title: Function of cytochrome P450 enzymes MycCI and MycG in Micromonospora griseorubida, a producer of the macrolide antibiotic mycinamicin.
abstract: The cytochrome P450 enzymes MycCI and MycG are encoded within the mycinamicin biosynthetic gene cluster and are involved in the biosynthesis of mycinamicin II (a 16-membered macrolide antibiotic produced by Micromonospora griseorubida). Based on recent enzymatic studies, MycCI is characterized as the C-21 methyl hydroxylase of mycinamicin VIII, while MycG is designated multifunctional P450, which catalyzes hydroxylation and also epoxidation at C-14 and C-12/13 on the macrolactone ring of mycinamicin. Here, we confirm the functions of MycCI and MycG in M. griseorubida. Protomycinolide IV and mycinamicin VIII accumulated in the culture broth of the mycCI disruption mutant; moreover, the mycCI gene fragment complemented the production of mycinamicin I and mycinamicin II, which are produced as major mycinamicins by the wild strain M. griseorubida A11725. The mycG disruption mutant did not produce mycinamicin I and mycinamicin II; however, mycinamicin IV accumulated in the culture broth. The mycG gene was located immediately downstream of the self-resistance gene myrB. The mycG gene under the control of mycGp complemented the production of mycinamicin I and mycinamicin II. Furthermore, the amount of mycinamicin II produced by the strain complemented with the mycG gene under the control of myrBp was approximately 2-fold higher than that produced by the wild strain. In M. griseorubida, MycG recognized mycinamicin IV, mycinamicin V, and also mycinamicin III as the substrates. Moreover, it catalyzed hydroxylation and also epoxidation at C-14 and C-12/13 on these intermediates. However, C-14 on mycinamicin I was not hydroxylated.
authors.affiliation: Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba, Japan. yanzai@phar.toho-u.ac.jp
authors.forename: Yojiro
authors.initials: Y
authors.lastname: Anzai
authors.forename: Shu-ichi
authors.initials: S
authors.lastname: Tsukada
authors.forename: Ayami
authors.initials: A
authors.lastname: Sakai
authors.forename: Ryohei
authors.initials: R
authors.lastname: Masuda
authors.forename: Chie
authors.initials: C
authors.lastname: Harada
authors.forename: Ayaka
authors.initials: A
authors.lastname: Domeki
authors.forename: Shengying
authors.initials: S
authors.lastname: Li
authors.forename: Kenji
authors.initials: K
authors.lastname: Kinoshita
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
authors.forename: Fumio
authors.initials: F
authors.lastname: Kato
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008847:Micromonospora / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D018942:Macrolides; C026686:mycinamicins; D003577:Cytochrome P-450 Enzyme System