Kiyoshi Shiga

A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis.

Blue light regulates processes such as the development of plants and fungi and the behaviour of microbes. Two types of blue-light receptor flavoprotein have been identified: cryptochromes, which have partial similarity to photolyases, and phototropins, which are photoregulated protein kinases. The former have also been found in animals with evidence of essential roles in circadian rhythms. Euglena gracilis, a unicellular flagellate, abruptly changes its swimming direction after a sudden increase or decrease in incident blue light intensity, that is, step-up or step-down photophobic responses, resulting in photoavoidance or photoaccumulation, respectively. Although these photobehaviours of Euglena have been studied for a century, the photoreceptor molecules mediating them have remained unknown. Here we report the discovery and biochemical characterization of a new type of blue-light receptor flavoprotein, photoactivated adenylyl cyclase, in the photoreceptor organelle of Euglena gracilis, with molecular genetic evidence that it mediates the step-up photophobic response.

Phosphorus nuclear magnetic resonance of perfused salivary gland

Phosphorus nuclear magnetic resonance (31P-NMR) was used for the sequential measurement of phosphorus energy metabolites in perfused canine submandibular gland. Under resting conditions, ATP and creatine phosphate levels were 0.42 +/- 0.11 mM and 0.62 +/- 0.16 mM (mean +/- S.D., in nine glands). When perfusion of the gland was stopped, the tissue contents of ATP and creatine phosphate decreased, that of ADP increased and tissue pH decreased. Restarting perfusion led to recoveries of the tissue content of the phosphorus compounds and tissue pH to normal. Acetylcholine administration induced secretion of saliva, decreased the level of ATP, creatine phosphate and tissue pH, and increased the ADP level.

A resonance Raman study on the nature of charge-transfer interactions in butyryl CoA dehydrogenase.

Butyryl-CoA dehydrogenase (BCD) (EC 1.3.99.2) is a flavoprotein that catalyses the first step in fatty acid p-oxidation. When isolated from various sources, the pure enzyme has a characteristic green colour [l-3], due to a long-wavelength absorption band centred at 710 nm. We have proposed that a new chemical species, CoA persulphide, tightly-bound at the enzyme’s active site, is the donor in a chargetransfer interaction with the FAD prosthetic group [4,5]. Complexes with long-wavelength absorption are also formed between the enzyme and various acyl-CoA compounds, including acetoacetyl-CoA, which gives a grey-green complex with an absorbance maximum at 580 nm [6]. Butyryl-CoA dehydrogenase was purified from Megasphaera elsdenii as in [ 121. Coenzyme A was purchased from Sigma. Other reagents were from BDH.

A reversible spin conversion of cytochrome b5 at high temperatures

Summary The thermodynamics for the reversible spin conversion of cytochrome b 5 have been studied by absorption and circular dichroic spectroscopy in the temperature range from 15 to as high as 83°C. The optical absorption spectrum of the oxidized cytochrome b 5 below 45°C, predominantly of low-spin type, changes to an essentially high-spin type on increasing the temperature. This spectral transition of cytochrome b 5 was found to take place in a relatively limited range from 45°C to 65°C. The circular dichroic spectrum of cytochrome b 5 at high temperatures is that of partially unfolded polypeptide with an exposed heme moiety. All these spectral changes were reversible.

Porcine kidney d-amino acid oxidase: the three-dimensional structure and its catalytic mechanism based on the enzyme–substrate complex model

Abstract The three-dimensional structure of porcine kidney d -amino acid oxidase (DAO), an FAD-dependent oxidase, has been solved by X-ray crystallography. The overall structure is a dimer, subunits of which are correlated by a non-crystallographic two-fold axis. Each subunit comprises two domains, ‘αβ domain’ and ‘pseudo-barrel domain’. The coenzyme FAD is in an elongated conformation and is bound at the N -terminal βαβ dinucleotide binding motif. The active site is located in the boundary region between the two domains. The crystal structure of DAO in complex with a substrate analog, o -aminobenzoate, was also solved and is used for modeling the DAO- d -leucine complex, i.e. Michaelis complex, by means of molecular mechanics simulation. The Michaelis-complex model provided structural information leading to two alternative hypothetical mechanisms for the reductive half-reaction of DAO. These two hypotheses characterize themselves by electron transfer from the lone-pair orbital of the substrate amino nitrogen to flavin C(4a) and by proton transfer from the substrate α-position to flavin N(5) which acts as a catalytic base.

Hydrogen-bonding dynamics of free flavins in benzene and FAD in electron-transferring flavoprotein upon excitation

The dynamic natures of two hydrogen-bonding model systems, riboflavin tetrabutylate (RFTB)-trichloroacetic acid (TCA) and RFTB-phenol in benzene, and of electron-transferring flavoprotein (ETF) from pig kidney upon excitation of flavins was investigated by means of steady state and time-resolved fluorescence spectroscopy. In both model systems fluorescence intensities of RFTB decreased as TCA or phenol was added. The spectral characteristics of ETF under steady state excitation were quite similar to those of the RFTB-TCA system, but not to those of the RFTB-phenol system. The observed fluorescence decay curves of ETF fit well with the calculated decay curves with two lifetime components, as in the model systems. Averaged lifetime was 0.9 ns. The time-resolved fluorescence spectrum of ETF shifted toward longer wavelength with time after pulsed excitation, which was also observed in the RFTB-TCA system. In the RFTB-phenol system the emission spectrum did not shift at all with time. These results reveal that the dynamic nature of ETF can be ascribed to aliphatic hydrogen-bonding(s) of the isoalloxazine ring with surrounding amino acid(s). From the fluorescence characteristics of ETF in comparison with the model systems, human ETF and other flavoproteins, it was suggested that ETF from pig kidney does not contain Tyr-16 in the beta subunit, unlike human ETF.

Resonance Raman study on the purple intermediates of the flavoenzyme D-amino acid oxidase

Abstract Resonance Raman (RR) spectra excited at 632.8 nm within a charge transfer absorption band were obtained for a catalytic intermediate, the purple complex of D-amino acid oxidase with D-proline or D-alanine as a substrate. The resonance enhanced Raman lines around 1605 and 1360 cm −1 in either of the complexes were suggested to be derived from vibrational modes of reduced flavin molecule. Since the highest energy band at 1692 cm −1 in the RR spectrum with D-alanine was shifted to 1675 cm −1 upon [ 15 N] substitution of alanine and ammonium, this Raman line in the spectrum with D-alanine or the line at 1658 cm −1 with D-proline is assigned to the CN stretching mode of an imino acid corresponding to each amino acid. These results confirm the concept that the purple intermediate of D-amino acid oxidase consists of reduced flavin and an imino acid.

Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FADs, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.

Crystallization and preliminary X-ray characterization of rat liver acyl-CoA oxidase

A recombinant form of the flavoenzyme acyl-CoA oxidase from rat liver has been crystallized by the hanging-drop vapour-diffusion technique using PEG 20 000 as a precipitating agent. The crystals grew as yellow prisms, with unit-cell parameters a = 71.05, b = 87.29, c = 213.05 A, alpha = beta = gamma = 90 degrees. The crystals exhibit the symmetry of space group P2(1)2(1)2(1) and are most likely to contain a dimer in the asymmetric unit, with a V(M) value of 2.21 A(3) Da(-1). The crystals diffract to a resolution of 2.5 A at beamline BL6A of the Photon Factory. Two heavy-atom derivatives have been identified.

A STUDY OF THE INTERACTION BETWEEN CHLORPROMAZINE AND RIBOFLAVIN BINDING PROTEIN

The interaction between chlorpromazine (CPZ) and riboflavin binding protein (RBP) was studied by the spectrophotometric and spectrofluorometric methods.