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However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD\u2010linked processes. A potential solution is to engineer FalDH to utilize non\u2010natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure\u2010guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150\u2010fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller\u2010sized cofactor. FalDH 9B2 together with other NCD\u2010linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.<\/jats:p>","DOI":"10.1002\/cbic.202100697","type":"journal-article","created":{"date-parts":[[2022,2,11]],"date-time":"2022-02-11T01:09:10Z","timestamp":1644541750000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Engineering Formaldehyde Dehydrogenase from <i>Pseudomonas putida<\/i> to Favor Nicotinamide Cytosine Dinucleotide"],"prefix":"10.1002","volume":"23","author":[{"given":"Junting","family":"Wang","sequence":"first","affiliation":[{"name":"Division of Biotechnology Dalian Institute of Chemical Physics Chinese Academy of Sciences  457 Zhongshan Road Dalian 116023 China"},{"name":"University of Chinese Academy of Sciences  19 Yuquan Road Shijingshan District, Beijing 100049 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaojia","family":"Guo","sequence":"additional","affiliation":[{"name":"Division of Biotechnology Dalian Institute of Chemical Physics Chinese Academy of Sciences  457 Zhongshan Road Dalian 116023 China"},{"name":"Dalian Key Laboratory of Energy Biotechnology Dalian Institute of Chemical Physics, CAS  Dalian 116023 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Wan","sequence":"additional","affiliation":[{"name":"Division of Biotechnology Dalian Institute of Chemical Physics Chinese Academy of Sciences  457 Zhongshan Road Dalian 116023 China"},{"name":"University of Chinese Academy of Sciences  19 Yuquan Road Shijingshan District, Beijing 100049 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuxue","family":"Liu","sequence":"additional","affiliation":[{"name":"Division of Biotechnology Dalian Institute of Chemical Physics Chinese Academy of Sciences  457 Zhongshan Road Dalian 116023 China"},{"name":"Present address: College of Life Sciences Henan Normal University  46 East Construction Road Xinxiang 453007 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haizhao","family":"Xue","sequence":"additional","affiliation":[{"name":"Division of Biotechnology Dalian Institute of Chemical Physics Chinese Academy of Sciences  457 Zhongshan Road Dalian 116023 China"},{"name":"University of Chinese Academy of Sciences  19 Yuquan Road Shijingshan District, Beijing 100049 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0654-1193","authenticated-orcid":false,"given":"Zongbao K.","family":"Zhao","sequence":"additional","affiliation":[{"name":"Division of Biotechnology Dalian Institute of Chemical Physics Chinese Academy of Sciences  457 Zhongshan Road Dalian 116023 China"},{"name":"Dalian Key Laboratory of Energy Biotechnology Dalian Institute of Chemical Physics, CAS  Dalian 116023 China"},{"name":"State Key Laboratory of Catalysis Dalian Institute of Chemical Physics, CAS  Dalian 116023 China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2022,2,19]]},"reference":[{"key":"e_1_2_8_1_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.redox.2020.101850"},{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/0041-008X(86)90201-2"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2015.01.007"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2019.10.002"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1128\/jb.178.5.1386-1393.1996"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.pep.2013.09.017"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1128\/jb.176.9.2483-2491.1994"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-06496-4"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2009.08.006"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.0c04862"},{"key":"e_1_2_8_11_1","unstructured":"\u00a0"},{"key":"e_1_2_8_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2012.10.010"},{"key":"e_1_2_8_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2020.101471"},{"key":"e_1_2_8_14_1","unstructured":"\u00a0"},{"key":"e_1_2_8_15_2","doi-asserted-by":"publisher","DOI":"10.1021\/ja2074032"},{"key":"e_1_2_8_16_2","first-page":"530","volume":"33","author":"Ji D.","year":"2012","journal-title":"Chin. 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