N-formimidoyl fortimicin A synthase, a unique oxidase involved in fortimicin A biosynthesis: purification, characterization and gene cloning.
Micromonospora olivasterospora, a fortimicin A (FTM A, astromicin) producer, was found to carry an enzyme that converts FTM A to N-formimidoyl FTM A (FI-FTM A). This enzyme (FI-FTMase) was purified to homogeneity and shown to be a flavin adenine dinucleotide (FAD) enzyme. Tracer experiments proved that the formimidoyl group was derived from C-2 of glycine via oxidation of the amino acid in the presence of FTM A and oxygen. The gene encoding this enzyme, fms 14, was cloned using a 26-mer oligonucleotide probe, designed according to the N-terminal amino acid sequence of purified FI-FTMase, from a cosmid clone pGLM990, which has been shown to contain a cluster of FTM A biosynthetic genes. The nucleotide sequence, and biochemical and genetic analysis revealed that FI-FTMase is composed of four identical subunits of mol. wt. 52,000, and contains at least one FAD per subunit. DNA regions homologous to fms14 were found in two other producers of the fortimicin group of antibiotics, Dactylosporangium matsuzakiense ATCC31570 and Micromonospora sp. SF-2098.
Dairi
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Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
Yamaguchi
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Hasegawa
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D000192:Actinomycetales / Q000235:genetics; D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002474:Cell-Free System; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003360:Cosmids; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D008847:Micromonospora / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid; D013302:Streptomyces / Q000235:genetics; D013379:Substrate Specificity
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; C023384:fortimicin A; D000594:Amino Acid Oxidoreductases; C079507:N-formimidoyl fortimicin A synthase