SpnH from Saccharopolyspora spinosa encodes a rhamnosyl 4'-O-methyltransferase for biosynthesis of the insecticidal macrolide, spinosyn A.
Deoxysugar, 2', 3', 4'-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4'-O-methylation of 2', 3'-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4'-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4'-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4'-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.
Huang
Ke-xue
KX
Key Lab of Microbial Molecular Biology of Hunan Province, College of Life Science, Hunan Normal University, 410081, Changsha, China. huangkx@hunnu.edu.cn
Zahn
James
J
Han
Lei
L
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004338:Drug Combinations; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D018942:Macrolides / Q000378:metabolism*; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004338:Drug Combinations; D018942:Macrolides; C114883:spinosyn A; C408426:spinosyn K; D008780:Methyltransferases; C415329:spinosad