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The recent discovery of two PLP\u2010dependent enzymes, CndF and Fub7, has unveiled unique C\u2212C bond forming capabilities, enabling the biocatalytic synthesis of alkyl\u2010 substituted pipecolic acids from <jats:italic>O\u2010<\/jats:italic>acetyl\u2010L\u2010homoserine and \u03b2\u2010keto acid or aldehyde derived enolates. This breakthrough presents fresh avenues for the biosynthesis of pipecolic acid derivatives. However, the catalytic mechanisms of these enzymes remain elusive, and a dearth of structural information hampers their extensive application. Here, we have broadened the catalytic scope of Fub7 by employing ketone\u2010derived enolates as carbon nucleophiles, revealing Fub7\u2019s capacity for substrate\u2010dependent regioselective \u03b1\u2010alkylation of unsymmetrical ketones. Through an integrated approach combining X\u2010ray crystallography, spectroscopy, mutagenesis, and computational docking studies, we offer a detailed mechanistic insight into Fub7 catalysis. Our findings elucidate the structural basis for its substrate specificity, stereoselectivity, and regioselectivity. Our work sets the stage ready for subsequent protein engineering effort aimed at expanding the synthetic utility of Fub7, potentially unlocking novel methods to access a broader array of noncanonical amino acids.<\/jats:p>","DOI":"10.1002\/anie.202317161","type":"journal-article","created":{"date-parts":[[2024,2,3]],"date-time":"2024-02-03T06:25:00Z","timestamp":1706941500000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Molecular and Structural Basis for C\u03b3\u2212C Bond Formation by PLP\u2010Dependent Enzyme Fub7"],"prefix":"10.1002","volume":"63","author":[{"given":"Shaonan","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry University of California Santa Barbara  93110 Santa Barbara CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher","family":"Yeh","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry University of California Santa Barbara  93110 Santa Barbara CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chloe","family":"Reavill","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry University of California Santa Barbara  93110 Santa Barbara CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin","family":"Jones","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry University of California Santa Barbara  93110 Santa Barbara CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yike","family":"Zou","sequence":"additional","affiliation":[{"name":"Physical and Life Sciences Directorate Lawrence Livermore National Laboratory  94550 Livermore CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2039-5367","authenticated-orcid":false,"given":"Yang","family":"Hai","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry University of California Santa Barbara  93110 Santa Barbara CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,2,20]]},"reference":[{"key":"e_1_2_8_1_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.9b00408"},{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00726-020-02890-9"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/gzac013"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0079-6468(10)49001-3"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.abl4784"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.6b00319"},{"key":"e_1_2_8_7_1","volume-title":"Handbook on syntheses of amino acids: General routes for the syntheses of amino acids","author":"Blaskovich M. 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