Biotransformation of sisomicin and verdamicin by Micromonospora sagamiensis.

Abstract

The resting cells of a 2-deoxystreptamine idiotrophic mutant of Micromonosporasagamiensis were found to transform sisomicin into gentamicin Cla and sagamicin. The biotransformation products were isolated by a combination of ion exchange and carbon column chromatographic procedures, and properly identified. Antibiotic G-52 (G'-A^methylsisomicin) was not detected in the transformation products. Gentamicin Cla was formed at an early stage of the biotransformation, followed by sagamicin formation. The (4',50-reduction of sisomicin mayoccur first, followed by 6'-iV-methylation.By use of a similar procedure, it was demonstrated that verdamicin was transformed into gentamicin C2a (the 6'-C epimer ofgentamicin C2), C2, and then Q. Carbon TLC clearly separated gentamicin C2a, C2, and verdamicin. In this biotransformation, the (4/,5/)-reduction of verdamicin occurred first, followed by 6/-C-epimerization, and then 6/-7V-methylation. In contrast with M. sagamiensis, M. zionensis NRRL5466and M. inyoensis NRRL3292did not possess any detectable activity of the (4',5 ')-re(iiiction of sisomicin or verdamicin. Alternatively, NRRL5466transformed sisomicin into antibiotic G-52, and verdamicin into a new antibiotic, VF3-1. The antibiotic VF3-1 was suggested as 6/-7V-methylverdamicin by chromatographic and mass spectrum data. The implications of these findings were discussed in relation to sagamicin biosynthesis in M. sagamiensis.In a previous report,1} it was demonstrated that resting cells of a 2-deoxystreptamine (DOS) idiotrophic mutant of Micromonospora sagamiensis transformed gentamicin minor components into sagamicin and gentamicin C. During these studies, transformation of sisomicin-group antibiotics was examined. As shown in Fig. 1 Sagamicin biosynthesis was discussed on the basis of these findings. MATERIALS AND METHODSMicroorgan isms. Micromonospora sagam iens is KYI1525 (a DOS idiotroph derived from a sagamicin producer, KY1 1505),5) M. inyoensis NRRL3292, and M. zionensis NRRL5466were used.Media and culture condition. The compositions of the seed and fermentation media and culture condition were described in a previous paper.1)

Specificity and Promiscuity at the Branch Point in Gentamicin Biosynthesis

“…Remarkably, purified GenB2 (but not the others) proved competent in vitro to catalyze the 6′-epimerization of gentamicin C2a into gentamicin C2 (and vice versa). This evidence strongly supports the original proposal (Kase et al., 1982b) that C2a is an obligatory precursor to C2, which in turn implies that two inversions of the configuration at the C-6′ position occur during the transformation of G418 into gentamicin C2.…”

The bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3′,4′-di-deoxygenation via reduction and transamination activities

“…S3b), which was consistent with that of 6′-N-methylsisomicin (5, G-52) [10][11][12]. Hence, we hypothesized that in the other parallel pathway of gentamicin biosynthesis, the genB4 disrupting strain would accumulate as verdamicin and 6′-N-methylverdamicin (VF3-1) [13][14][15][16]. Interestingly, the exact mass of intermediate (2) S4b), both of which were consistent with that of VF3-1.…”

“…Then, compound (8) was treated with NaBH 4 or NaBD 4 to clarify the expected aldehyde formation [18]. Mass, 1 H NMR, and 13 C NMR spectroscopic analyses demonstrated that compound (8) was 6′-deamino-6′-oxogentamicin C1a (Fig. 4 and Additional file 9: Fig.…”

The Bifunctional Enzyme, GenB4, Catalyzes the Last Step of Gentamicin 3’,4’-Di-Deoxygenation via Reduction and Transamination Activities

“…S3b), which was consistent with that of 6'-N-methylsisomicin (5, G-52) [10,11,12]. Hence, we hypothesized that in the other parallel pathway of gentamicin biosynthesis, the genB4 disrupting strain would accumulate as verdamicin and 6'-N-methylverdamicin (VF3-1) [14][15][16][17]. Interestingly, the exact mass of intermediate (2) from sisomicin in vitro in 2008, at which time this synthesis was first mentioned and its chiral isomerization structure was named [13].…”