β-Aspartyl compounds, such as β-aspartyl hydroxamate (serine racemase inhibitor), β-aspartyl-l-lysine (moisture retention), and β-aspartyl-l-tryptophan (immunomodulator) are physiologically active compounds. There is limited literature on the development of effective methods of production of β-aspartyl compounds. In this study, we describe the biochemical characterization of asparagine synthetase (AS) from Streptococcus thermophilus NBRC 13957 (StAS) and the enzymatic synthesis of β-aspartyl compounds using StAS.
View Article and Find Full Text PDFA convenient method for the determination of plant sphingolipids (glycosylinositol phosphoceramide, GIPC; glucosylceramide, GluCer; phytoceramide 1-phosphate, PC1P and phytoceramide, PCer) was developed. This method includes the extraction of lipids using 1-butanol, alkali hydrolysis with methylamine and separation by TLC. The amounts of sphingolipids in the sample were determined based on the relative intensities of standard sphingolipids visualized by primulin/UV on TLC.
View Article and Find Full Text PDFCrystal structures of Pseudomonas veroniil-arginine dehydrogenase (l-ArgDH), belonging to the μ-crystallin/ornithine cyclodeaminase family, were determined for the enzyme in complex with l-lysine and NADP and with l-arginine and NADPH. The main chain coordinates of the P. veroniil-ArgDH monomer showed notable similarity to those of Archaeoglobus fulgidusl-AlaDH, belonging to the same family, and pro-R specificity similar to l-AlaDH for hydride transfer to NADP was postulated.
View Article and Find Full Text PDFBiochim Biophys Acta Mol Cell Biol Lipids
February 2023
One of the major functions of peroxisomes in mammals is oxidation of very long-chain fatty acids (VLCFAs). Genetic defects in peroxisomal β-oxidation result in the accumulation of VLCFAs and lead to a variety of health problems, such as demyelination of nervous tissues. However, the mechanisms by which VLCFAs cause tissue degeneration have not been fully elucidated.
View Article and Find Full Text PDFGlycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots.
View Article and Find Full Text PDFIntroduction: Semi-extended tibial nailing techniques include the extra-articular technique (EAT) and the patellar eversion technique (PET). These approaches differ regarding the exposure of the patellar retinaculum and the size of the surgical field. This study compared the postoperative alignment and intramedullary nailing entry points between the EAT and PET for tibial fractures.
View Article and Find Full Text PDFOrnithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5'-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P.
View Article and Find Full Text PDFBiochim Biophys Acta Mol Cell Biol Lipids
February 2022
Fatty acids (FAs) longer than C20 are classified as very long-chain fatty acids (VLCFAs). Although biosynthesis and degradation of VLCFAs are important for the development and integrity of the myelin sheath, knowledge on the incorporation of extracellular VLCFAs into the cells is limited due to the experimental difficulty of solubilizing them. In this study, we found that a small amount of isopropanol solubilized VLCFAs in aqueous medium by facilitating the formation of the VLCFA/albumin complex.
View Article and Find Full Text PDFL-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2.
View Article and Find Full Text PDFThe amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu.
View Article and Find Full Text PDFγ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their γ-glutamyl moiety to acceptor substrates. Pseudomonas nitroreducens GGT (PnGGT) is used for the industrial synthesis of theanine, thus it is important to determine the structural basis of hydrolysis and transfer reactions and identify the acceptor site of PnGGT to improve the efficient of theanine synthesis. Our previous structural studies of PnGGT have revealed that crucial interactions between three amino acid residues, Trp385, Phe417, and Trp525, distinguish PnGGT from other GGTs.
View Article and Find Full Text PDFα-1,3-Glucan is a homopolymer composed of D-glucose (Glc) and it is an extracellular polysaccharide found in dental plaque due to Streptococcus species. α-1,3-Glucanase from Streptomyces thermodiastaticus strain HF3-3 (Agl-ST) has been identified as a thermostable α-1,3-glucanase, which is classified into glycoside hydrolase family 87 (GH87) and specifically hydrolyzes α-1,3-glucan with an endo-action. The enzyme has a potential to inhibit the production of dental plaque and to be used for biotechnological applications.
View Article and Find Full Text PDFSphingomyelin (SM) with N-α-hydroxy fatty acyl residues (hSM) has been shown to occur in mammalian skin and digestive epithelia. However, the metabolism and physiological relevance of this characteristic SM species have not been fully elucidated yet. Here, we show methods for mass spectrometric characterization and quantification of hSM.
View Article and Find Full Text PDFJ Chromatogr B Analyt Technol Biomed Life Sci
September 2020
Glycosylinositol phosphoceramide (GIPC) is a sphingophospholipid in plants. Recently, we identified that GIPC is hydrolyzed to phytoceramide 1-phosphate (PC1P) by an uncharacterized phospholipase D activity following homogenization of certain plant tissues. We now developed methods for isolation of GIPC and PC1P from plant tissues and characterized their chemical stabilities.
View Article and Find Full Text PDFThe α-1,3-glucanase from Paenibacillus glycanilyticus FH11 (Agl-FH1), a member of the glycoside hydrolase family 87 (GH87), hydrolyzes α-1,3-glucan with an endo-action. GH87 enzymes are known to degrade dental plaque produced by oral pathogenic Streptococcus species. In this study, the kinetic analyses revealed that this enzyme hydrolyzed α-1,3-tetraglucan into glucose and α-1,3-triglucan with β-configuration at the reducing end by an inverting mechanism.
View Article and Find Full Text PDFA gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C.
View Article and Find Full Text PDFPreviously, we found an unidentified sphingolipid in cabbage, and determined it as phytoceramide 1-phosphate (PC1P). PC1P is found to be produced from glycosylinositol phosphoceramide (GIPC) by the action of phospholipase D (PLD) activity. Although GIPC is abundant sphingolipid, especially in cruciferous vegetables, amount of daily intake, digestibility and nutritional activity of GIPC are not well understood.
View Article and Find Full Text PDFGlycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipid in plants and fungi. Recently, we detected GIPC-specific phospholipase D (GIPC-PLD) activity in plants. Here, we found that GIPC-PLD activity in young cabbage leaves catalyzes transphosphatidylation.
View Article and Find Full Text PDFThe purpose of this study was to examine motor imagery ability in patients with peripheral nerve disorder using the hand mental rotation task. Five patients with left total avulsion brachial plexus palsy (BPP) and 16 healthy age-matched adults participated in this study. The mean±SD time from the injury was 103.
View Article and Find Full Text PDFα-1,3-Glucanase hydrolyzes α-1,3-glucan, an insoluble linear α-1,3-linked homopolymer of glucose that is found in the extracellular polysaccharides produced by oral streptococci in dental plaque and in fungal cell walls. This enzyme could be of application in dental care and the development of fungal cell-wall lytic enzymes, but its three-dimensional structure has not been available to date. In this study, the recombinant catalytic domain of α-1,3-glucanase FH1 from Paenibacillus glycanilyticus FH11, which is classified into glycoside hydrolase family 87, was prepared using a Brevibacillus choshinensis expression system and purified in a soluble form.
View Article and Find Full Text PDFStenotrophomonas maltophilia HS1 exhibits l-amino acid ester hydrolase (SmAEH) activity, which can synthesize dipeptides such as Ile-Trp, Val-Gly, and Trp-His from the corresponding amino acid methyl esters and amino acids. The gene encoding SmAEH was cloned and expressed in Escherichia coli and was purified and characterized. SmAEH shared 77% sequence identity with a known amino acid ester hydrolase (AEH) from Xanthomonas citri, which belongs to a class of β-lactam antibiotic acylases.
View Article and Find Full Text PDFA gene-encoding a dye-linked D-lactate dehydrogenase (Dye-DLDH) homolog was identified in the genome of the hyperthermophilic archaeon Thermoproteus tenax. The gene was expressed in Escherichia coli and the product was purified to homogeneity. The recombinant protein exhibited highly thermostable Dye-DLDH activity.
View Article and Find Full Text PDFThe orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH.
View Article and Find Full Text PDFMany kinds of NAD(P)-dependent L-amino acid dehydrogenases have been so far found and effectively used for synthesis of L-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of D-amino acid dehydrogenase (D-AADH) has not been achieved because useful D-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP-dependent D-AADH was created from -diaminopimelate dehydrogenase (-DAPDH).
View Article and Find Full Text PDFChitinase 1 (Chi1) is an acidic and thermostable hydrolytic enzyme capable of the breakdown of chitin, a resilient biopolymer that is the primary building block of fungi cell walls and marine exoskeletons. In this study, Chi1 was purified from the bacterium Streptomyces thermodiastaticus HF 3-3, and its properties were carefully characterized. The molecular mass of Chi1 was estimated to be approximately 46 kDa and, through sequencing, its N-terminal amino acid sequence was identified as ADSGKVKL.
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