Yoshiyuki Tsujimoto

Characteristic Features in the Structure and Collagen-Binding Ability of a Thermophilic Collagenolytic Protease from the Thermophile Geobacillus collagenovorans MO-1

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, extracellularly produces a collagenolytic protease with a large molecular mass. Complete nucleotide sequencing of this gene after gene cloning revealed that the collagenolytic protease is a member of the subtilisin family of serine proteases and consists of a signal sequence for secretion, a prosequence for maturation, a catalytic region, 14 direct repeats of 20 amino acids at the C terminus, and a region with unknown function intervening between the catalytic region and the numerous repeats. Since the unusual repeats are most likely to be cleaved in the secreted form of the enzyme, the intervening region was investigated to determine whether it participates in collagen binding to facilitate collagen degradation. It was found that the mature collagenolytic protease containing the intervening region at the C terminus bound collagen but not the other insoluble proteins, elastin and keratin. Furthermore, the intervening region fused with glutathione S-transferase showed a collagen-binding ability comparable to that of the mature collagenolytic protease. The collagen-binding ability was finally attributed to two-thirds of the intervening region which is rich in beta-strands and is approximately 35 kDa in molecular mass. In the collagenolytic protease from strain MO-1, hydrogen bonds most likely predominate over the hydrophobic interaction for collagen binding, since a higher concentration of NaCl released collagen from the enzyme surface but a nonionic detergent could not. To the best of our knowledge, this is the first report of a thermophilic collagenolytic protease containing the collagen-binding segment.

Cooperative Regulation of DOG2, Encoding 2-Deoxyglucose-6-Phosphate Phosphatase, by Snf1 Kinase and the High-Osmolarity Glycerol–Mitogen-Activated Protein Kinase Cascade in Stress Responses of Saccharomyces cerevisiae

We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZ transposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression of DOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels of DOG2 gene expression were increased in a mig1Delta mutant, while the derepression of DOG2 was not observed in a snf1Delta mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptants pbs2Delta, hog1Delta, and snf1Delta. However, the osmotic induction was completely abolished in both the snf1Delta pbs2Delta mutant and the snf1Delta hog1Delta mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.

An Aneurinibacillus sp. strain AM‐1 produces a proline‐specific aminopeptidase useful for collagen degradation

Aims:  We have been for a species of thermophilic bacteria that can effectively decompose collagen and collagen peptides that tend to be hard‐to‐degrade proteins because of their high content of proline residues. This study focused upon the enzymatic degradation of prolyl peptides by thermophilic bacteria.

Oxidative stress response in yeast: glutathione peroxidase of Hansenula mrakii is bound to the membrane of both mitochondria and cytoplasm

The yeast Hansenula mrakii IFO 0895 induces glutathione peroxidase (GPx) when the cells are exposed to the oxidative stress such as lipid hydroperoxide, superoxide- and hydroxy radical-generating conditions. To clarify the localization of GPx in H. mrakii cell, distribution of the enzyme was investigated. After centrifugation of the yeast protoplast homogenates at 2500 x g for 10 min, 67% of total GPx activity was recovered from the supernatant (Sup. 1) and 33% was from the pellet (Pellet 1). When the Sup. 1 was fractionated by sucrose density gradient ultracentrifugation, GPx activity was essentially recovered from the mitochondria fraction. Submitochondrial localization of the enzyme showed that 95% and 2.5% of the enzyme was recovered from the inner and outer membrane, respectively. No GPx activity was detected neither in intermembrane space nor in matrix of mitochondria. On the other hand, at least 12% of total GPx activity was recovered from the purified plasma membrane which was obtained from the Pellet 1 by successive sucrose density gradient centrifugation. Thus, the GPx of H. mrakii is present in the inner and outer membrane of mitochondria as well as the plasma membrane.

Identification of a Helix-Turn-Helix Motif of Bacillus thermoglucosidasius HrcA Essential for Binding to the CIRCE Element and Thermostability of the HrcA-CIRCE Complex, Indicating a Role as a Thermosensor

In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in B. thermoglucosidasius HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from Bacillus subtilis and B. thermoglucosidasius, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of B. thermoglucosidasius and B. subtilis and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.

Two Thimet Oligopeptidase-Like Pz Peptidases Produced by a Collagen- Degrading Thermophile, Geobacillus collagenovorans MO-1

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, was found to produce two metallopeptidases that hydrolyze the synthetic substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPR), containing the collagen-specific sequence -Gly-Pro-X-. The peptidases, named Pz peptidases A and B, were purified to homogeneity and confirmed to hydrolyze collagen-derived oligopeptides but not collagen itself, indicating that Pz peptidases A and B contribute to collagen degradation in collaboration with a collagenolytic protease in G. collagenovorans MO-1. There were many similarities between Pz peptidases A and B in their catalytic properties; however, they had different molecular masses and shared no antigenic groups against the respective antibodies. Their primary structures clarified from the cloned genes showed lower identity (22%). From homology analysis for proteolytic enzymes in the database, the two Pz peptidases belong to the M3B family. In addition, Pz peptidases A and B shared high identities of over 70% with unassigned peptidases and oligopeptidase F-like peptidases of the M3B family, respectively. Those homologue proteins are putative in the genome database but form two distinct segments, including Pz peptidases A and B, in the phylogenic tree. Mammalian thimet oligopeptidases, which were previously thought to participate in collagen degradation and share catalytic identities with Pz peptidases, were found to have lower identities in the overall primary sequence with Pz peptidases A and B but a significant resemblance in the vicinity of the catalytic site.

Multidrug resistance transporters Snq2p and Pdr5p mediate caffeine efflux in Saccharomyces cerevisiae.

SNQ2 was identified as a caffeine-resistance gene by screening a genomic library of Saccharomyces cerevisiae in a multicopy vector YEp24. SNQ2 encodes an ATP-binding cassette transporter and is highly homologous to PDR5. Multicopy of PDR5 also conferred resistance to caffeine, while its resistance was smaller than that of SNQ2. Residual caffeine contents were analyzed after transiently exposing cells to caffeine. The ratios of caffeine contents were 21.3 ± 8.8% (YEp24-SNQ2) and 81.9 ± 8.7% (YEp24-PDR5) relative to control (YEp24, 100%). In addition, multicopies of SNQ2 or PDR5 conferred resistance to rhodamine 6G (R6G), which was widely used as a substrate for transport assay. R6G was exported by both transporters, and their efflux activities were inhibited by caffeine with half-maximal inhibitory concentrations of 5.3 ± 1.9 (YEp24-SNQ2) and 17.2 ± 9.6 mM (YEp24-PDR5). These results demonstrate that Snq2p is a more functional transporter of caffeine than Pdr5p in yeast cells. Graphical Abstract Multicopies of SNQ2 or PDR5 conferred caffeine resistance; single knockout (snq2Δ, pdr5Δ) did not lead to caffeine sensitivity, although snq2Δ pdr5Δ double knockout led to significant sensitivity.

Oxidative stress response in yeast: Induction of glucose-6-phosphate dehydrogenase by lipid hydroperoxide in Hansenula mrakii

Abstract Hansenula mrakii IFO 0895 induces glutathione peroxidase as an oxidative stress response. Activities of other antioxidant enzymes—superoxide dismutase, catalase and cytochrome c peroxidase—were assayed. The superoxide dismutase activity did not change when the cells were cultured in the presence of lipid hydroperoxide, whereas the catalase and cytochrome c peroxidase activities decreased. The intracellular glutathione content and the activities of enzymes involved in glutathione biosynthesis were not changed by oxidative stress. Glucose-6-phosphate dehydrogenase, which is involved in the glutathione recycling system, was induced by oxidative stress to supply NADPH which is consumed by glutathione reductase coupling with the glutathione peroxidase reaction.

Enzymatic degradation of fibroin fiber by a fibroinolytic enzyme of Brevibacillus thermoruber YAS-1.

The employment of thermophiles and their enzymes is effective for the degradation of hard-to-degrade proteins. In this report, we describe the efficient degradation of fibroin, one major protein of silk fiber, by a thermophile and its enzymes. Brevibacillus thermoruber YAS-1, a thermophile which produces a proteinase with high action on fibroin fiber, was screened from samples obtained at the Manza hot springs, Gunma, Japan. The time course of fibroin degradation by the strain was investigated with a culture supernatant or concentrated enzyme sample. The breakdown of fibroin molecules was confirmed by the use of scanning electron microscopy, and the fibroin solubilized by the enzyme action was examined by amino acid composition analyses together with SDS-PAGE analysis. Herein, we demonstrate the significant degradation of fibroin fiber by strain YAS-1 and the potentials of its fibroinolytic enzyme(s).

Evaluation of Catechin and its Derivatives as Antioxidant: Recovery of Growth Arrest of Escherichia coli under Oxidative Conditions

Effects of (+)-catechin, (-)-epigallocatechin gallate and gallic acid on the growth of Escherichia coli K-12 under oxidative conditions were examined. (+)-Catechin did not protect the bacterial growth which was inhibited by H 2 O 2 , lipid hydroperoxide, paraquat, Cu 2+ , H 2 O 2 + Cu 2+ and lipid hydroperoxide + Cu 2+ . Both (-)-epigallocatechin gallate and gallic acid could restore the growth inhibition of E coli K-12 under the same conditions. Radical scavenging activity of (-)-epigallocatechin gallate, which is one of the most abundant components in Japanese green tea (Camellia sinensis L), is likely to be dependent upon the gallic acid group of the compound.