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RosC, a cytochrome P450 enzyme involved in the biosynthesis of the 16-membered ring macrolide antibiotic rosamicin, catalyzes stepwise oxidation of the ethyl group at C-20 via hydroxylation to an alcohol, followed by successive oxidation to the corresponding aldehyde and carboxylic acid. The P107S\/L176Q mutant produces the hydroxylated intermediate in the first oxidation step, but the efficiency of the subsequent conversion to aldehyde and carboxylic acid is significantly reduced. To elucidate the factors responsible for the reduced efficiency of the second and subsequent oxidation steps in the P107S\/L176Q mutant and to understand how RosC facilitates multistep oxidative modification, we compared the reaction time courses and substrate-binding affinities of RosC and the P107S\/L176Q mutant. The mutant exhibited a reduced reaction rate for the initial hydroxylation and showed reduced substrate-binding affinities for both the hydroxylated and aldehydic intermediates.<\/jats:p>\n                    <jats:p>Furthermore, crystallographic analysis revealed that Leu-176 played a key role in binding to the desosamine moiety of the substrate, and its mutation resulted in the loss of this function. Ser-248 was presumed to play a role in re-anchoring the modification site to the active center through hydrogen bonding with the hydroxyl and aldehyde groups generated in the first and second reactions. These findings are expected to contribute to the multifunctionalization of cytochrome P450 enzymes and the regulation of their reactivity.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Key points<\/jats:title>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>RosC-catalyzed three-step oxidation is limited to one step in the P107S\/L176Q mutant.<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>P107S\/L176Q mutations impair substrate binding by increasing structural fluctuations.<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>Leu-176 and Ser-248 position the substrate for RosC-catalyzed iterative oxidation.<\/jats:italic>\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Graphical Abstract<\/jats:title>\n                  <\/jats:sec>","DOI":"10.1007\/s00253-025-13648-2","type":"journal-article","created":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T18:57:23Z","timestamp":1765306643000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Enzyme engineering of cytochrome P450 RosC provides mechanistic insights into factors controlling iterative oxidation"],"prefix":"10.1007","volume":"109","author":[{"given":"Yohei","family":"Iizaka","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hironori","family":"Suzuki","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nanako","family":"Sasa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kihika","family":"Ishiuchi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuta","family":"Kumakiri","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haruki","family":"Kawasaki","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hayato","family":"Sato","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kanon","family":"Fujimoto","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuji","family":"Noguchi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yojiro","family":"Anzai","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,12,9]]},"reference":[{"key":"13648_CR1","doi-asserted-by":"publisher","first-page":"950","DOI":"10.1016\/j.chembiol.2008.07.014","volume":"15","author":"Y Anzai","year":"2008","unstructured":"Anzai Y, Li S, Chaulagain MR, Kinoshita K, Kato F, Montgomery J, Sherman DH (2008) Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics. 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