Hiroyuki Hori

Identification and characterization of tRNA (Gm18) methyltransferase from Thermus thermophilus HB8: domain structure and conserved amino acid sequence motifs

Background: Transfer RNAs from an extreme thermophile, Thermus thermophilus, commonly possess 2′‐O‐methylguanosine at position 18 (Gm18) in the D‐loop. This modification is post‐transcriptionally introduced by tRNA (Gm18) methyltransferase.

Characterization of Mouse nNOS2, a Natural Variant of Neuronal Nitric-oxide Synthase Produced in the Central Nervous System by Selective Alternative Splicing

Mouse neuronal nitric-oxide synthase 2 (nNOS2) is a unique natural variant of constitutive neuronal nitric-oxide synthase (nNOS) specifically expressed in the central nervous system having a 105-amino acid deletion in the heme-binding domain as a result of in-frame mutation by specific alternative splicing. The mouse nNOS2 cDNA gene was heterologously expressed in Escherichia coli, and the resultant product was characterized spectroscopically in detail. Purified recombinant nNOS2 contained heme but showed no l-arginine- and NADPH-dependent citrulline-forming activity in the presence of Ca2+-promoted calmodulin, elicited a sharp electron paramagnetic resonance (EPR) signal at g = 6.0 indicating the presence of a high spin ferriheme as isolated and showed a peak at around 420 nm in the CO difference spectrum, instead of a 443-nm peak detected with the recombinant wild-type nNOS1 enzyme. Thus, although the heme domain of nNOS2 is capable of binding heme, the heme coordination geometry is highly abnormal in that it probably has a proximal non-cysteine thiolate ligand both in the ferric and ferrous states. Moreover, negligible spectral perturbation of the nNOS2 ferriheme was detected upon addition of either l-arginine or imidazole. These provide a possible rational explanation for the inability of nNOS2 to catalyze the cytochrome P450-type monooxygenase reaction.

Selective inhibition of human inducible nitric oxide synthase by S‐alkyl‐L‐isothiocitrulline‐containing dipeptides

The aim of this study was to investigate the structure‐activity relationship of S‐alkyl‐L‐isothiocitrulline‐containing dipeptides towards three partially purified recombinant human nitric oxide synthase (NOS) isozymes, as well as the effects of these compounds on cytokine‐induced NO production by human DLD‐1 cells. In an in vitro assay, S‐methyl‐L‐isothiocitrulline (L‐MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L‐amino acid (L‐Phe, L‐Leu or L‐Trp) with L‐MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L‐MIT‐L‐Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S‐alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS. These results suggest that the human NOS isozymes have different‐sized cavities in the binding site near the position to which the C‐terminal of L‐arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L‐MIT‐D‐Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well‐defined direction from the α carbon atom. NO production in cytokine‐stimulated human DLD‐1 cells was measured with a fluorescent indicator, DAF‐FM. MILF, L‐MIT‐L‐Trp(‐CHO) (MILW) and L‐MIT‐L‐Tyr (MILY) showed more potent activity than L‐MIT in this whole‐cell assay. Thus, S‐alkyl‐L‐isothiocitrulline‐containing dipeptides are selective inhibitors of human iNOS, and work efficiently in cell‐based assay.

MODULATION OF THE REMOTE HEME SITE GEOMETRY OF RECOMBINANT MOUSE NEURONAL NITRIC-OXIDE SYNTHASE BY THE N-TERMINAL HOOK REGION

The role of two essential residues at the N-terminal hook region of neuronal nitric-oxide synthase (nNOS) in nitric-oxide synthase activity was investigated. Full-length mouse nNOS proteins containing single-point mutations of Thr-315 and Asp-314 to alanine were produced in the Escherichia coli and baculovirus-insect cell expression systems. The molecular properties of the mutant proteins were analyzed in detail by biochemical, optical, and electron paramagnetic resonance spectroscopic techniques and compared with those of the wild-type enzyme. Replacement of Asp-314 by Ala altered the geometry around the heme site and the substrate-binding pocket of the heme domain and abrogated the ability of nNOS to form catalytically active dimers. Replacement of Thr-315 by Ala reduced the protein stability and altered the geometry around the heme site, especially in the absence of bound (6R)-5,6,7,8-tetrahydro-l-biopterin cofactor. These results suggest that Asp-314 and Thr-315 both play critical structural roles in stabilizing the heme domain and subunit interactions in mouse nNOS.

Transfer RNA Modification Enzymes from Thermophiles and Their Modified Nucleosides in tRNA

To date, numerous modified nucleosides in tRNA as well as tRNA modification enzymes have been identified not only in thermophiles but also in mesophiles. Because most modified nucleosides in tRNA from thermophiles are common to those in tRNA from mesophiles, they are considered to work essentially in steps of protein synthesis at high temperatures. At high temperatures, the structure of unmodified tRNA will be disrupted. Therefore, thermophiles must possess strategies to stabilize tRNA structures. To this end, several thermophile-specific modified nucleosides in tRNA have been identified. Other factors such as RNA-binding proteins and polyamines contribute to the stability of tRNA at high temperatures. Thermus thermophilus, which is an extreme-thermophilic eubacterium, can adapt its protein synthesis system in response to temperature changes via the network of modified nucleosides in tRNA and tRNA modification enzymes. Notably, tRNA modification enzymes from thermophiles are very stable. Therefore, they have been utilized for biochemical and structural studies. In the future, thermostable tRNA modification enzymes may be useful as biotechnology tools and may be utilized for medical science.

The Mechanism of Conversion of Xanthine Dehydrogenase to Xanthine Oxidase

Xanthine dehydrogenase and xanthine oxidase are complex metalloflavoproteins that represent alternate forms of the same gene product. The cDNAs encoding the enzymes have been cloned from several sources, and structural information is becoming available. Using purified enzyme, comparative analyses between the two forms were attempted by spectroscopic and kinetics methods. The most significant difference between the two forms is the protein conformation around flavin adenine dinucleotide (FAD), which changes the redox potential of the flavin and the reactivity of FAD with the electron acceptors, nicotinamide adenine dinucleotide (NAD) and molecular oxygen. The flavin semiquinone is thermodynamically stable in xanthine dehydrogenase but is unstable in xanthine oxidase. Detailed analyses by stopped-flow techniques suggest that the flavin semiquinone reacts with oxygen to form superoxide anion while the fully reduced flavin reacts to form hydrogen peroxide. Although xanthine dehydrogenase can produce greater amounts of superoxide anion than xanthine oxidase during xanthine oxygen reaction, it seems not to be physiologically significant in the cell, where excess NAD exists under normal conditions.