Analysis of the loading and hydroxylation steps in lankamycin biosynthesis in Streptomyces rochei.
The biosynthetic gene cluster of lankamycin (LM), a 14-member macrolide antibiotic, is encoded on the 210-kb linear plasmid pSLA2-L in Streptomyces rochei 7434AN4. LM contains a 3-hydroxy-2-butyl group at the C-13 position, which is different from an ethyl group in erythromycin. The following two possibilities could be considered for the origin of this starter moiety of LM biosynthesis: (i) an extra module exists in the biosynthetic gene cluster and loads an additional acetate molecule, or (ii) 3-hydroxy-2-butyrate or its equivalent is loaded and incorporated as a starter. The former possibility was eliminated by the complete sequencing of pSLA2-L, which showed no extra module. On the other hand, the latter was confirmed by incorporation of deuterium in [3-(2)H]dl-isoleucine into the C-14 position of LM. The timing of hydroxylation reactions at the C-15 and C-8 positions of LM was studied by constructing disruptants of two P450 hydroxylase genes, lkmF (orf26) and lkmK (orf37). The lkmF disruptant produced 8-deoxylankamycin, while the lkmK disruptant produced both 15-deoxylankamycin and 8,15-dideoxylankamycin. These results clearly showed that LkmF is a C-8 hydroxylase and LkmK is a C-15 hydroxylase in LM biosynthesis and in addition suggested the order of hydroxylation steps; namely, hydroxylation may occur at first at C-15 by LkmK and then at C-8 by LkmF.
Arakawa
Kenji
K
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Japan.
Kodama
Kazuya
K
Tatsuno
Satoshi
S
Ide
Sayoko
S
Kinashi
Haruyasu
H
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D017665:Hydroxyl Radical / Q000737:chemistry; D006900:Hydroxylation; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D005810:Multigene Family / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D018942:Macrolides; D017665:Hydroxyl Radical; D004917:Erythromycin; C100149:kujimycin B; D006899:Mixed Function Oxygenases