Structural basis of L-glucose oxidation by scyllo-inositol dehydrogenase: Implications for a novel enzyme subfamily classification

For about 70 years, L-glucose had been considered non-metabolizable by either mammalian or bacterial cells. Recently, however, an L-glucose catabolic pathway has been discovered in Paracoccus laeviglucosivorans, and the genes responsible cloned. Scyllo-inositol dehydrogenase is involved in the first step in the pathway that oxidizes L-glucose to produce L-glucono-1,5-lactone with concomitant reduction of NAD+ dependent manner. Here, we report the crystal structure of the ternary complex of scyllo-inositol dehydrogenase with NAD+ and L-glucono-1,5-lactone at 1.8 Å resolution. The enzyme adopts a homo-tetrameric structure, similar to those of the inositol dehydrogenase family, and the electron densities of the bound sugar was clearly observed, allowing identification of the residues responsible for interaction with the substrate in the catalytic site. In addition to the conserved catalytic residues (Lys106, Asp191, and His195), another residue, His318, located in the loop region of the adjacent subunit, is involved in substrate recognition. Site-directed mutagenesis confirmed the role of these residues in catalytic activity. We also report the complex structures of the enzyme with myo-inositol and scyllo-inosose. The Arg178 residue located in the flexible loop at the entrance of the catalytic site is also involved in substrate recognition, and plays an important role in accepting both L-glucose and inositols as substrates. On the basis of these structural features, which have not been identified in the known inositol dehydrogenases, and a phylogenetic analysis of IDH family enzymes, we suggest a novel subfamily of the GFO/IDH/MocA family. Since many enzymes in this family have not biochemically characterized, our results could promote to find their activities with various substrates

Structural basis for the substrate recognition of aminoglycoside 7′′-phosphotransferase-Ia from Streptomyces hygroscopicus

Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular-biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7′′-phosphotransferase-Ia [APH(7′′)-Ia] from Streptomyces hygroscopicus and aminoglycoside 4-phosphotransferase-Ia [APH(4)-Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7′′ and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)-Ia was reported as a ternary complex with HygB and 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). To investigate the differences in the substrate-recognition mechanism between APH(7′′)-Ia and APH(4)-Ia, the crystal structure of APH(7′′)-Ia complexed with HygB is reported. The overall structure of APH(7′′)-Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)-Ia, and consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7′′)-Ia and APH(4)-Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C-lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7′′)-Ia and APH(4)-Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes

Effects of Triazole Derivatives on Strigolactone Levels and Growth Retardation in Rice

We previously discovered a lead compound for strigolactone (SL) biosynthesis inhibitors, TIS13 (2,2-dimethyl-7-phenoxy-4-(1H-1,2,4-triazol-1-yl)heptan-3-ol). Here, we carried out a structure-activity relationship study of TIS13 to discover more potent and specific SL biosynthesis inhibitor because TIS13 has a severe side effect at high concentrations, including retardation of the growth of rice seedlings. TIS108, a new TIS13 derivative, was found to be a more specific SL biosynthesis inhibitor than TIS13. Treatment of rice seedlings with TIS108 reduced SL levels in both roots and root exudates in a concentration-dependent manner and did not reduce plant height. In addition, root exudates of TIS108-treated rice seedlings stimulated Striga germination less than those of control plants. These results suggest that TIS108 has a potential to be applied in the control of root parasitic weeds germination

Nitrogen oxide cycle regulates nitric oxide levels and bacterial cell signaling

Nitric oxide (NO) signaling controls various metabolic pathways in bacteria and higher eukaryotes. Cellular enzymes synthesize and detoxify NO; however, a mechanism that controls its cellular homeostasis has not been identified. Here, we found a nitrogen oxide cycle involving nitrate reductase (Nar) and the NO dioxygenase flavohemoglobin (Fhb), that facilitate inter-conversion of nitrate, nitrite, and NO in the actinobacterium Streptomyces coelicolor. This cycle regulates cellular NO levels, bacterial antibiotic production, and morphological differentiation. NO down-regulates Nar and up-regulates Fhb gene expression via the NO-dependent transcriptional factors DevSR and NsrR, respectively, which are involved in the auto-regulation mechanism of intracellular NO levels. Nitrite generated by the NO cycles induces gene expression in neighboring cells, indicating an additional role of the cycle as a producer of a transmittable inter-cellular communication molecule

(Short Paper) Evidence Collection and Preservation System with Virtual Machine Monitoring

In a system audit and verification, it is important to securely collect and preserve evidence of execution environments, execution processes, and program execution results. Evidence-based verification of program processes ensures their authenticity; for example, the processes include no altered/infected program library. This paper proposes a solution for collection of evidence on program libraries based on Virtual Machine Monitor (VMM). The solution can solve semantic gap by obtaining library file path names. This paper also shows a way to obtain hash values of library files from a guest OS. Furthermore, this paper provides examples of evidence on program xecution and the overhead of the solution