Mycinamicin II

Mycinamicin is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida A11725, which shows strong antimicrobial activity against gram-positive bacteria. Recently, the nucleotide sequences of the mycinamicn biosynthetic gene cluster in M. griseorubida have been completely determined. Mycinamicin non-producer M7A21 was isolated by mycAV inactivation, which encodes the module 7 of mycinamicin polyketide synthase (PKS) required for the biosynthesis of the mycinamicin biosynthetic intermediate protomycinolide-IV (PML-IV). When the bioconversion to mycinamicin II (M-II) from PML-IV was performed using M7A21 and the feeding culture method, the productivity of M-II was the same as that of M-II in wild-type strain A11725. p446M7 containing mycAV was constructed using the Escherichia coli-Streptomyces shuttle vector pGM446. The mycinamicin productivity of M7A21 was restored by the introduction of p446M7 into the M7A21 cell, but almost all p446M7 was integrated into the chromosome of M7A21 because the plasmid was unstable in M7A21.

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 majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites.

Mycinamicin, a 16-membered macrolide antibiotic produced by Micromonospora griseorubida, comprises a macrolactone and two deoxysugars: desosamine and mycinose. Mycinose is synthesized through two modification steps: the methylation of 6-deoxyallose in mycinamicin VI and of javose in mycinamicin III. To confirm the role of mycE and mycF genes in mycinamicin biosynthesis in M. griseorubida, disruption mutants of mycE and mycF were constructed by disruption plasmids containing attB in the disruption cassette FRT-neo-oriT-FRT-attB for the integration of phiC31-derivative vector plasmids; the disruption mutants were complemented through the integration of pSET152 derivatives containing intact mycE or mycF into the artificially inserted attB site. These disruption mutants did not produce mycinamicin II, but mainly accumulated mycinamicins VI and III, indicating that MycE and MycF methylated the C2''-OH group of 6-deoxyallose in mycinamicin VI and the C3''-OH group of C2''-methylated 6-deoxyallose in mycinamicin III, respectively.