Hiromasa Mori

On the molecular embrace of bleomycin with helical DNA

The active centre model of bleomycin, proposed in our recent paper, has developed in such a way as to reveal the precise physical structure of the bleomycin molecule as a whole and to clarify the mode of binding interaction of this antibiotic molecule with the helically arranged DNA. The thorough model building researches have shown that the three strong hydrogen bonds are formed between the bleomycin molecule and the double helical DNA. The partial intercalation of a thiazole ring in the former molecule into the neighbouring bases on one strand of DNA also contributes to the binding of bleomycin with DNA. These interactions, together with the previously predicted one ionic interaction, establish many points of fixation of the bleomycin molecule with DNA and constitute the origin of the specific selectivity of this molecule with the helical DNA as the partner of molecular embrace. The concept of partial intercalation, introduced in the above-mentioned paper, has also been developed so as to elucidate the essential feature of electronic interaction between the thiazole ring moiety of the bleomycin molecule and the thymine base of DNA, in contact with one thiazole ring. This electronic interaction can be expressed as the intermolecular electron transfer from the 2pπ-orbital of the ring nitrogen atoms N3 in the thymine base to the 3dπ- and/or 4s-orbital(s) of sulphur atom contained in the thiazole ring. As a result of this electron transfer, the release of thymine base is plainly explained. The liberation of a thymine base from DNA results in the restabilization of the double helical DNA with which the bleomycin molecule is still linked. This finding has given the resolution of the existing most puzzling experimental results. Then as a consequence of this resolution, the correctness of our active centre model of the bleomycin molecule is verified with sufficient confidence.

Active centre model on the structure and function of the bleomycin molecule as the prototype of esteratic enzymes

Abstract The unique interactions of the bleomycin molecule with DNA are analysed from biophysical and stereochemical points of view. According to our model, bleomycin has a functional part which includes threonine hydroxyl and histidine imidazole groups. These are arranged in a way essentially the same as those in the active centres of esterases, especially in α-chymotrypsin and ribonuclease although the hydroxyl group in the latter comes from the serine residue or the bound substrate. Taking into account this situation and also the participation of sulphydryl compounds, the ability of the bleomycin molecule to introduce scission in one strand of DNA is clearly explained. The proposed relative conformation of the bleomycin molecule with DNA quite naturally predicts the “in-line” mechanism as the mode of scission of the phosphodiester linkage. The central part of the theory, especially the requirement of a histidine imidazole ring for the activity of bleomycin, is shown experimentally by the application of sensitized photo-oxidation in the presence of rose bengal.

A demetallation method for IMP-1 metallo-β-lactamase with restored enzymatic activity upon addition of metal ion(s)

Metallo-b-lactamases (MBLs) are Zn-dependent enzymes that hydrolyze most b-lactams and pose a potential threat in clinical environments due to their wide substrate specificity and lack of clinically available inhibitors. MBLs are classified into three subclasses, B1, B2, and B3, according to the set of metal ion ligands in the active center. Most recently, a new metallob-lactamase has been found in India, Pakistan, and also in the United Kingdom and many other countries. Further proliferation of the types of metallo-b-lactamases is becoming a grave worldwide public health concern. Among the currently known MBLs, IMP-1, belonging to subclass B1, is one of the most serious threats because the gene encoding it is located in an integron structure on a plasmid, which is horizontally transferable between bacterial strains. The X-ray crystal structure of IMP-1 has been determined and two Zn ions (termed Zn1 and Zn2) were found in the active center. Zn1 is tetrahedrally coordinated by His116, His118, and His196, whereas Zn2 is trigonal–pyramidally coordinated by Asp120, Cys221, His263, and a water molecule (Figure 1). In addition, OH2 or OH is thought to be bridged between Zn1 and Zn2, although no electron density for this molecule is observed due to the low resolution of the crystal structure. Correlation between Zn content and catalytic efficiency continues to be of great controversy, despite the fact that Zn ligands are well-conserved in other subclass B1 MBLs, for example, BcII from Bacillus cereus and CcrA from Bacteroides fragilis. BcII has one Zn ion under physiological conditions but achieves maximum activity when two Zn ions are bound to the active center. Unlike BcII, CcrA and IMP-1 contain two Zn ions under physiological conditions and require two Zn ions for catalysis. To investigate the metal coordination environment in the active center, spectroscopic studies on Co-substituted MBLs have been extensively carried out, due to the spectroscopically silent Zn. Success in these studies requires preparation of apo-enzymes. In BcII and CcrA, demetallations of Zn and metal substitution with Co have been performed successfully. Similar efforts have been attempted for IMP-1, but complete demetallation with various chelating agents, such as EDTA, yields samples that cannot be reactivated in the presence of excess Zn ions. Herein, we describe the kinetic behavior of the inactivation of IMP-1 by EDTA, and successful preparation of the apoenzyme (apo-IMP-1). In addition, spectroscopic characterization of Co-substituted IMP-1 derived from apo-IMP-1 with Co, in situ, is also described. Temperature dependency for inactivation of IMP-1 (10 mm) by EDTA (50 mm) in MOPS (50 mm ; pH 7.0) and NaCl (1.0 m) was examined by changing the temperature of the incubation samples (0, 10, 20, and 30 8C, Figure S1 in the Supporting Information). Figure 2 shows the plot of the residual activity against incubation time at 10 8C, where the residual activity, kt/k0, (i.e. , the relative activity) is the ratio of the molecular activity, kt, of the activated IMP-1 at each incubation time of IMP-1 with EDTA to the molecular activity, k0, of IMP-1 before the addition of EDTA at t = 0 (control). From Figure 2, it is apparent that the inactivation reaction proceeds in two steps, namely fast and slow. A plausible explanation for this biphasic behavior is successive demetallation of the two Zn ions from the active center (Scheme 1). Figure 1. Schematic representation of the active site of IMP-1 from Pseudomonas aeruginosa (PDB ID: 1DDK). The Zn (Zn1, Zn2) atoms and a water (Wat) molecule are shown as spheres.

Modes of DNA replication primed with the oligomer of palindrome

Abstract What is the precise molecular mechanism of semi-conservative DNA replication? After the great efforts of the past 20 years, molecular biology has now established the discontinuous syntheses of daughter DNA on both of the parental strands. In order to explain this type of discontinuous replication, we introduce the concept of a palindromic primer. First we focus our attention on various oligomers (RNA or DNA) which appear usually or occasionally in the process of replication. Then we propose the palindromic nature of these oligomers so as to serve as the primer of DNA synthesis. This postulation gives a theoretical reasoning for the discontinuities of both new strands in the fork region of replication. Subsequently we consider Watsons concatemeric intermediate theory, proposed for the explanation of replicative synthesis of phage T7 DNA. By considering the contribution of some sequence-specific endonuclease(s), we suggest the existence of partial palindromic sequences of bases at the connecting region(s) in which the redundant ends of the respective phage DNA molecules are overlapping. Another theory on the replication of linear chromosomal DNA including the concept of the terminal palindromic sequence of bases is also analyzed from the viewpoint of palindromic primer. Further, some recent experimental approaches, especially on the origin(s) of DNA replication, are shown to favour the concept of a palindromic primer.