55715-66-7Relevant academic research and scientific papers
Crystal Structure of GenD2, an NAD-Dependent Oxidoreductase Involved in the Biosynthesis of Gentamicin
De Araújo, Natalia Cerrone,Bury, Priscila Dos Santos,Tavares, Maurício Temotheo,Huang, Fanglu,Parise-Filho, Roberto,Leadlay, Peter,Dias, Marcio Vinicius Bertacine
, (2019)
Gentamicins are clinically relevant aminoglycoside antibiotics produced by several Micromonospora species. Gentamicins are highly methylated and functionalized molecules, and their biosynthesis include glycosyltransferases, dehydratase/oxidoreductases, aminotransferases, and methyltransferases. The biosynthesis of gentamicin A from gentamicin A2 involves three enzymatic steps that modify the hydroxyl group at position 3″ of the unusual garosamine sugar to provide its substitution for an amino group, followed by an N-methylation. The first of these reactions is catalyzed by GenD2, an oxidoreductase from the Gfo/Idh/MocA protein family, which reduces the hydroxyl at the C3″ of gentamicin A to produce 3′′-dehydro-3′′-oxo-gentamicin A2 (DOA2). In this work, we solved the structure of GenD2 in complex with NAD+. Although the structure of GenD2 has a similar fold to other members of the Gfo/Idh/MocA family, this enzyme has several new features, including a 3D-domain swapping of two β-strands that are involved in a novel oligomerization interface for this protein family. In addition, the active site of this enzyme also has several specialties which are possibly involved in the substrate specificity, including a number of aromatic residues and a negatively charged region, which is complementary to the polycationic aminoglycoside-substrate. Therefore, docking simulations provided insights into the recognition of gentamicin A2 and into the catalytic mechanism of GenD2. This is the first report describing the structure of an oxidoreductase involved in aminoglycoside biosynthesis and could open perspectives into producing new aminoglycoside derivatives by protein engineering.
Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical S-Adenosyl- l -methionine Methyltransferase in the Biosynthetic Pathway of Gentamicin, Proceeds with Retention of Configuration
Kim, Hak Joong,Liu, Yung-Nan,McCarty, Reid M.,Liu, Hung-Wen
supporting information, p. 16084 - 16087 (2017/11/22)
Many cobalamin (Cbl)-dependent radical S-adenosyl-l-methionine (SAM) methyltransferases have been identified through sequence alignment and/or genetic analysis; however, few have been studied in vitro. GenK is one such enzyme that catalyzes methylation of the 6′-carbon of gentamicin X2 (GenX2) to produce G418 during the biosynthesis of gentamicins. Reported herein, several alternative substrates and fluorinated substrate analogs were prepared to investigate the mechanism of methyl transfer from Cbl to the substrate as well as the substrate specificity of GenK. Experiments with deuterated substrates are also shown here to demonstrate that the 6′-pro-R-hydrogen atom of GenX2 is stereoselectively abstracted by the 5′-dAdo· radical and that methylation occurs with retention of configuration at C6′. Based on these observations, a model of GenK catalysis is proposed wherein free rotation of the radical-bearing carbon is prevented and the radical SAM machinery sits adjacent rather than opposite to the Me-Cbl cofactor with respect to the substrate in the enzyme active site.
