Synthesis and Characterization of Anguibactin To Reveal Its Competence To Function as a Thermally Stable Surrogate Siderophore for a Gram-Negative Pathogen, Acinetobacter baumannii.

Total synthesis of anguibactin was accomplished for the first time, and the following biochemical characterizations allowed for the determination of its Fe(III) binding mode as well as the demonstration of its iron delivery capability for Acinetobacter baumannii. These properties, in addition to the thermal stability over acinetobactin, render anguibactin as a competent surrogate siderophore that can be useful for the future development of a siderophore-based antibiotic delivery system against A. baumannii.

Crystal structure of the C-terminal domain of Bacillus subtilis GabR reveals a closed conformation by γ-aminobutyric acid binding, inducing transcriptional activation.

Bacillus subtilis GabR (BsGabR) is involved in the γ-aminobutyric acid (GABA) catabolism as a transcriptional regulator, consisting of an N-terminal helix-turn-helix DNA-binding domain and a C-terminal aminotransferase-like (AT-like) domain. Research on the C-terminal AT-like domain of BsGabR (BsGabR-CTD) has focused on the interaction with GABA as an effector, but most its functional details remain unclear. To understand the underlying mechanism, we report the crystal structure of BsGabR-CTD in complex with pyridoxal 5'-phosphate (PLP) and GABA at 2.0 Å resolution. The structure of ligand-bound BsGabR-CTD revealed two distinct monomeric states in a homodimer. One subunit is a closed-form containing the PLP-GABA adduct, and the other subunit is a PLP-bound open-form. Our structural studies provide a detailed mechanism indicating that the open-to-closed transition by the binding of GABA induces the conformational rearrangement of BsGabR-CTD, which may trigger the activation of transcription.

Structure-based functional studies for the cellular recognition and cytolytic mechanism of pneumolysin from Streptococcus pneumoniae.

Cholesterol-dependent cytolysins (CDCs) contribute to various pathogenesis by Gram-positive bacterial pathogens. Among them, pneumolysin (PLY) produced by Streptococcus pneumoniae is a major contributor to pneumococcal infections. Despite numerous studies of the cytolytic mechanism of PLY, little structural information on its interactions with a specific receptor of the cell membrane is available. We report here the first crystal structures of PLY in an apo-form and in a ternary complex with two mannoses at 2.8Å and 2.5Å resolutions, respectively. Both structures contained one monomer in an asymmetric unit and were comprised of four discontinuous domains, similar to CDC structures reported previously. The ternary complex structure showed that loop 3 and the undecapeptide region in domain 4 might contribute to cellular recognition by binding to mannose, as a component of a specific cell-surface receptor. Moreover, mutational studies and docking simulations for four residues (Leu431, Trp433, Thr459, and Leu460) in domain 4 indicated that Leu431 and Trp433 in the undecapeptide might be involved in the binding of cholesterol, together with the Thr459-Leu460 pair in loop 1. Our results provide structure-based molecular insights into the interaction of PLY with the target cell membrane, including the binding of mannose and cholesterol.

Kinetic characterization and molecular modeling of NAD(P)(+)-dependent succinic semialdehyde dehydrogenase from Bacillus subtilis as an ortholog YneI.

Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde (SSA) into succinic acid in the final step of γ-aminobutyric acid degradation. Here, we characterized Bacillus subtilis SSADH (BsSSADH) regarding its cofactor discrimination and substrate inhibition. BsSSADH showed similar values of the catalytic efficiency (kcat/Km) in both NAD(+) and NADP(+) as cofactors, and exhibited complete uncompetitive substrate inhibition at higher SSA concentrations. Further analyses of the sequence alignment and homology modeling indicated that the residues of catalytic and cofactor-binding sites in other SSADHs were highly conserved in BsSSADH.