Purification and characterization of a new fungalysin-like metallopeptidase from the culture filtrate of a plant worm,Nomuraea atypicola

A new protease was purified from the culture filtrate of a plant worm, Nomuraea atypicola. The activity of the protease was suppressed by metalloprotease inhibitors such as EDTA and 1,10-phenanthroline, suggesting that it might be a metalloprotease. Its molecular mass was estimated to be 48 kDa by SDS-PAGE, and its optimal pH and temperature were pH 8.5–9.0 and 40 °C, respectively. The N-terminal amino acid sequence of the metalloprotease was similar to those of fungalysin metallopeptidases of the M36 family from fungi such as Coccidioides posadasii, Pyrenophora tritici-repentis, and Arthroderma gypseum, supporting the idea that it is a fungalysin-like metallopeptidase.     

Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D3 whose mutation causes vitamin D-dependent rickets type 1

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1alpha-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1alpha-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.     

Substrate-induced conformational changes of the truncated catalytic domain of Geobacillus stearothermophilus lysyl-tRNA synthetase as examined by fluorescence

The substrate-induced conformational change of the truncated C-terminal catalytic domain (CAT) of Geobacillus stearothermophilus lysyl-tRNA synthetase was examined by measuring tryptophan fluorescence of the truncated CAT domain in the presence or absence of the truncated N-terminal tRNA anticodon-binding domain (TAB). The fluorescence spectrum of CAT was not changed by the addition of l-lysine or ATP, whereas the intensity increased by adding a lysyl-adenylate analogue, suggesting that the CAT fluorescence increases when lysyl-adenylate is formed in the active site of CAT in l-lysine activation. In the presence of TAB, the addition of l-lysine to CAT decreased the fluorescence, and the subsequent addition of ATP recovered partially the decreased intensity, as is similar to the case of the intact enzyme. The static parameters of the CAT-TAB complex were similar to those of the intact enzyme, suggesting that a somewhat impaired structure of CAT is repaired on the formation of the complex with TAB. The mutational analysis of the fluorescence showed that Trp314 but not Trp332 is responsible for the observed fluorescence changes. The role of the TAB domain in the intact enzyme is considered to enhance the binding efficiency of lysyl-adenylate to the CAT domain.     

Catalytic properties of an organic solvent-resistant tyrosinase from Streptomyces sp. REN-21 and its high-level production in E. coli

An organic solvent-resistant tyrosinase (OSRT) from Streptomyces sp. REN-21 is a unique enzyme showing high activity in the presence of organic solvents. The OSRT-catalyzed oxidation of monophenols such as tyrosine-containing peptides and proteins was examined. The catalytic properties of OSRT were compared with those of mushroom tyrosinase. OSRT was shown to oxidize Gly-l-Tyr most effectively among four peptide substrates tested. On the other hand, mushroom tyrosinase showed the highest activity toward l-Tyr-Gly under the condition of 1 mM substrate. OSRT oxidized several proteins, including casein and hemoglobin, with relatively higher activity compared with mushroom tyrosinase under the condition of 1% (w/v) substrate. Thus, it was clarified that the catalytic properties of OSRT toward tyrosine-containing peptides and proteins are different from those of mushroom tyrosinase under these conditions. The OSRT-encoding gene operon was cloned, and found to consist of two genes, designated ORF-OSRT and ORF-393. The former encodes apo-OSRT, and the latter encodes the putative activator protein of apo-OSRT. A binuclear copper-binding site (type-3 copper site) characteristic of tyrosinases is contained in the deduced amino acid sequence for apo-OSRT. A high-level production system for the OSRT was constructed using pET20b(+) and Escherichia coli BL21(DE3)pLysS. Approximately 54 mg of active OSRT was synthesized in a 1-liter broth culture by this system. The properties of the recombinant OSRT were similar to those of the wild-type enzyme. In conclusion, we succeeded in constructing a high-level production system for OSRT.     

Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYP1A subfamily in the metabolism of dioxins

Metabolism of polychlorinated dibenzo-p-dioxins by CYP1A subfamily was examined by using the recombinant yeast microsomes. In substrate specificity and reaction specificity, considerable species differences between rats and humans were observed in both CYP1A1- and CYP1A2-dependent metabolism of dioxins. Among four CYPs, rat CYP1A1 showed the highest activity toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. To reveal the mechanism of dioxin metabolism, we examined rat CYP1A1-dependent metabolism of 2-chloro-dibenzo-p-dioxin. In addition to hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and cleavage of an ether linkage of the dioxin ring were observed. In particular, the cleavage of an ether linkage of the dioxin ring appeared most important for the detoxication of dioxins. Based on these results, the metabolic pathways of 2-chloro-dibenzo-p-dioxin by rat CYP1A1 were proposed. The metabolic pathways contain most of the metabolites observed in vivo using experimental animals, suggesting that P450 monooxygenase systems including CYP1A1 are greatly responsible for dioxin metabolism in vivo.     

Secreted expression of pseudozymogen forms of recombinant matriptase in Pichia pastoris

Matriptase is a transmembrane serine protease expressed in vertebrates. This enzyme is synthesized as a zymogen form and is converted to an active form by cleavage at the N-terminus of the serine protease catalytic domain. In a mammalian cell-based expression system, we have produced pseudozymogen forms of recombinant matriptase (r-matriptase) that are activated by cleavage with a recombinant enterokinase (r-EK) in vitro. In the present study, four different pseudozymogen forms of r-matriptase containing a site for activation by r-EK and a hexahistidine tag (His₆-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His₆-tag at their C-termini formed multimers linked by intermolecular disulfide bonds. After treatment with r-EK, they exhibited no detectable hydrolytic activity toward a chromogenic substrate. A pseudozymogen form of matriptase catalytic domain with N-terminal His₆-tag (designated His₆t-S-CD) was secreted as a monomer. His₆t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His₆t-S-CD could be purified from culture medium in milligram quantities. The expression in the yeast offers an efficient method of producing larger amounts of r-matriptase.     

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k_cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s⁻¹, respectively. The k_cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s⁻¹, respectively, at 1.0 M. On the other hand, the k_cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s⁻¹. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K_m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K_m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.     

Diverse genes of cellulase homologues of glycosyl hydrolase family 45 from the symbiotic protists in the hindgut of the termite Reticulitermes speratus

Diverse genes encoding cellulase homologues belonging to glycosyl hydrolase family 45 were identified from the symbiotic protists in the hindgut of the termite Reticulitermes speratus through the use of consensus PCR and the screening of a cDNA library. Fifteen full-length cDNA clones were isolated and sequenced, which encoded polypeptides consisting of 218–221 amino acid residues showing up to 63% identity to known family 45 cellulases. The cellulase sequences of the termite symbiotic protists were phylogenetically monophyletic, showing more than 75% amino acid identity with each other. These enzymes consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. By whole-cell in situ hybridization using oligonucleotide probes specific for regions conserved in some of the sequences, the origin of the genes was identified as symbiotic hypermastigote protists. The presence of diverse cellulase homologues suggests that symbiotic protists of termites may be rich reservoirs of novel cellulase sequences. Received: July 10, 2000 / Accepted: August 15, 2000     

Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists

Some species of protists inhabiting the hindgut of lower-termites have a large number of ectosymbiotic spirochetes on the cell surface. The phylogenetic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha sp. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated without cultivation of these organisms. Protist fractions carefully collected with a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two clusters. One was grouped together with the spirochetal sequences reported previously from the gut of termites and the other was related to the Treponema bryantii subgroup of treponemes (denoted as termite Treponema clusters I and II, respectively). Whole-cell in situ hybridization using a fluorescent-labeled oligonucleotide probe specific for the group of sequences in cluster II identified most of the ectosymbiotic spirochetes of the oxymonad protists in the gut of R. speratus and H. sjoestedti. However, not all of the ectosymbiotic spirochetes could be detected by means of this cluster II group-specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a portion of the ectosymbiotic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spirochetes of cluster II and probably those of a part of cluster I can be assigned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes.     

Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences, with emphasis on the Hypermastigea

Small subunit rRNA gene sequences were identified without cultivation from parabasalid symbionts of termites belonging to the hypermastigid orders Trichonymphida (the genera Hoplonympha, Staurojoenina, Teranympha, and Eucomonympha) and Spirotrichonymphida (Spirotrichonymphella), and from four yet-unidentified parabasalid symbionts of the termite Incisitermes minor. All these new sequences were analyzed by Bayesian, likelihood, and parsimony methods in a broad phylogeny including all identified parabasalid sequences available in databases and some as yet unidentified sequences probably derived from hypermastigids. A salient point of our study focused on hypermastigids was the polyphyly of this class. We also noted a clear dichotomy between Trichonymphida and the other parabasalid taxa. However, this hypermastigid order was apparently polyphyletic, probably reflecting its morphological diversity. Among Trichonymphida, Teranympha (Teranymphidae) grouped together with the members of the family Eucomonymphidae, suggesting that its family status is ambiguous. The monophyletic lineage composed by Spirotrichonymphida exhibited a narrower branching pattern than Trichonymphida. The root of parabasalids was examined but could not be discerned accurately.