Ken-ichi Hatano

Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds.

Abstract A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH2-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins.

Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean.

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution.

Novel strategy using an adsorbent-column chromatography for effective ethanol production from sugarcane or sugar beet molasses

Molasses-based distilleries generate large volumes of a highly polluted and dark brown-colored wastewater. The present work describes the way in which an adsorbent-column chromatography can effectively remove the colorant and produce biomass ethanol from sugarcane or sugar beet molasses. It was found that the color and chemical oxygen demand of the resulting wastewater was respectively reduced by approximately 87% and 28% as compared with conventional molasses fermentation. Gas chromatography showed that the decolorized molasses maintained good ethanol productivity almost equal to that of the original molasses. Furthermore, it was revealed that the colorant concentrations of about 5 mg ml(-1) in the medium were the most favorable for ethanolic fermentation. In summary, we have concluded that this method is the most effective when the adsorbent chromatography is performed just before molasses fermentation and that the decolorized molasses is an ideal substrate for fuel ethanol production.

Proteinase Inhibitor from Ginkgo Seeds Is a Member of the Plant Nonspecific Lipid Transfer Protein Gene Family

A 9-kD proteinase inhibitor was isolated from the seeds of ginkgo (Ginkgo biloba) and purified to homogeneity. This protein was revealed to partial-noncompetitively inhibit the aspartic acid proteinase pepsin and the cysteine proteinase papain (inhibition constant = 10−5–10−4 m). The cDNA of the inhibitor was revealed to contain a 357-bp open reading frame encoding a 119-amino acid protein with a potential signal peptide (27 residues), indicating that this protein is synthesized as a preprotein and secreted outside the cells. Semiquantitative reverse transcription-polymerase chain reaction revealed that this gene expresses only in seeds, not in stems, leaves, and roots, suggesting that the protein is involved in seed development and/or germination. The inhibitor showed about 40% sequence homology with type-I nonspecific lipid transfer protein (nsLTP1) from other plant species. Actually, this inhibitor exerted both lipid transfer activity and lipid-binding activity, while the protein did not show any antifungal and antibacterial activities. Furthermore, the site-directed mutagenesis study using a recombinant ginkgo nsLTP1 revealed that proline (Pro)-79 and phenylalanine-80 are important on phospholipid transfer activity and that Pro-79 and isoleucine-82 are essential for the binding activity toward cis-unsaturated fatty acids. On the other hand, the α-helical content of P79A and F80A mutants was significantly lower than that of the wild-type protein. It was noteworthy that the papain-inhibitory activity of P79A and F80A mutants was elevated twice as much as that of the wild-type protein. In summary, we concluded that Pro-79 plays a critical role in both the lipid transfer and binding activities of ginkgo nsLTP1.

Crystal structure of ginkbilobin-2 with homology to the extracellular domain of plant cysteine-rich receptor-like kinases.

Fungi are an extremely diverse group of organisms with about 250,000 species and are found in all ecosystems.1 They are also proficient in colonization and infection of plants, and often cause harm to the host organisms. Most plants have evolved a variety of potent defense mechanisms against these pathogens, such as hypersensitive responses, reinforcement of cell walls, and synthesis of phytoalexins and antifungal proteins.2 To date, hundreds of antifungal proteins have been discovered in a wide variety of plants and are classified into the following groups: cyclophilins, defensins, pathogenesis-related proteins, ribosome-inactivating proteins, and so on.1 Recently, we discovered a novel antifungal protein, ginkbilobin-2 (Gnk2), in the endosperm of Ginkgo seeds.3 Gnk2 consists of 108 amino acids as a mature protein and inhibits the growth of phytopathogenic fungi such as Fusarium oxysporum.3 This antifungal protein shows no sequence similarity to other antifungal proteins.3 On the other hand, Gnk2 has considerable homology ( 85%) to embryo-abundant proteins (EAP) from the gymnosperms Picea abies and P. glauca, which suggests that Gnk2-like proteins are widely conserved in the seeds of gymnosperms. Plant EAP are expressed in the late stage of seed maturation and are involved in protection against environmental stresses such as drought.4 However, there are no reports suggesting that these proteins help defend against fungal pathogens. The sequence of Gnk2 is also 28–31% identical to the extracellular domain of cysteine-rich receptor-like kinases (CRK) from the angiosperm Arabidopsis. CRK has been classified as a member of the plant receptor-like kinases subfamily. There are more than 40 members of CRK in Arabidopsis and they contain 1–4 copies of domain 26 of unknown function (DUF26) with a C-X8-C-X2-C motif in their extracellular regions.5,6 The CRK members are induced by pathogen infection and treatment with reactive oxygen species or salicylic acid7,8 and are involved in the hypersensitive reaction, which is a typical system of programmed cell death.9 The C-X8-C-X2-C motif is completely conserved in the sequences of both Gnk2 and gymnosperms EAP. In addition, there are at least 60 genes in Arabidopsis encoding the cysteine-rich secreted

Separation and characterization of the colored material from sugarcane molasses

The colored material (X) was effectively separated from sugarcane molasses using reversed-phase chromatography. Characterization of the molecular structure of sample X was performed using infrared absorption (IR) spectrometry, mass spectrometry (MS), and dynamic light scattering (DLS). The IR spectrum was similar to that of commercial humic acid, and the MS analysis showed that the sample possessed relatively small heterogeneous molecules with molecular masses around 234, 446, 657, 868, and 1079 Da. On the other hand, X sample showed an inhibitory activity toward the cysteine proteinase papain. Furthermore, the inhibitory (G-1) and weak inhibitory (G-2) fractions were separated from sample X using gel permeation chromatography. Samples G-1 and G-2 inhibited papain partial-noncompetitively and had the inhibition constants of 5.01 x 10(-5) and 1.08 x 10(-3)M, respectively. Interestingly, in the DLS experiment, the Stokes radius of sample G-1 was approximately 2 nm, about twice one of sample G-2.

Characterization of genomic sequence coding for bromelain inhibitors in pineapple and expression of its recombinant isoform

Bromelain inhibitor (BI) is a cysteine proteinase inhibitor isolated from pineapple stem (Reddy, M. N., Keim, P. S., Heinrikson, R. L., and Kézdy, F. J. (1975)J. Biol. Chem. 250, 1741–1750). It consists of eight isoinhibitors, and each isoinhibitor has a two-chain structure. In this study, the genomic DNA has been cloned and found to encode a precursor protein with 246 amino acids (M r = ∼27,500) containing three isoinhibitor domains (BI-III, -VI, and -VII) that are 93% identical to one another in amino acid sequences. The gene structure indicated that these isoinhibitors are produced by removal of the N-terminal pre-peptide (19 residues), 3 interchain peptides (each 5 residues), 2 interdomain peptides (each 19 residues), and the C-terminal pro-peptide (18 residues). Moreover, all the amino acid sequences of bromelain isoinhibitors could be explained by removal of one or two amino acids from BI-III, -VI, and -VII with exopeptidases. A recombinant single-chain BI-VI with and without the interchain peptide showed the same and no bromelain inhibitory activity as compared with the native BI-VI, respectively. These results indicate that the interchain peptide plays an important role of the folding process of the mature isoinhibitors.

A secreted protein with plant-specific cysteine-rich motif functions as a mannose-binding lectin that exhibits antifungal activity.

Ginkbilobin2 is shown to exert antifungal activity through its interaction with a1,2-linked mannose chains of fungal cell wall mannan, which provides clues to the molecular function of the DUF26 protein family. Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the α1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family.

Structure-Function Relationship of Bromelain Isoinhibitors from Pineapple Stem

Abstract Bromelain isoinhibitors from pineapple stem (BIs) are unique doublechain inhibitors and inhibit the cysteine proteinase bromelain competitively. The threedimensional structure was shown to be composed of two distinct domains, each of which is formed by a threestranded antiparallel βsheet. Unexpectedly, BIs were found to share similar folding and disulfidebond connectivities not with the cystatin superfamily, but with BowmanBirk trypsin/chymotrypsin inhibitor (BBI). The structural similarity between them suggests that BIs and BBI have evolved from a common ancestor and differentiated in function during the course of molecular evolution.

Crystal structure of the YjgF/YER057c/UK114 family protein from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7

ammoniumacetate, and 20% (v/v) glycerol and were then flash-cooled ina nitrogen stream. Diffraction data were collected at 100 Kwith an R-AXIS VII image plate detector mounted on aRigaku FR-E rotating-anode X-ray generator (Rigaku, Ja-pan) using the operation software CrystalClear (Rigaku/MSC) and were processed with MOSFLM