金澤 宏樹

The aminoglycoside resistance conferred by an N1-methylation of A1408 in 16S rRNA by a novel plasmid-mediated methyltransferase NpmA can be a future health threat. In the present study, we have determined crystal structures of the bacterial ribosomal decoding A site with an A1408m(1)A antibiotic-resistance mutation both in the presence and absence of aminoglycosides. G418 and paromomycin both possessing a 6 '-OH group specifically bind to the mutant A site and disturb its function as a molecular switch in the decoding process. On the other hand, binding of gentamicin with a 6 '-NH3+ group to the mutant A site could not be observed in the present crystal structure. These observations agree with the minimum inhibitory concentration of aminoglycosides against Escherichia coli. In addition, one of our crystal structures suggests a possible conformational change of A1408 during the N1-methylation reaction by NpmA. The structural information obtained explains how bacteria acquire resistance against aminoglycosides along with a minimum of fitness cost by the N1-methylation of A1408 and provides novel information for designing the next-generation aminoglycoside.

We have determined a crystal structure of an RNA duplex containing a novel metal-binding motif. The motif is composed of two sheared GOA base pairs, two unpaired A residues and four phosphate groups in close proximity. Four A residues make an A-A-A-A stacking column at the minor groove side and two G bases are highly inclined, thereby forming the pocket-shaped motif at the major groove side. In the present structure, a hydrated Sr2+ ion exists in the pocket and binds to the O6 and N7 atoms of the two G bases and four phosphate groups. According to the previously-reported metal-binding properties to RNA molecules, many of divalent cations, such as Mg2+ Mn2+ Co2+ Zn2+ Ba2+ Pb2+ and Cd2+, may bind to the motif. This metal-binding motif can be used as a modular building block that allows for precise positioning of a single metal ion in functional nucleic acid molecules.

D-3-Hydroxybutyrate dehydrogenase catalyzes the reversible conversion of acetoacetate and D-3-hydroxybutyrate. These ketone bodies are both energy-storage forms of acetyl-CoA. In order to clarify the structural mechanisms of the catalytic reaction with the cognate substrate D-3-hydroxybutyrate and of the inhibition of the reaction by inhibitors, the enzyme from Alcaligenes faecalis has been analyzed by X-ray crystallography in liganded states with the substrate and with two types of inhibitor: malonate and methylmalonate. In each subunit of the tetrameric enzyme, the substrate is trapped on the nicotinamide plane of the bound NAD+. An OMIT map definitively shows that the bound ligand is D-3-hydroxybutyrate and not acetoacetate. The two carboxylate O atoms form four hydrogen bonds to four conserved amino-acid residues. The methyl group is accommodated in the nearby hydrophobic pocket so that the formation of a hydrogen bond from the OH group of the substrate to the hydroxy group of Tyr155 at the active centre is facilitated. In this geometry, the H atom attached to the C-3 atom of the substrate in the sp(3) configuration is positioned at a distance of 3.1 angstrom from the nicotinamide C-4 atom in the direction normal to the plane. In addition, the donor-acceptor relationship of the hydrogen bonds suggests that the Tyr155 OH group is allowed to ionize by the two donations from the Ser142 OH group and the ribose OH group. A comparison of the protein structures with and without ligands indicates that the Gln196 residue of the small movable domain participates in the formation of additional hydrogen bonds. It is likely that this situation can facilitate H-atom movements as the trigger of the catalytic reaction. In the complexes with inhibitors, however, their principal carboxylate groups interact with the enzyme in a similar way, while the interactions of other groups are changed. The crucial determinant for inhibition is that the inhibitors have no active H atom at C-3. A second determinant is the Tyr155 OH group, which is perturbed by the inhibitors to donate its H atom for hydrogen-bond formation, losing its nucleophilicity.

Aminoglycoside antibiotics are pseudosaccharides decorated with ammonium groups that are critical for their potent broad-spectrum antibacterial activity. Despite over three decades of speculation whether or not modulation of pK(a) is a viable strategy to curtail aminoglycoside kidney toxicity, there is a lack of methods to systematically probe amine-RNA interactions and resultant cytotoxicity trends. This study reports the first series of potent aminoglycoside antibiotics harboring fluorinated N1-hydroxyaminobutyryl acyl (HABA) appendages for which fluorine-RNA contacts are revealed through an X-ray cocrystal structure within the RNA A-site. Cytotoxicity in kidney-derived cells was significantly reduced for the derivative featuring our novel beta,beta-difluoro-HABA group, which masks one net charge by lowering the pK(a) without compromising antibacterial potency. This novel side-chain assists in evasion of aminoglycoside-modifying enzymes, and it can be easily transferred to impart these properties onto any number of novel analogs.

This study reports the synthesis, antibacterial evaluation and nature of fluorine-rRNA contacts revealed by an X-ray co-crystal structure of a series of 4'-deoxy-4'-fluoro B-neomycin analogs. 4'-Deoxyfluorination improves the inhibition profile towards resistant enzymes and renders equally potent antibiotics compared to the parent neomycin B. The 4'-deoxy-4'-fluoro-4'-epi neomycin analogs showed a preferential inhibition over the 4'-deoxy-4'-fluoro neomycin counterpart against the strains of P. aeruginosa carrying a chromosomal APH(3')-IIb enzyme, known to inactivate the parent aminoglycoside. To the best of our knowledge, this is the first example of a neighboring-group aminoglycoside-modifying enzyme evasion by fluorine substitution. A unique F-G1491 stacking was observed in a co-crystal structure of 4'-deoxy-4'-fluoro-4'-epi neomycin with a bacterial ribosomal RNA A-site.