Yoshiaki Oyama

A single amino acid substitution converts cytochrome P45014DM to an inactive form, cytochrome P450SG1: Complete primary structures deduced from cloned DNAs

Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.

Humanization of a mouse neutralizing monoclonal antibody against tumor necrosis factor-α (TNF-α)

An anti-human tumor necrosis factor-α (TNF-α) monoclonal antibody, designated as 3B10, inhibits the biological activity of human TNF-α. In the present study, we constructed humanized version of the antibody by grafting its complementarity-determining regions (CDRs) onto a human antibody, HBS-1. Using a molecular model of mouse 3B10, framework residues affecting the CDR conformation were identified. Thus, these residues were also introduced into the framework together with the CDRs in a stepwise manner, depending on the degree of the possible importance of the residues. As a result, one humanized version (h3B10-9) which possesses nine mouse framework residues showed the same binding activity as that of the chimeric version. This humanized anti-TNF-α antibody is expected to be less immunogenic and thus more suitable for possible clinical use.

Notch signaling regulates the differentiation of bone marrow-derived cells into smooth muscle-like cells during arterial lesion formation.

Bone marrow- (BM-) derived cells can differentiate into smooth muscle-like cells (SMLC), resulting in vascular pathogenesis. However, the molecular mechanism of the differentiation remains unknown. We have recently reported that Notch signaling promotes while a Notch target HERP1 inhibit the differentiation of mesenchymal cells to SMC. During the differentiation of BM-derived mononuclear cells into smooth muscle alpha-actin (SMA)-positive cells, expression of Jagged1 and SMC-specific Notch3 was increased. Blocking Notch with gamma-secretase inhibitor prevented the induction of SMA. Wire-mediated vascular injury was produced in femoral arteries in mice transplanted with green fluorescent protein (GFP)-positive cells. Many double-positive cells for GFP/Jagged1 or GFP/Notch3 were detected in the thickened neointima. In contrast, only a few SMA-positive cells were positive for GFP in neointima where HERP1, a suppressor for Notch, were abundantly expressed. In conclusion, Notch-HERP1 pathway plays an important role in differentiation of BM-derived mononuclear cells into SMLC.

pKa measurements from nuclear magnetic resonance of tyrosine-150 in class C beta-lactamase.

13C-NMR spectroscopy was used to estimate the p K a values for the Tyr(150) (Y150) residue in wild-type and mutant class C beta-lactamases. The tyrosine residues of the wild-type and mutant lactamases were replaced with (13)C-labelled L-tyrosine ([ phenol -4-(13)C]tyrosine) in order to observe the tyrosine residues selectively. Spectra of the wild-type and K67C mutant (Lys(67)-->Cys) enzyme were compared with the Y150C mutant lactamase spectra to identify the signal originating from Tyr(150). Titration experiments showed that the chemical shift of the Tyr(150) resonance in the wild-type enzyme is almost invariant in a range of 0.1 p.p.m. up to pH 11 and showed that the p K (a) of this residue is well above 11 in the substrate-free form. According to solvent accessibility calculations on X-ray-derived structures, the phenolic oxygen of Tyr(150), which is near the amino groups of Lys(315) and Lys(67), appears to have low solvent accessibility. These results suggest that, in the native enzyme, Tyr(150) in class C beta-lactamase of Citrobacter freundii GN346 is protonated and that when Tyr(150) loses a proton, a proton from Lys(67) would replace it. Consequently, Tyr(150) would be protonated during the entire titration.

Revisit to the sulfonation of pyrroles: is the sulfonation position correct?

Abstract Sulfonation of pyrrole and its 1-methyl derivatives with a sulfur trioxide–pyridine complex was found to give 3-sulfonated pyrroles, but not 2-sulfonates as described in textbooks. The replacement of 1-methyl-2-tri- n -butylstannylpyrrole with trimethylsilyl chlorosulfonate, followed by quenching with aq. NaHCO 3 also generated sodium 1-methylpyrrole-3-sulfonate, not 2-sulfonate.

Structural Models of the Photointermediates in the Rhodopsin Photocascade, Lumirhodopsin, Metarhodopsin I, and Metarhodopsin II

Model building of the two photointermediates, lumirhodopsin and metarhodopsin I, and the activated form of rhodopsin, metarhodopsin II, is described. An outward swing of the C‐terminal portion of transmembrane segment 3, pivoting on Cys110 at the N‐terminal end of transmembrane segment 3, led to structural models of lumirhodopsin and metarhodopsin I. The conformation of the chromophore in the lumirhodopsin and metarhodopsin I models is controlled by the motion of transmembrane segment 3 and agreed closely with the hydrogen‐bonding states of the protonated Schiff base in lumirhodopsin and metarhodopsin I as deduced from their FTIR and resonance Raman spectra and with the negative and positive CD bands of lumirhodopsin and metarhodopsin I, respectively. The structure of metarhodopsin II was constructed by an outward swing of transmembrane segment 3 and the rigid‐body motion of transmembrane segment 6. The arrangement of the entire transmembrane segment of the metarhodopsin II model closely agreed with the electron paramagnetic resonance spectra of spin‐labeled rhodopsin mutants and provided a structural basis for the protonation of Glu134, which is a key process in transducin activation.

Modelling of photointermediates suggests a mechanism of the flip of the β-ionone moiety of the retinylidene chromophore in the rhodopsin photocascade

Light causes an extremely rapid 11-cis-to-all-trans isomerization of the retinylidene chromophore of rhodopsin. This isomerization leads to bleaching intermediates in the photoactivation cascade. An early photointermediate, bathorhodopsin (Batho), which already contains a photoisomerized all-trans retinylidene chromophore, slowly ( 1 sec) decays by conformational changes to metarhodopsin I (Meta I) through lumirhodopsin (Lumi). The cis-trans photoisomerization of the retinylidene chromophore of rhodopsin occurs within the limited space of opsin, which results in a highly strained conformation of the chromophore. In a photoaffinity labeling experiment, Nakanishi et al. showed that a modified -ionone moiety cross-linked Trp265 on transmembrane segment 6 (TM6) both in the rhodopsin and Batho states, which suggests that the cyclohexenyl moiety remains unchanged in the rhodopsin-to-Batho transition. In the subsequent Batho-to-Lumi transition, the moiety flipped from TM6 towards TM4. The flip of the modified -ionone moiety suggests that TM3 and TM4 rearrange to accommodate the modified -ionone moiety, as schematically shown in Figure 1, while the helix

Constraints of Opsin Structure on the Ligand-binding Site: Studies with Ring-fused Retinals¶

Ring‐fused retinal analogs were designed to examine the hula‐twist mode of the photoisomerization of the 9‐cis retinylidene chromophore. Two 9‐cis retinal analogs, the C11–C13 five‐membered ring–fused and the C12–C14 five‐membered ring–fused retinal derivatives, formed the pigments with opsin. The C11–C13 ring‐fused analog was isomerized to a relaxed all‐trans chromophore (λmax > 400 nm) at even −269°C and the Schiff base was kept protonated at 0°C. The C12–C14 ring‐fused analog was converted photochemically to a bathorhodopsin‐like chromophore (λmax= 583 nm) at −196°C, which was further converted to the deprotonated Schiff base at 0°C. The model‐building study suggested that the analogs do not form pigments in the retinal‐binding site of rhodopsin but form pigments with opsin structures, which have larger binding space generated by the movement of transmembrane helices. The molecular dynamics simulation of the isomerization of the analog chromophores provided a twisted C11–C12 double bond for the C12–C14 ring‐fused analog and all relaxed double bonds with a highly twisted C10–C11 bond for the C11–C13 ring‐fused analog. The structural model of the C11–C13 ring‐fused analog chromophore showed a characteristic flip of the cyclohexenyl moiety toward transmembrane segments 3 and 4. The structural models suggested that hula twist is a primary process for the photoisomerization of the analog chromophores.

Structure–activity relationships for mini atrial natriuretic peptide by proline-Scanning mutagenesis and shortening of peptide backbone

MiniANP is a synthetic pentadecapeptide analogue of atrial natriuretic polypeptide (ANP). We have used the proline-scanning mutagenesis and the analogue peptides with shorter backbones to characterize the turn-like conformation at residue 6-9 and an extended structure of Gly5-Gly6 as the receptor-bound structure of miniANP. A docking study of miniANP at the binding site of the type A natriuretic peptide receptor (NPR-A) supported the deduced conformation in the receptor-bound structure.

Discovery and dimeric approach of novel Natriuretic Peptide Receptor A (NPR-A) agonists

Novel agonists of the Natriuretic Peptide Receptor A (NPR-A) were obtained through random screening and subsequent structural modification of triazine derivatives. The key structural feature to improve in vitro activity was the dimerization of triazine monomer derivatives. The non peptide derivative 7c and 13a showed highly potent NPR-A agonistic activity in vitro and diuretic activity in vivo. These results implied that non-peptidic small molecules open the possibility of new therapy for congestive heart failure.