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Subtilisin-like Serine Proteases (subtilisin-like + serine_protease)
Selected AbstractsPurification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leavesPHYSIOLOGIA PLANTARUM, Issue 4 2003Irma N. Roberts A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent Km and Vmax for the peptide were 1.18 mm and 2.27 mmol pNA mg,1 h,1, respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60°C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65,75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process. [source] Crystallization and preliminary X-ray analysis of carnein, a serine protease from Ipomoea carneaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Ashok Kumar Patel Carnein is an 80,kDa subtilisin-like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the first crystal structure of a plant subtilisin and to gain insight into the structural determinants underlying its remarkable stability, carnein was isolated from I. carnea latex, purified and crystallized by the hanging-drop vapour-diffusion method. A data set was collected to 2.0,Å resolution in-house from a single crystal at 110,K. The crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 126.9, c = 84.6,Å, , = , = 90, , = 120°. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient is 2.46,Å3,Da,1, corresponding to a solvent content of 50%. Structure determination of the enzyme is in progress. [source] Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leavesPHYSIOLOGIA PLANTARUM, Issue 4 2003Irma N. Roberts A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent Km and Vmax for the peptide were 1.18 mm and 2.27 mmol pNA mg,1 h,1, respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60°C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65,75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process. [source] A subtilisin-like serine protease essential for mucilage release from Arabidopsis seed coatsTHE PLANT JOURNAL, Issue 3 2008Carsten Rautengarten Summary During Arabidopsis seed development large quantities of mucilage, composed of pectins, are deposited into the apoplast underneath the outer wall of the seed coat. Upon imbibition of mature seeds, the stored mucilage expands through hydration and breaks the outer cell wall that encapsulates the whole seed. Mutant seeds carrying loss-of-function alleles of AtSBT1.7 that encodes one of 56 Arabidopsis thaliana subtilisin-like serine proteases (subtilases) do not release mucilage upon hydration. Microscopic analysis of the mutant seed coat revealed no visible structural differences compared with wild-type seeds. Weakening of the outer primary wall using cation chelators triggered mucilage release from the seed coats of mutants. However, in contrast to mature wild-type seeds, the mutant's outer cell walls did not rupture at the radial walls of the seed coat epidermal cells, but instead opened at the chalazal end of the seed, and were released in one piece. In atsbt1.7, the total rhamnose and galacturonic acid contents, representing the backbone of mucilage, remained unchanged compared with wild-type seeds. Thus, extrusion and solubility, but not the initial deposition of mucilage, are affected in atsbt1.7 mutants. AtSBT1.7 is localized in the developing seed coat, indicating a role in testa development or maturation. The altered mode of rupture of the outer seed coat wall and mucilage release indicate that AtSBT1.7 triggers the accumulation, and/or activation, of cell wall modifying enzymes necessary either for the loosening of the outer primary cell wall, or to facilitate swelling of the mucilage, as indicated by elevated pectin methylesterase activity in developing atsbt1.7 mutant seeds. [source] Crystallization of the virulent and benign subtilisin-like proteases from the ovine footrot pathogen Dichelobacter nodosusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Wilson Wong Dichelobacter nodosus is the principal causative agent of ovine footrot, a disease of significant economic importance to the sheep industry. D. nodosus secretes a number of subtilisin-like serine proteases which mediate tissue damage and presumably contribute to the pathogenesis of footrot. Strains causing virulent footrot secrete the proteases AprV2, AprV5 and BprV and strains causing benign footrot secrete the closely related proteases AprB2, AprB5 and BprB. Here, the cloning, purification and crystallization of AprV2, AprB2, BprV and BprB are reported. Crystals of AprV2 and AprB2 diffracted to 2.0 and 1.7,Å resolution, respectively. The crystals of both proteases belonged to space group P1, with unit-cell parameters a = 43.1, b = 46.0, c = 47.2,Å, , = 97.8, , = 115.2, , = 115.2° for AprV2 and a = 42.7, b = 45.8, c = 45.7,Å, , = 98.4, , = 114.0, , = 114.6° for AprB2. Crystals of BprV and BprB diffracted to 2.0 and 1.8,Å resolution, respectively. The crystals of both proteases belonged to space group P21, with unit-cell parameters a = 38.5, b = 89.6, c = 47.7,Å, , = 113.6° for BprV and a = 38.5, b = 90.5, c = 44.1,Å, , = 109.9° for BprB. The crystals of all four proteases contained one molecule in the asymmetric unit, with a solvent content ranging from 36 to 40%. [source] Preliminary crystallographic study of two cuticle-degrading proteases from the nematophagous fungi Lecanicillium psalliotae and Paecilomyces lilacinusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Fengping Ye Cuticle-degrading proteases are extracellular subtilisin-like serine proteases that are secreted by entomopathogenic and nematophagous fungi. These proteases can digest the host cuticle during invasion of an insect or nematode and serve as a group of important virulence factors during the infection of nematodes by nematophagous fungi. To elucidate the mechanism of interaction between the proteases and the nematode cuticle, two cuticle-degrading proteases, Ver112 from Lecanicillium psalliotae (syn. Verticillium psalliotae) and PL646 from Paecilomyces lilacinus, were studied. The Ver112 protein and the complex between PL646 and the substrate-like tetrapeptide inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MSU-AAPV) were crystallized using the hanging-drop vapour-diffusion method at 289,K. The crystals were analyzed by X-ray diffraction to resolutions of 1.65 and 2.2,Å, respectively. These analyses identified that crystals of Ver112 belonged to space group P212121, with unit-cell parameters a = 43.7, b = 67.8, c = 76.3,Å, , = , = , = 90°. In contrast, crystals of the PL646,MSU-AAPV complex belonged to space group P21, with unit-cell parameters a = 65.1, b = 62.5, c = 67.6,Å, , = 92.8°. [source] | ||||||||||