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The recent characterization of the tiacumicin B biosynthetic gene cluster from <jats:italic>Dactylosporangium aurantiacum<\/jats:italic> subsp. <jats:italic>hamdenensis<\/jats:italic> NRRL 18085 revealed the functions of two glycosyltransferases, a C\u2010methyltransferase, an acyltransferase, two cytochrome P450s, and a tailoring dihalogenase in tiacumicin biosynthesis. Here we report the genetic confirmation and biochemical characterization of TiaS5 as a sugar\u2010O\u2010methyltransferase, requisite for tiacumicin B biosynthesis. The <jats:italic>tiaS5<\/jats:italic>\u2010inactivation mutant is capable of producing 14 tiacumicin analogues (11 of which are new), all lacking the 2\u2032\u2010O\u2010methyl group on the internal rhamnose moiety. Notably, two tiacumicin analogues exhibit improved antibacterial properties. We have also biochemically verified TiaS5 as an <jats:italic>S<\/jats:italic>\u2010adenosyl\u2010<jats:sc>L<\/jats:sc>\u2010methionine\u2010dependent O\u2010methyltransferase, requiring divalent metal ions for activity. Substrate probing revealed TiaS5 to be a promiscuous enzyme, recognizing 12 tiacumicin analogues. These findings unequivocally establish that TiaS5 functions as a 2\u2032\u2010O\u2010methyltransferase and provide direct biochemical evidence that TiaS5\u2010catalyzed methylation is a tailoring step after glycosyl coupling in tiacumicin B biosynthesis.<\/jats:p>","DOI":"10.1002\/cbic.201100129","type":"journal-article","created":{"date-parts":[[2011,6,1]],"date-time":"2011-06-01T09:00:08Z","timestamp":1306918808000},"page":"1740-1748","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":27,"title":["Characterization of a Sugar\u2010O\u2010methyltransferase TiaS5 Affords New Tiacumicin Analogues with Improved Antibacterial Properties and Reveals Substrate Promiscuity"],"prefix":"10.1002","volume":"12","author":[{"given":"Siwen","family":"Niu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sumei","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liang","family":"Ma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangtao","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haibo","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaohong","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianhua","family":"Ju","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changsheng","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2011,6]]},"reference":[{"key":"e_1_2_6_1_2","doi-asserted-by":"publisher","DOI":"10.1038\/emboj.2010.135"},{"key":"e_1_2_6_2_2","doi-asserted-by":"publisher","DOI":"10.1345\/aph.1M351"},{"key":"e_1_2_6_3_2","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.042010-0"},{"key":"e_1_2_6_4_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00090-10"},{"key":"e_1_2_6_5_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkn269"},{"key":"e_1_2_6_6_2","unstructured":"M. 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