\napplications, metabolism, and biotechnological production of L-xylulose","volume":"100","author":"Meng","year":"2016","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"2026102109084027900_cit3","doi-asserted-by":"publisher","first-page":"116058","DOI":"10.1016\/j.foodres.2025.116058","article-title":"Green biotechnological\nsynthesis of rare sugars\/alcohols: D-allulose,\nallitol, D-tagatose, L-xylulose, L-ribose","volume":"206","author":"Wen","year":"2025","journal-title":"Food\nRes. Int."},{"issue":"6","key":"2026102109084027900_cit4","doi-asserted-by":"publisher","first-page":"1021","DOI":"10.1007\/s00449-020-02505-3","article-title":"Construction of recombinant Escherichia coli expressing xylitol-4-dehydrogenase\nand optimization for enhanced\nL-xylulose biotransformation from xylitol","volume":"44","author":"Tesfay","year":"2021","journal-title":"Bioprocess\nBiosyst. Eng."},{"key":"2026102109084027900_cit5","doi-asserted-by":"publisher","first-page":"92","DOI":"10.1016\/j.procbio.2021.03.007","article-title":"Characterization\nof\nxylitol 4-dehydrogenase from Erwinia aphidicola and\nits co-expression with NADH oxidase in Bacillus subtilis","volume":"104","author":"Li","year":"2021","journal-title":"Process Biochem."},{"issue":"5","key":"2026102109084027900_cit6","doi-asserted-by":"publisher","first-page":"1206","DOI":"10.1016\/j.enzmictec.2006.09.009","article-title":"Production of L-xylulose from xylitol\nby a newly isolated\nstrain of Bacillus pallidus Y25 and characterization\nof its relevant enzyme xylitol dehydrogenase","volume":"40","author":"Poonperm","year":"2007","journal-title":"Enzyme Microb. Technol."},{"key":"2026102109084027900_cit7","doi-asserted-by":"publisher","first-page":"134528","DOI":"10.1016\/j.biortech.2026.134528","article-title":"Computational-guided\nsemi-rational design to reprogram phospholipase D into industrially\nrobust enzyme","volume":"451","author":"Kong","year":"2026","journal-title":"Bioresour. Technol."},{"key":"2026102109084027900_cit8","doi-asserted-by":"publisher","first-page":"543","DOI":"10.1016\/S0009-2797(02)00217-X","article-title":"Identification of amino\nacid residues involved in substrate recognition\nof L-xylulose reductase by site-directed mutagenesis","volume":"143-144","author":"Ishikura","year":"2003","journal-title":"Chem. Biol. Interact."},{"key":"2026102109084027900_cit9","doi-asserted-by":"publisher","first-page":"118552","DOI":"10.1016\/j.foodres.2026.118552","article-title":"Computational design\nof a highly active and stable\nalkaline pectate lyase: Unraveling the molecular mechanisms for superior\nperformance","volume":"230","author":"Zhao","year":"2026","journal-title":"Food Res. Int."},{"issue":"41","key":"2026102109084027900_cit10","doi-asserted-by":"publisher","first-page":"26303","DOI":"10.1021\/acs.jafc.5c08213","article-title":"Surface charge engineering unlocks dual enhancement of thermostability\nand catalytic efficiency in a bacterial \u03b2-Glucosidase for sustainable\nresveratrol production","volume":"73","author":"Qi","year":"2025","journal-title":"J. Agric. Food Chem."},{"key":"2026102109084027900_cit11","doi-asserted-by":"publisher","first-page":"106","DOI":"10.1016\/j.enzmictec.2017.07.010","article-title":"Coupling\nxylitol dehydrogenase with NADH oxidase improves\nL-xylulose production in Escherichia coli culture","volume":"106","author":"Han","year":"2017","journal-title":"Enzyme Microb. Technol."},{"key":"2026102109084027900_cit12","doi-asserted-by":"publisher","first-page":"103593","DOI":"10.1016\/j.ifset.2024.103593","article-title":"Recent research\nand prospects of non-thermal physical technologies in green and high-efficient\nextraction of natural pigments: A review","volume":"92","author":"Zhang","year":"2024","journal-title":"Innovative\nFood Sci. Emerging Technol."},{"key":"2026102109084027900_cit13","doi-asserted-by":"publisher","first-page":"108137","DOI":"10.1016\/j.bej.2021.108137","article-title":"Efficient L-xylulose production using whole-cell biocatalyst with\nNAD+ regeneration system through co-expression of xylitol\ndehydrogenase and NADH oxidase in Escherichia coli","volume":"175","author":"Tesfay","year":"2021","journal-title":"Biochem. Eng. J."},{"issue":"1","key":"2026102109084027900_cit14","doi-asserted-by":"publisher","first-page":"7031","DOI":"10.1038\/s41467-022-34850-0","article-title":"Identification\nof purine\nbiosynthesis as an NADH-sensing pathway to mediate energy stress","volume":"13","author":"Yang","year":"2022","journal-title":"Nat. Commun."},{"issue":"5","key":"2026102109084027900_cit15","doi-asserted-by":"publisher","first-page":"590","DOI":"10.1080\/10826068.2021.1975299","article-title":"Cloning, expression,\nand characterization of an arabitol\ndehydrogenase and coupled with NADH oxidase for effective production\nof L-xylulose","volume":"52","author":"Zhu","year":"2022","journal-title":"Prep. Biochem. Biotechnol."},{"key":"2026102109084027900_cit16","doi-asserted-by":"publisher","first-page":"128251","DOI":"10.1016\/j.biortech.2022.128251","article-title":"High-level production\nof D-arabitol by Zygosaccharomyces rouxii from glucose:\nMetabolic engineering and process optimization","volume":"367","author":"Li","year":"2023","journal-title":"Bioresour. Technol."},{"key":"2026102109084027900_cit17","doi-asserted-by":"publisher","first-page":"130162","DOI":"10.1016\/j.biortech.2023.130162","article-title":"Sustainable\nbio-manufacturing of D-arabitol through combinatorial engineering\nof Zygosaccharomyces rouxii, bioprocess optimization\nand downstream separation","volume":"393","author":"Li","year":"2024","journal-title":"Bioresour. Technol."},{"issue":"21","key":"2026102109084027900_cit18","doi-asserted-by":"publisher","first-page":"12769","DOI":"10.3390\/ijms232112769","article-title":"A heat shock transcription\nfactor TrHSFB2a of White Clover negatively regulates\ndrought, heat and salt stress tolerance in transgenic arabidopsis","volume":"23","author":"Iqbal","year":"2022","journal-title":"Int. J. Mol. Sci."},{"issue":"12","key":"2026102109084027900_cit19","doi-asserted-by":"publisher","first-page":"10430","DOI":"10.1021\/acs.jafc.6c00416","article-title":"Engineering distant allosteric networks to remodel the substrate\naccess channel for efficient \u03b2-Arbutin biosynthesis","volume":"74","author":"Zhang","year":"2026","journal-title":"J. Agric. Food Chem."},{"key":"2026102109084027900_cit20","doi-asserted-by":"publisher","first-page":"112848","DOI":"10.1016\/j.measurement.2023.112848","article-title":"Digital\nelectronic based portable device for colorimetric quantification of\nketones and glucose level in human urine","volume":"214","author":"Mitra","year":"2023","journal-title":"Measurement"},{"issue":"1","key":"2026102109084027900_cit21","doi-asserted-by":"publisher","first-page":"4548","DOI":"10.1038\/s41467-019-12497-8","article-title":"Galactose to tagatose\nisomerization at moderate temperatures\nwith high conversion and productivity","volume":"10","author":"Bober","year":"2019","journal-title":"Nat.\nCommun."},{"key":"2026102109084027900_cit22","doi-asserted-by":"publisher","first-page":"313","DOI":"10.1016\/j.ymben.2026.04.005","article-title":"Enzyme engineering\nfor optimizing biosynthesis of 2,4-dihydroxybutyric\nacid via the synthetic threose-dependent glycolaldehyde assimilation\n(STEGA) pathway","volume":"96","author":"Wen","year":"2026","journal-title":"Metab. Eng."},{"issue":"20","key":"2026102109084027900_cit23","doi-asserted-by":"publisher","first-page":"17206","DOI":"10.1021\/acscatal.5c06238","article-title":"Reprogramming\nthe catalytic pocket of Baeyer\u2013Villiger monooxygenase for environmentally\ncompatible synthesis of a chiral sulfoxide pharmaceutical","volume":"15","author":"Zhao","year":"2025","journal-title":"ACS Catal."},{"issue":"5","key":"2026102109084027900_cit24","doi-asserted-by":"publisher","first-page":"777","DOI":"10.1006\/jmbi.1994.1334","article-title":"Satisfying hydrogen\nbonding potential in proteins","volume":"238","author":"McDonald","year":"1994","journal-title":"J. Mol. 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Commun."},{"issue":"30","key":"2026102109084027900_cit27","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.abo0696","article-title":"ATP-competitive inhibitors\nmodulate the substrate binding cooperativity of a kinase by altering\nits conformational entropy","volume":"8","author":"Olivieri","year":"2022","journal-title":"Sci. Adv."},{"issue":"11","key":"2026102109084027900_cit28","doi-asserted-by":"publisher","first-page":"203","DOI":"10.1016\/j.eng.2023.04.009","article-title":"Pocket modification of co-Amine transaminase\nAtATA for overcoming the trade-off between activity and stability\ntoward 1-Acetonaphthone","volume":"30","author":"Cao","year":"2023","journal-title":"Engineering"},{"key":"2026102109084027900_cit29","doi-asserted-by":"publisher","first-page":"103119","DOI":"10.1016\/j.fbio.2023.103119","article-title":"Improving the thermostability of glycosyltransferase UGT76G1 by computer-aided\ntarget analysis for highly efficient biosynthesis of rebaudioside\nM","volume":"56","author":"Yang","year":"2023","journal-title":"Food Biosci."},{"key":"2026102109084027900_cit30","doi-asserted-by":"publisher","first-page":"8122","DOI":"10.1021\/acscatal.5c09239","article-title":"Computational study on key factors controlling the regioselectivity\nof the CYP11A1-Catalyzed sequential hydroxylations of cholesterol","volume":"16","author":"Yue","year":"2026","journal-title":"ACS Catal."},{"issue":"12","key":"2026102109084027900_cit31","doi-asserted-by":"publisher","first-page":"1020","DOI":"10.1038\/s41929-020-00539-0","article-title":"A strategic\nSer\/Cys\nexchange in the catalytic triad unlocks an acyltransferase-mediated\nsynthesis of thioesters and tertiary amides","volume":"3","author":"Contente","year":"2020","journal-title":"Nat. Catal."},{"issue":"1","key":"2026102109084027900_cit32","doi-asserted-by":"publisher","first-page":"8508","DOI":"10.1038\/s41467-023-44333-5","article-title":"Pervasive epistasis\nexposes intramolecular\nnetworks in adaptive enzyme evolution","volume":"14","author":"Buda","year":"2023","journal-title":"Nat.\nCommun."},{"issue":"12","key":"2026102109084027900_cit33","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2313513121","article-title":"Network of\nepistatic\ninteractions in an enzyme active site revealed by large-scale deep\nmutational scanning","volume":"121","author":"Judge","year":"2024","journal-title":"Proc. Natl. Acad. Sci.\nU. S. A."},{"issue":"1","key":"2026102109084027900_cit34","doi-asserted-by":"publisher","first-page":"1058","DOI":"10.1038\/s41598-017-01235-z","article-title":"Distant Phe345\nmutation compromises the stability and activity of Mycobacterium tuberculosis isocitrate lyase by modulating its structural\nflexibility","volume":"7","author":"Shukla","year":"2017","journal-title":"Sci. Rep."},{"issue":"24","key":"2026102109084027900_cit35","doi-asserted-by":"publisher","first-page":"5243","DOI":"10.1002\/cctc.202101558","article-title":"Iterative\nalanine scanning mutagenesis confers aromatic ketone specificity and\nactivity of L-Amine dehydrogenases","volume":"13","author":"Mu","year":"2021","journal-title":"ChemCatChem"},{"key":"2026102109084027900_cit36","doi-asserted-by":"publisher","first-page":"148465","DOI":"10.1016\/j.ijbiomac.2025.148465","article-title":"Multi-strategy computational screening\nand single-point saturation mutation optimize catalytic efficiency\nand thermal stability of glucose oxidase","volume":"331","author":"Miao","year":"2025","journal-title":"Int.\nJ. Biol. Macromol."},{"issue":"3","key":"2026102109084027900_cit37","doi-asserted-by":"publisher","first-page":"211","DOI":"10.1016\/j.ijbiomac.2006.02.017","article-title":"Alkaline unfolding and salt-induced\nfolding of arginine kinase from Shrimp feneropenaeus chinensis under high pH conditions","volume":"38","author":"Liu","year":"2006","journal-title":"Int. J.\nBiol. Macromol."},{"issue":"1","key":"2026102109084027900_cit38","doi-asserted-by":"publisher","first-page":"368","DOI":"10.1128\/AEM.72.1.368-377.2006","article-title":"Cloning and\nexpression of a xylitol-4-dehydrogenase gene from Pantoea ananatis","volume":"72","author":"Aarnikunnas","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"issue":"8","key":"2026102109084027900_cit39","doi-asserted-by":"publisher","first-page":"707","DOI":"10.1016\/j.jmgm.2010.01.004","article-title":"Molecular modeling studies of L-arabinitol 4-dehydrogenase\nof Hypocrea jecorina: its binding interactions with\nsubstrate and cofactor","volume":"28","author":"Tiwari","year":"2010","journal-title":"J. Mol. Graphics Modell."},{"issue":"1","key":"2026102109084027900_cit40","doi-asserted-by":"publisher","first-page":"173","DOI":"10.1016\/j.bmcl.2013.11.047","article-title":"pH-rate profiles of L-arabinitol\n4-dehydrogenase from Hypocrea jecorina and its application\nin L-xylulose production","volume":"24","author":"Tiwari","year":"2014","journal-title":"Bioorg. Med. Chem.\nLett."},{"issue":"8","key":"2026102109084027900_cit41","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.01150-26","article-title":"Pathway remodeling\nand adaptive evolution\nenable efficient co-utilization of glucose and xylose in Escherichia coli","volume":"17","author":"Tan","year":"2026","journal-title":"mBio"}],"container-title":["Journal of Agricultural\nand Food Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jafc.6c11391","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/41\/32562\/68763851\/acs.jafc.6c11391.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/41\/32562\/68763851\/acs.jafc.6c11391.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T13:14:54Z","timestamp":1792588494000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jafcau\/article\/74\/41\/32562\/5445632\/Structure-Guided-Engineering-of-Xylitol-4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,6]]},"references-count":41,"journal-issue":{"issue":"41","published-online":{"date-parts":[[2026,10,6]]},"published-print":{"date-parts":[[2026,10,21]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jafc.6c11391","relation":{},"ISSN":["0021-8561","1520-5118"],"issn-type":[{"value":"0021-8561","type":"print"},{"value":"1520-5118","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,6]]}}}