These enzymes all perform the cobalamin-dependent N-methyltransferase that transforms gentamicin A into gentamicin X2.
Kim JY, Suh JW, Kang SH, Phan TH, Park SH, Kwon HJ
Gene inactivation study of gntE reveals its role in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis
▸ Abstract
A gene inactivation study was performed on gntE, a member of the gentamicin biosynthetic gene cluster in Micromonospora echinospora. Computer-aided homology analysis predicts a methyltransferase-related cobalamin-binding domain and a radical S-adenosylmethionine domain in GntE. It is also found that there is no gntE homolog within other aminoglycoside biosynthetic gene clusters. Inactivation of gntE was achieved in both M. echinospora ATCC 15835 and a gentamicin high-producer GMC106. High-performance liquid chromatographic analysis, coupled with mass spectrometry, revealed that gntE mutants accumulated gentamicin A2 and its derivative with a methyl group installed on the glucoamine moiety. This result substantiated that GntE participates in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis, though the catalytic nature of this unusual oxidoreductase/methyltransferase candidate is not resolved. The present gene inactivation study also demonstrates that targeted genetic engineering can be applied to produce specific gentamicin structures and potentially new gentamicin derivatives in M. echinospora.
Biochem Biophys Res Commun
2008;372(4):730-734
| PubMed ID:
18533111
Huang C, Huang F, Moison E, Guo J, Jian X, Duan X, Deng Z, Leadlay PF, Sun Y
Delineating the biosynthesis of gentamicin x2, the common precursor of the gentamicin C antibiotic complex
▸ Abstract
Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.
Previous studies have suggested that these enzymes performed the the conversion of an OH group into an NHMe group. However, more recent studies that took into account the analysis of gentamicin-related metabolites accumulated in strains bearing single and multiple specific gene deletions, analysis following the bioconversion of specific intermediates fed to such mutants, and reconstituting all of the steps using purified recombinant enzymes, it was demonstrated that the respective (and essential) catalytic contributions of the dehydrogenase GenD2, the pyridoxal phosphate-dependent aminotransferase GenS2, the SAM-dependent N-methyltransferase GenN, and the radical SAM-dependent C-methyltransferase GenD1 are all required for this transformation.
