title: Characterization of a key aminoglycoside phosphotransferase in gentamicin biosynthesis.
abstract: Gentamicin is an aminoglycoside antibiotic obtained from cultures of Micromonospora as the important anti-infective agents. Gentamicin which lacks 3'-hydroxyl group can avoid the attack from the modification enzymes of antibiotic-resistant bacteria in clinic. Consequently, C-3' dehydroxylation is the key step in gentamicins biosynthesis. We suppose that there are some enzymes responsible for converting intermediate JI-20A to 3',4'-bisdehydroxylated final product gentamicin C(1a), while phosphorylation of 3'-OH is possibly the first step for C-3' dehydroxylation. The gentamicin biosynthetic gene gntI, encoding an aminoglycoside phosphotransferase, was cloned from Micromonospora echinospora ATCC15835 and overexpressed in Escherichia coli. The resulting phosphotransferase was purified, and the kinetic parameters for Kanamycin A, Kanamycin B, Neomycin B and Amikacin were determined. Elucidation of NMR data of phosphorylated kanamycin B has unambiguously demonstrated a regiospecific phosphorylation of 3'-hydroxyl of the 6-aminohexose ring. The results described here partly confirm that the 3'-dehydroxylation step is preceded by a 3' phosphorylation step. It is predicted that GntI belongs to a new aminoglycoside phosphotransferase group involved with aminoglycoside antibiotics biosynthesis pathway.
authors: State Key Lab of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 1320 West Beijing Rd., Shanghai 200040, China.
authors: Lei
authors: L
authors: Shao
authors: Junsheng
authors: J
authors: Chen
authors: Chunxia
authors: C
authors: Wang
authors: Ji-an
authors: JA
authors: Li
authors: Yumin
authors: Y
authors: Tang
authors: Daijie
authors: D
authors: Chen
authors: Wen
authors: W
authors: Liu
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D005839:Gentamicins / Q000096:biosynthesis* / Q000378:metabolism; D007612:Kanamycin / Q000096:biosynthesis / Q000378:metabolism; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism*; D008847:Micromonospora / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016415:Sequence Alignment
chemical_list: D000900:Anti-Bacterial Agents; D005839:Gentamicins; D007612:Kanamycin; D019868:Kanamycin Kinase