Crystal Structure of GenD2, an NAD-Dependent Oxidoreductase Involved in the Biosynthesis of Gentamicin

Abstract

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.

Biochemical and Structural Analysis of a Dehydrogenase, KanD2, and an Aminotransferase, KanS2, That Are Responsible for the Construction of the Kanosamine Moiety in Kanamycin Biosynthesis

“…These observations indicate that the well-studied CH−π interactions between carbohydrates and aromatic amino acid residues , are used by KanD2 to stack 3″-hks in the active site. Our observation also supports the results from the docking studies of GenD2 with gentamicin A2 . Overall, the protein structural analysis and mutational analysis clearly illustrate that KanD2 selectively recognizes the pseudotrisaccharide structure of 3″-hks.…”

“…The N-terminal domain adopts a typical Rossmann-type α/β fold and interacts with NAD + . KanD2 is structurally similar to GenD2 [Protein Data Bank (PDB) entry 6NOR, root-mean-square deviation (rmsd) of 1.1 Å for chain A Cα atoms, sequence identity of 45%], Sinorhizobium morelense 1,5-ahhydro- d -fructose reductase (PDB entry 2GLX, rmsd of 1.9 Å for chain A Cα atoms, sequence identity of 24%), and Caulobacter crescentus aldose–aldose oxidoreductase (PDB entry 5A03, rmsd of 2.2 Å for chain A Cα atoms, sequence identity of 25%) . KanD2 exists as a homotetramer in the crystal structure (Figure S13A) as observed in the GenD2 structure .…”

“…His182 is completely conserved among the KanD2/GenD2 family of dehydrogenases (Figure S15) and commonly functions as a base to abstract the proton of the hydroxy group at C3″ during dehydrogenation. It was hypothesized that Lys96 of GenD2 functions as a base to abstract a proton from the C3″ hydroxy group because the side chain of Lys96 was observed to be oriented toward the substrate binding pocket in the GenD2 structure . However, the side chain of the corresponding Lys97 of KanD2 points away from the substrate binding pocket in the KanD2 structure (Figure S16).…”

Pyridoxal-5'-Phosphate-Dependent Enzyme GenB3 Catalyzes C-3',4'-Dideoxygenation in Gentamicin Biosynthesis

“…Gentamicin is a broad-spectrum aminoglycoside bactericidal antibiotic featuring the C-3',4' dideoxygenation moity. As with other 2-deoxystreptamine-containing AGAs, significant progress has been made in gentamicin biosynthesis [16][17][18][19][20] . However, little detail of the featured C-3',4' dideoxygenation process is known.…”

Mechanistic Insights into Dideoxygenation in Gentamicin Biosynthesis

“…Since the biosynthetic gene cluster of gentamicin was identified more than a decade ago, we and others have elucidated most of the gentamicin biosynthetic pathway in Micromonospora echinospora ATCC15835 [8][9][10][11][12][13][14][15][16][17][18][19] and revealed crystal structures of several of the enzymes involved in the biosynthesis 14,20,21,22 . GenD2, GenN, GenS2 and GenD1 have been demonstrated to be involved in the formation of gentamicin X2 16,21,22 . At X2 there is a branching of the pathway: methylation of X2 at C-6ʹ by GenK produces G418, precursor of gentamicins C1, C2 and C2a, 13,14,16 whereas this methylation does not occur on route to gentamicins C1a and C2b (Fig.…”