Crystallization and Preliminary X-Ray Analysis of Subtilisin DY, a Natural Mutant of Subtilisin Carlsberg

Betzel, Christian; Visanji, Marcia; Eschenburg, Susanne; Wilson, K.S.; Peters, K.; Fittkau, S.; Singh, T. P.; Genov, Nicolay

doi:10.1006/abbi.1993.1245

Cited by 6 publications

(5 citation statements)

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“…A sequence analysis indicated that the DNA sequence encoding the 26‐kDa protein in Fig. 2b (DDBJ ) exhibited the greatest homology with the alkaline protease, subtilisin DY (Betzel et al. 1993).…”

Section: Resultsmentioning

confidence: 99%

Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein

et al. 2007

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Aims: The purpose of this paper was to screen candidate bacterial strains for the production of proteases suitable for application to the degradation of pathogenic forms of prion protein (PrPSc). This paper describes the biochemical characteristics and proteolytic activity of the isolated protease. Methods and Results: After screening more than 200 bacterial proteases for keratinolytic activity, we identified a Bacillus stain that produced a protease exhibiting high‐degradation activity against a scrapie PrPSc. Sequence analysis indicated that this serine‐protease belonged to the Subtilisin family and had optimum pH and temperature ranges of 9–10 and 60–70°C. Western blotting analysis revealed that the protease was also capable of decomposing bovine spongiform encephalopathy‐infected brain homogenate. In addition, the protease was demonstrated to degrade dried PrPSc that had become firmly attached to a plastic surface considerably more effectively than proteinase K or PWD‐1, a previously reported keratinase. Conclusions: These results indicate that the isolated protease exhibited higher activity for PrPSc degradation compared with other proteases examined. Significance and Impact of the Study: This protease could be used under moderate conditions for the decontamination of precision instruments that are susceptible to PrPSc contamination.

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Section: Resultsmentioning

confidence: 99%

Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein

et al. 2007

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“…TA41, subtilisin from Antarctic B. subtilis strain TA41 (Davail et al, 1992); SUBT. CARLSBERG, subtilsin Carlsberg (Betzel et al, 1993); SUBT. BACMS, subtilisin Bacms (Svendsen et al, 1986); SUBT.…”

Section: Phvsgalaliksyeeesfqrk--lsesevfaqlirrtlpldiakmentioning

confidence: 99%

“…J, subtilisin J (Jang et al, 1992); SUBT. DY, subtilisin DY (Betzel et al, 1993); SUBT. BPN', subtilisin BPN' (Pantoliano et al, 1989); ISP-I, ISP-1 (Rufo et al, 1990); B. ALCALOPHILUS, subtilisin from Bacillus alcalophilus (van der Laan et al, 1992); ELASTASE YAB, elastase YaB (Kaneko et al, 1989); BSUB MINOR PROT., minor protease from B. subtilis (Sloma et al, 1991); Aqualysin I, aqualysin I from Thermus aquaticus (Terada et al, 1990); PROTEINASE K, proteinase K from Tritirachium album (Gunkel & Gassen, 1989); Predictprotein, secondary structure prediction of Predictprotein algorithm for aerolysin; Thermitase 2", Proteinase K 2", Carlsberg 2", BPN' 2", secondary structures of thermitase, proteinase K, subtilisin Carlsberg, and subtilisin BPN'.…”

Section: Phvsgalaliksyeeesfqrk--lsesevfaqlirrtlpldiakmentioning

confidence: 99%

“…a Numbering is based on the first amino acid in the P. aerophilum aerolysin sequence alignment, rather than the position of the initiator methionine. The corresponding positions in thermitase (TM) from 7: vulgaris (Meloun et al, 1985), subtilisin Carlsberg (CB; after Betzel et al, 1993), and subtilisin TA41 from the Antarctic B. subtilis strain TA41 (Davail et al, 1992) are given.…”

Section: Phvsgalaliksyeeesfqrk--lsesevfaqlirrtlpldiakmentioning

confidence: 99%

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The sequence of a subtilisin‐type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability

et al. 1994

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The hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 "C and pH 7.0. Cell homogenates exhibit strong proteolytic activity within a temperature range of 80-130 "C. During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacihs sp. The total DNA sequence of the gene encoding the protease (named "aerolysin") was determined. Multiple sequence alignment with 15 different serine-type proteases showed greatest homology with subtilisins from gram-positive bacteria rather than archaeal or eukaryal serine proteases.Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P. aerophilurn subtilisin. This allowed identification of sites potentially contributing to the thermostability of the protein. One common transition put alanines at the beginning and end of surface alpha-helices. Aspartic acids were found at the N-terminus of several surface helices, possibly increasing stability by interacting with the helix dipole. Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.

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“…Crystallisation. As described earlier [22] crystals of subtilisin DY inhibited by N-benzyloxy-carbonyl-Ala-Pro-Phe-chloromethyl ketone were grown by hanging-drop vapour diffusion. The drops contained initially 20 mg/ml protein, 4% poly(ethylene glycol) 4000, 0.33 M NaCl, 1.5 mM CaCl 2 in 18 mM sodium citrate buffer, pH 6.0, and were equilibrated against reservoirs containing 10% poly(ethylene glycol) 4000, 1 M NaCl, 5 mM CaCl 2 in 50 mM sodium citrate, pH 6.0.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of subtilisin DY, a random mutant of subtilisin Carlsberg

Eschenburg

Genov

Peters³

et al. 1998

European Journal of Biochemistry

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The crystal structure of subtilisin DY inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone has been solved by molecular replacement with subtilisin Carlsberg as the starting model. The model has been refined to a crystallographic R factor (ϭ Σ | |F o | Ϫ |F c | | / Σ |F o |) of 15.1% using Xray diffraction data to 0.175 nm resolution. Subtilisin DY is an alkaline proteinase from the X-irradiated Japanese strain DY of Bacillus licheniformis, which normally produces subtilisin Carlsberg. It has very similar properties to subtilisin Carlsberg, with a slightly enhanced resistance to heat and guanidine hydrochloride-induced denaturation, in spite of the fact that the sequences of the two enzymes differ in 31 positions out of 274 residues. The close similarity in overall three-dimensional structure of subtilisins DY and Carlsberg and also their physicochemical properties, such as activity and stability, shows that nature aided by X-irradiation for rapid 'evolution' is able to accommodate considerable changes in sequence without substantial changes in property.Keywords : subtilisin; X-ray structure ; sequence variability; X-irradiation; mutagenesis.Subtilisins form a well-characterised family of extracellular alkaline serine proteinases, which have been found in various species of Bacillus [1], as well as in fungi and yeasts and even in higher eukaryotes, such as worms, insects and plants [2]. The increasing interest over the last decades in these enzymes has been due to the biological and industrial significance of their proteolytic activity. Since subtilisins have a very broad substrate specificity and are highly stable at neutral and alkaline pH, they are of considerable industrial importance as protein-degrading additives to detergents. Subtilisin DY is secreted by an X-ray irradiated strain of Bacillus licheniformis [12] and was isolated in a homogeneous state [13]. Sequence studies show high similarity of subtilisin DY to subtilisin Carlsberg [14], with 29 substitutions out of 274 residues (but see below). Comparing the physicochemical properties of the two enzymes, subtilisin DY is less susceptible to oxidation in the presence of hematoporphyrin [15] and shows slightly enhanced stability to heat and guanidine hydrochlorideinduced denaturation [16]. The melting temperature of subtilisin DY is 4°C higher than that of the wild-type Carlsberg. The kinetic parameters for the hydrolysis of succinyl-Ala 2 -Phe-methylcoumarylamide [17] and several peptidyl-4-nitroanilides [18] do not differ significantly from the kinetic constants for subtilisin Carlsberg. Co-crystallisation experiments with subtilisin DY and the proteinase inhibitor eglin-c [19] suggested that DY hydrolyses the peptide bond between residues 45 and 46 in the reactive centre of eglin-c, unlike other known subtilisins including Carlsberg.In this paper, we describe the 0.175-nm resolution analysis of the crystal structure of subtilisin DY in a 1:1 complex with the synthetic inhibitor N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone. For sub...

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Crystallization and Preliminary X-Ray Analysis of Subtilisin DY, a Natural Mutant of Subtilisin Carlsberg

Cited by 6 publications

References 0 publications

Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein

Characterization of a proteolytic enzyme derived from a Bacillus strain that effectively degrades prion protein

The sequence of a subtilisin‐type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability

Crystal structure of subtilisin DY, a random mutant of subtilisin Carlsberg

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