Structural Characterization of the Chlorophyllide a Oxygenase (CAO) Enzyme Through an In Silico Approach.

Chlorophyllide a oxygenase (CAO) is responsible for converting chlorophyll a to chlorophyll b in a two-step oxygenation reaction. CAO belongs to the family of Rieske-mononuclear iron oxygenases. Although the structure and reaction mechanism of other Rieske monooxygenases have been described, a member of plant Rieske non-heme iron-dependent monooxygenase has not been structurally characterized. The enzymes in this family usually form a trimeric structure and electrons are transferred between the non-heme iron site and the Rieske center of the adjoining subunits. CAO is supposed to form a similar structural arrangement. However, in Mamiellales such as Micromonas and Ostreococcus, CAO is encoded by two genes where non-heme iron site and Rieske cluster localize on the distinct polypeptides. It is not clear if they can form a similar structural organization to achieve the enzymatic activity. In this study, the tertiary structures of CAO from the model plant Arabidopsis thaliana and the Prasinophyte Micromonas pusilla were predicted by deep learning-based methods, followed by energy minimization and subsequent stereochemical quality assessment of the predicted models. Furthermore, the chlorophyll a binding cavity and the interaction of ferredoxin, which is the electron donor, on the surface of Micromonas CAO were predicted. The electron transfer pathway was predicted in Micromonas CAO and the overall structure of the CAO active site was conserved even though it forms a heterodimeric complex. The structures presented in this study will serve as a basis for understanding the reaction mechanism and regulation of the plant monooxygenase family to which CAO belongs.

The Non-Mendelian Green Cotyledon Gene in Soybean Encodes a Small Subunit of Photosystem II.

Chlorophyll degradation plays important roles in leaf senescence including regulation of degradation of chlorophyll-binding proteins. Although most genes encoding enzymes of the chlorophyll degradation pathway have been identified, the regulation of their activity has not been fully understood. Green cotyledon mutants in legume are stay-green mutants, in which chlorophyll degradation is impaired during leaf senescence and seed maturation. Among them, the soybean (Glycine max) green cotyledon gene cytG is unique because it is maternally inherited. To isolate cytG, we extensively sequenced the soybean chloroplast genome, and detected a 5-bp insertion causing a frame-shift in psbM, which encodes one of the small subunits of photosystem II. Mutant tobacco plants (Nicotiana tabacum) with a disrupted psbM generated using a chloroplast transformation technique had green senescent leaves, confirming that cytG encodes PsbM. The phenotype of cytG was very similar to that of mutant of chlorophyll b reductase catalyzing the first step of chlorophyll b degradation. In fact, chlorophyll b-degrading activity in dark-grown cytG and psbM-knockout seedlings was significantly lower than that of wild-type plants. Our results suggest that PsbM is a unique protein linking photosynthesis in presenescent leaves with chlorophyll degradation during leaf senescence and seed maturation. Additionally, we discuss the origin of cytG, which may have been selected during domestication of soybean.

Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues.

Cathepsins B and L belong to the papain superfamily of cysteine proteases and play important roles in various physiological and pathological processes. In the course of studies on their inhibitors, we examined the inhibitory effects of the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and its analogues. As a result, rat liver cathepsins B and L were shown to be strongly inhibited by them. The concentration required for 50% inhibition (IC(50)) by ZLLLal was 88 nM for cathepsin B and 163 nM for cathepsin L. Moreover, various analogues of ZLLLal, including 2-furancarbonyl-, nicotinyl-, isonicotinyl- and 4-morpholinylsuccinyl-LLLals, and some acetyl-Pro (AcP)-containing analogues, AcPLLLal and AcPPLLLal, were shown to inhibit both enzymes more strongly than ZLLLal. Among them, isonicotinyl-LLLal was most inhibitory against both cathepsins B (IC(50), 12 nM) and L (IC(50), 20 nM). Several of these inhibitors were indicated to be somewhat more soluble in aqueous media than ZLLLal.

Structural and phylogenetic comparison of three pepsinogens from Pacific bluefin tuna: molecular evolution of fish pepsinogens.

The amino acid sequences of three pepsinogens (PG1, PG2 and PG3) of Pacific bluefin tuna (Thunnus orientalis) were deduced by cloning and nucleotide sequencing of the corresponding cDNAs. The amino acid sequences of the pre-forms of PG1, PG2 and PG3 were composed of a signal peptide (16 residues each), a propeptide (41, 37 and 35 residues, respectively) and a pepsin moiety (321, 323 and 332 residues, respectively). Amino acid sequence comparison and phylogenetic analysis indicated that PG1 and PG2 belong to the pepsinogen A family and PG3 to the pepsinogen C family. Homology modeling of the three-dimensional structure suggested that the remarkably high specific activity of PG2 toward hemoglobin, which had been found previously, was partly due to a characteristic deletion of several residues in the S1'-loop region that widens the space of the active site cleft region so as to accommodate protein and larger polypeptide substrates more efficiently. Including the tuna and all other fish pepsinogen sequences available to date, the molecular phylogenetic comparison was made with reference to evolution of fish pepsinogens. It was suggested that functional divergences of pepsinogens (pepsins) occurring in fishes as well as in mammals, correlated with differences in various aspects of fish physiology.

Novel peptide-based pepsin inhibitors containing an epoxide group.

1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP) is known to inhibit pepsin A and other aspartic proteinases by reacting with the active site aspartic acid residue(s). However, the reaction is considerably slow in general, and therefore, it is desirable to develop similar reagents that are capable of inhibiting these enzymes more rapidly. In the present study, we synthesized a series of novel inhibitors which have a reactive epoxide group linked with peptide by a hydrazide bond, with a general structure: Iva-L-Val-L-Val-(L-AA)(n)-N2H2-ES-OEt (n = 0 approximately 2) (Iva, isovaleryl; AA, bulky hydrophobic or aromatic amino acid residue; ES, epoxysuccinyl). These inhibitors were shown to inhibit porcine pepsin A remarkably faster than EPNP.

Substrate specificities of porcine and bovine enteropeptidases toward the peptide Val-(Asp)4-Lys-Ile-Val-Gly and its analogs.

The substrate specificities of porcine and bovine enteropeptidases were investigated using the peptide Val-(Asp)(4)-Lys-Ile-Val-Gly and its various analogs with mutations in the (Asp)(4)-Lys sequence as substrates. The results indicated that in addition to P1 Lys, P2 Asp in the substrates is most important, that P3 Asp is additionally important, and that P5 Asp contributes somewhat to the susceptibility, and that P4 Asp is the least important. These results were essentially identical as between porcine and bovine enteropeptidases.

[An adult case of congenital fiber type disproportion (CFTD) with cardiomyopathy].

A 30 year-old man with CFTD was reported. He had normal motor milestone during infancy but had been poor at sports. At 28, he experienced exertional and nocturnal dyspnea and had been diagnosed as having dilated cardiomyopathy. At 29, a cardiac pace-maker was implanted because of the complete atrio-ventricular block. Around that time, he began to notice limb muscle weakness. Examination at 30 showed mild diffuse muscle atrophy and weakness at the torso and limbs. No dysmorphic features or joint contractures were noted. His serum CK was normal. A histochemical study of his muscle biopsy showed type 1 fiber predominancy (64.6%) and that the mean diameter of type 1 fibers was smaller than that of type 2 by 14.6% (36.9 microm vs. 42.3 microm). Results of immunostaining of dystrophin, emerin, laminA/C, alpha, beta, gamma, delta-sarcoglycan or dysferlin were normal. He was diagnosed as having CFTD because there were no histochemical abnormalities which characterize other congenital myopathies except for the type 1 predominancy and atrophy.

Tetrapeptides on N- and C-terminal regions of mastoparan inhibit catecholamine release from chromaffin cells by blocking nicotinic acetylcholine receptor.

Mastoparan (MP), a tetradecapeptide in wasp venom, has been reported to evoke catecholamine release, but also reported to inhibit secretory response upon nicotinic stimulation in adrenal chromaffin cells. To elucidate the inhibitory mechanism of MP, we examined the effect of two MP fragments (INLK-NH2 and KKIL-NH2) on catecholamine release in bovine adrenal chromaffin cells. These MP fragments inhibited catecholamine release induced by nicotinic stimulation in a noncompetitive manner. These fragments did not affect catecholamine release evoked by high [K+] or by other secretagogues, neither caused catecholamine release by themselves. Replacement by hydrophobic and basic amino acids of the MP fragments enhanced the inhibitory effects on ACh-evoked catecholamine release. Among 23 analogs of the MP fragments, (Nle)3-R-NH2 showed the most potent inhibition with IC50 = 541 microM. These results suggested that the MP fragments selectively inhibit the secretory response to nicotinic stimulation by attacking nAChR on the site(s) made up of hydrophobic and acidic amino acids but other than ACh-binding sites. This mechanism may explain the inhibitory action of MP on nicotine-evoked catecholamine release.

The sugar-metabolic enzymes aldolase and triose-phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana: detection using biotinylated glutathione.

GSH has multiple actions in physiological responses of plants, but the molecular mechanisms are not fully understood. GSH plays an important role in functional alteration of proteins by reversible covalent incorporation (glutathionylation) in vertebrate cells. To investigate the function of glutathionylation in plant cells, we examined glutathionylated proteins in the suspension-cultured cells of Arabidopsis using biotinylated GSH. Biotinylated GSH was incorporated into about 20 proteins. Two of these proteins were identified as the key enzymes for sugar metabolism, triose-phosphate isomerase (TPI) and putative plastidic aldolase. Recombinant TPI was inactivated by GSSG, and it was reactivated by GSH. The physiological roles of glutathionylation of TPI and aldolase in sugar metabolism are discussed.