Comprehensive Experimental System for a Promising Model Organism Candidate for Marine Teleosts.

A comprehensive experimental system for Japanese anchovy, a promising candidate model organism for marine teleosts, was established. Through the design of a rearing/spawning facility that controls the photoperiod and water temperature, one-cell eggs were continuously obtained shortly after spawning throughout the rearing period. The stages of eggs are indispensable for microinjection experiments, and we developed an efficient and robust microinjection system for the Japanese anchovy. Embryos injected with GFP mRNA showed strong whole-body GFP fluorescence and the survival rates of injected- and non-injected embryos were not significantly different, 87.5% (28 in 32 embryos) and 90.0% (45 in 50 embryos), respectively. We verified that the Tol2 transposon system, which mediates gene transfer in vertebrates, worked efficiently in the Japanese anchovy using the transient transgenesis protocol, with GFP or DsRed as the reporter gene. Finally, we confirmed that genome-editing technologies, namely Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas9, were applicable to the Japanese anchovy. In practice, specific gene-disrupted fishes were generated in the F1 generation. These results demonstrated the establishment of a basic, yet comprehensive, experimental system, which could be employed to undertake experiments using the Japanese anchovy as a model organism for marine teleost fish.

Closed complex of the D-3-hydroxybutyrate dehydrogenase induced by an enantiomeric competitive inhibitor.

D-3-Hydroxybutyrate dehydrogenase (HBDH) from Pseudomonas fragi showed a strict stereospecificity to the d-enantiomer of 3-hydroxybutyrate (d-3-HB) as a substrate. The l-enantiomer acts as a competitive inhibitor, with a K(i) value comparable to the K(m) value for d-3-HB. We have determined the crystal structures of the ternary complex of HBDH-NAD(+)-l-3-HB and the binary complex of HBDH-NAD(+). The former structure showed a so-called closed-form conformation, which is considered an active form for catalysis, while the latter stayed mostly in a open-form conformation. The determined structures along with the site-directed mutagenesis confirmed the substrate recognition mechanism that we proposed previously. The hydrogen bonding interaction between Gln196, located in the moving helix, and the carboxyl group of the substrate/inhibitor is important for the stable ternary complex formation. Finally, the crystal structures of the Thr190 mutants, T190S and T190A, indicate that the Thr190 is a key residue for the open-closed conformational change. T190S retained 37% of the activity. In T190A, however, the activity decreased to 0.1% that of the wild-type enzyme. Fixing the position of the hydroxyl group of Thr190 to form hydrogen bonds to the pyrophosphate moiety and the carboxamide of NAD(+) seems to be a significant factor for the open-closed conformational change.

Crystal structure of aminopeptidase N (proteobacteria alanyl aminopeptidase) from Escherichia coli and conformational change of methionine 260 involved in substrate recognition.

Aminopeptidase N from Escherichia coli is a broad specificity zinc exopeptidase belonging to aminopeptidase clan MA, family M1. The structures of the ligand-free form and the enzyme-bestatin complex were determined at 1.5- and 1.6-A resolution, respectively. The enzyme is composed of four domains: an N-terminal beta-domain (Met(1)-Asp(193)), a catalytic domain (Phe(194)-Gly(444)), a middle beta-domain (Thr(445)-Trp(546)), and a C-terminal alpha-domain (Ser(547)-Ala(870)). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal-binding motif (HEXXHX(18)E) is found in the domain. The zinc ion is coordinated by His(297), His(301), Glu(320), and a water molecule. The groove on the catalytic domain that contains the active site is covered by the C-terminal alpha-domain, and a large cavity is formed inside the protein. However, there exists a small hole at the center of the C-terminal alpha-domain. The N terminus of bestatin is recognized by Glu(121) and Glu(264), which are located in the N-terminal and catalytic domains, respectively. Glu(298) and Tyr(381), located near the zinc ion, are considered to be involved in peptide cleavage. A difference revealed between the ligand-free form and the enzyme-bestatin complex indicated that Met(260) functions as a cushion to accept substrates with different N-terminal residue sizes, resulting in the broad substrate specificity of this enzyme.

Crystallization and preliminary X-ray characterization of aminopeptidase N from Escherichia coli.

A recombinant form of aminopeptidase N (molecular weight 99 kDa) from Escherichia coli was crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitating agent. The crystals belong to the hexagonal space group P3(1)21, with unit-cell parameters a = b = 120.5, c = 171.0 angstroms. The crystals are most likely to contain one molecule in the asymmetric unit, with a V(M) value of 3.62 angstroms3 Da(-1). Diffraction data were collected to 2.0 angstroms resolution using Cu Kalpha radiation from a rotating-anode X-ray generator.

Unusual extra space at the active site and high activity for acetylated hydroxyproline of prolyl aminopeptidase from Serratia marcescens.

The prolyl aminopeptidase complexes of Ala-TBODA [2-alanyl-5-tert-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-tert-butyl-(1, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 angstroms resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the prolyl aminopeptidase. Moreover, 4-acetyloxyproline-betaNA (4-acetyloxyproline beta-naphthylamide) was a better substrate than Pro-betaNA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyr150, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria.

D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme.

The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme-NAD+ complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and was refined to 2.0 A resolution. The overall structure of P.fragi HBDH, including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme-NAD+ complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NAD+-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate.

Preparation of primary amine-modified gold nanoparticles and their transfection ability into cultivated cells.

Cationic gold nanoparticles, prepared by reducing HAuCl(4) with NaBH(4) in the presence of 2-aminoethanethiol, formed a complex with plasmid DNA, and could be transfected into cultivated cells.