Tadatake Oku

Design and Screening Strategies for α-Glucosidase Inhibitors Based on Enzymological Information

α-Glucosidase inhibitors are marketed as therapeutic drugs for diabetes that act through the inhibition of carbohydrate metabolism. Inhibitors of the α-glucosidases that are involved in the biosynthesis of N-linked oligosaccharide chains have been reported to have antitumor, antiviral, and apoptosis-inducing activities, and some have been used clinically. α-Glucosidase inhibitors have interesting biological activities, and their design, synthesis, and screening are being actively performed. In quite a few reports, however, α-glucosidases with different origins than the target α-glucosidases, have been used to evaluate inhibitory activities. There might be confusion regarding the naming of α-glucosidases. For example, the term α-glucosidase is sometimes used as a generic name for α-glucoside hydrolases. Moreover, IUBMB recommends the use of “α-glucosidase” (EC 3.2.1.20) for exo-α-1,4-glucosidases, which are further classified into four families based on amino acid sequence similarities. Accordingly, substrate specificity and susceptibility to inhibitors varies markedly among enzymes in the IUBMB α-glucosidases. The design and screening of inhibitors without consideration of these differences is not efficient. For the development of a practical inhibitor that is operational in cells, HTS using the target α-glucosidase and the computer-aided design of inhibitors based on enzymatic information concerning the same α-glucosidase are essential.

Glycon specificity profiling of α-glucosidases using monodeoxy and mono-O-methyl derivatives of p-nitrophenyl α-d-glucopyranoside

Hydrolysis of probe substrates, eight possible monodeoxy and mono-O-methyl analogs of p-nitrophenyl alpha-D-glucopyranoside (pNP alpha-D-Glc), modified at the C-2, C-3, C-4, and C-6 positions, was studied as part of investigations into the glycon specificities of seven alpha-glucosidases (EC 3.2.1.20) isolated from Saccharomyces cerevisiae, Bacillus stearothermophilus, honeybee (two enzymes), sugar beet, flint corn, and Aspergillus niger. The glucosidases from sugar beet, flint corn, and A. niger were found to hydrolyze the 2-deoxy analogs with substantially higher activities than against pNP alpha-D-Glc. Moreover, the flint corn and A. niger enzymes showed hydrolyzing activities, although low, for the 3-deoxy analog. The other four alpha-glucosidases did not exhibit any activities for either the 2- or the 3-deoxy analogs. None of the seven enzymes exhibited any activities toward the 4-deoxy, 6-deoxy, or any of the methoxy analogs. The hydrolysis results, with the deoxy substrate analogs, demonstrated that alpha-glucosidases having remarkably different glycon specificities exist in nature. Further insight into the hydrolysis of deoxyglycosides was obtained by determining the kinetic parameters (k(cat) and K(m)) for the reactions of sugar beet, flint corn, and A. niger enzymes.

Hydrolytic activity of α-galactosidases against deoxy derivatives of p-nitrophenyl α-d-galactopyranoside

The four possible monodeoxy derivatives of p-nitrophenyl (PNP) alpha-D-galactopyranoside were synthesized, and hydrolytic activities of the alpha-galactosidase of green coffee bean, Mortierella vinacea and Aspergillus niger against them were elucidated. The 2- and 6-deoxy substrates were hydrolyzed by the enzymes from green coffee bean and M. vinacea, while they scarcely acted on the 3- and 4-deoxy compounds. On the other hand, A. niger alpha-galactosidase hydrolyzed only the 2-deoxy compound in these deoxy substrates, and the activity was very high. These results indicate that the presence of two hydroxyl groups (OH-3 and -4) is essential for the compounds to act as substrates for the enzymes of green coffee bean and M. vinacea, while the three hydroxyl groups (OH-3, -4, and -6) are necessary for the activity of the A. niger enzyme. The kinetic parameters (K(m) and Vmax) of the enzymes for the hydrolysis of PNP alpha-D-galactopyranoside and its deoxy derivatives were obtained from kinetic studies.

Structure of cytochrome c6 from the red alga Porphyra yezoensis at 1.57 Å resolution

The crystal structure of cytochrome c(6) from the red alga Porphyra yezoensis has been determined at 1.57 A resolution. The crystal is tetragonal and belongs to space group P4(3)2(1)2, with unit-cell parameters a = b = 49.26 (3), c = 83.45 (4) A and one molecule per asymmetric unit. The structure was solved by the molecular-replacement method and refined with X-PLOR to an R factor of 19.9% and a free R factor of 25.4%. The overall structure of cytochrome c(6) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys14 and Cys17, and the iron has an octahedral coordination with His18 and Met58 as the axial ligands. The sequence and the structure of the eukaryotic red algal cytochrome c(6) are very similar to those of a prokaryotic cyanobacterial cytochrome c(6) rather than those of eukaryotic green algal c(6) cytochromes.

Synthesis of Monomethyl Derivatives of P-Nitrophenyl α-D-Gluco, Galacto, and Mannopyranosides and their Hydrolytic Properties Against α-Glycosidases

ABSTRACT All possible monomethyl derivatives of p-nitrophenyl α-D-gluco, galacto, and mannopyranosides were synthesized. Hydrolytic activities of α-glucosidase (rice), α-galactosidases (green coffee bean, Mortierella vinacea, and Aspergillus niger), and α-mannosidases (almond and jack bean) against them were elucidated. The 6-O-methyl galactopyranoside and mannopyranoside were hydrolyzed by the M. vinacea α-galactosidase and the almond and jack bean α-mannosidases, respectively, while these enzymes did not act on the 2-, 3-, and 4-O-methyl derivatives. On the other hand, rice α-glucosidase and green coffee bean and A. niger α-galactosidases had no hydrolyzing activities at all against the respective four monomethylated substrates.

Increasing the conformational stability by replacement of heme axial ligand in c‐type cytochrome

To investigate the role of the heme axial ligand in the conformational stability of c‐type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c 6 in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (ΔG°unf=1.48 kcal/mol) was lower than that of the wild‐type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a ΔG°unf of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild‐type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild‐type according to the heme‐bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c‐type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.

Heterodisaccharide 4-O-(N-acetyl-β-D-glucosaminyl)-D-glucosamine is a specific inducer of chitinolytic enzyme production in Vibrios harboring chitin oligosaccharide deacetylase genes

Vibrio parahaemolyticus KN1699 produces 4-O-(N-acetyl-beta-d-glucosaminyl)-d-glucosamine (GlcNAc-GlcN) as a major end product from chitin using two extracellular hydrolases: glycoside hydrolase family 18 chitinase, which produces (GlcNAc)(2) from chitin, and carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), which hydrolyzes the N-acetyl group at the reducing-end GlcNAc residue of (GlcNAc)(2). In this study, we clarified that this heterodisaccharide functions as an inducer of the production of the two above-mentioned chitinolytic enzymes, particularly chitinase. Similar results for chitinase production were obtained with other chitin-decomposing Vibrio strains harboring the CE family 4 COD gene; however, such an increase in chitinase production was not observed in chitinolytic Vibrio strains that did not harbor the COD gene. These results suggest that GlcNAc-GlcN is a unique inducer of chitinase production in Vibrio bacteria that have the COD-producing ability and that the COD involved in the synthesis of this signal compound is one of the key enzymes in the chitin catabolic cascade of these bacteria.

Crystal structure of oxidized cytochrome c6A from Arabidopsis thaliana

Compared with algal and cyanobacterial cytochrome c 6, cytochrome c 6A from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c 6A from Arabidopsis thaliana at 1.5 Å resolution in order to help elucidate its function. The overall structure of cytochrome c 6A follows the topology of class I c‐type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c 6A. Our model provides a chemical basis for the known low redox potential of cytochrome c 6A which makes it an unsuitable electron carrier between cytochrome b 6 f and PSI.

Design and synthesis of an ER-specific fluorescent probe based on carboxylesterase activity with quinone methide cleavage process

CEs are important enzymes that catalyze the hydrolysis of prodrugs. In this Letter, we present a new mechanistic ER-specific fluorescent probe 1 based on CE activity. Permeation of 1 into cells and subsequent hydrolytic activation by CEs causes spontaneously quinone methide cleavage, resulting in bright red fluorescence in ER with high specificity. Probe 1 was developed for CE activity imaging and inhibitor screening at the cellular level.

Recognition properties of processing α-glucosidase I and α-glucosidase II

Abstract All four possible monodeoxy derivatives of p‐nitrophenyl α‐D‐glucopyranoside (PNP Glc) and 1‐amino‐2,6‐anhydro‐1‐deoxy‐D‐glycero‐D‐ido‐heptitol derivatives were prepared and used as substrates and inhibitors of rat liver processing α‐glucosidases. α‐Glucosidase II hydrolyzed the 2‐deoxy derivative of PNP Glc (1); the hydrolysis of 1 was more rapid than that of PNP Glc. These results indicate that the presence of a C‐2 hydroxyl group is not essential for the action of α‐glucosidase II. In contrast, PNP Glc and all of the deoxy derivatives of PNP Glc 1–4 inhibited α‐glucosidase I. These results indicate that α‐glucosidase I does not necessarily need all of the hydroxyl groups of the glycon moiety for binding to the enzyme. 2,6‐Anhydro‐1‐benzamide‐D‐glycero‐D‐ido‐heptitol (11), with a terminal phenyl group, inhibited α‐glucosidase I and α‐glucosidase II. Both α‐glucosidase I and II showed the same aglycon specificities. When probes 5–12 were assayed for their ability to inhibit processing by α‐glucosidases at the cellular level, no effects on glycoprotein processing were observed.