Crystallization and preliminary X-ray diffraction studies of an alkaline protease from Bacillus lentus

Betzel, Christian; Dauter, Zbigniew; Dauter, Mirosława; Ingelman, Margareta; Papendorf, Gerlind; Wilson, K.S.; Branner, Sven

doi:10.1016/0022-2836(88)90372-5

Cited by 24 publications

(10 citation statements)

References 13 publications

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“…(2011). D67, 331-337 (Calderone, 2004), 1rgg (Sevcik et al, 1996), 1m32 (Chen et al, 2002) and a subtilisin data set (Betzel et al, 1988;Dauter et al, 2002). Data where a program terminated abnormally in either pipeline were excluded from the statistics and graphs presented, resulting in 116 data sets.…”

Section: Appendix a Data Setsmentioning

confidence: 99%

Recent advances in theCRANKsoftware suite for experimental phasing

Pannu

Waterreus

Skubák

et al. 2011

Acta Crystallogr D Biol Crystallogr

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For its first release in 2004, CRANK was shown to effectively detect and phase anomalous scatterers from single‐wavelength anomalous diffraction data. Since then, CRANK has been significantly improved and many more structures can be built automatically with single‐ or multiple‐wavelength anomalous diffraction or single isomorphous replacement with anomalous scattering data. Here, the new algorithms that have been developed that have led to these substantial improvements are discussed and CRANK's performance on over 100 real data sets is shown. The latest version of CRANK is freely available for download at http://www.bfsc.leidenuniv.nl/software/crank/ and from CCP4 (http://www.ccp4.ac.uk/).

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Section: Appendix a Data Setsmentioning

confidence: 99%

Recent advances in theCRANKsoftware suite for experimental phasing

Pannu

Waterreus

Skubák

et al. 2011

Acta Crystallogr D Biol Crystallogr

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“…This crystal form was first reported by Betzel et al (1988) and a three-dimensional structure (PDB entry 1ndq) was solved at 1.8 Å resolution by Pan et al (2003). The present structure of Savinase in the 1ndq-like crystal form is, as expected, very similar to others published for the wild-type enzyme, with r.m.s.d.s of 0.25 Å with A1, 0.44 Å with A2 and, not surprisingly, the smallest, 0.19 Å with PDB entry 1ndq.…”

Section: Structure Of the 1ndq-like Crystalmentioning

confidence: 55%

“…The highly purified Savinase was buffered in 50 mM H 3 BO 3 , 5 mM dimethylglutaric acid, 1 mM CaCl 2 , 100 mM NaCl pH 6.0. The conditions reported by Betzel et al (1988) were used. Crystals were grown in 15 ml hanging drops consisting of 20 mg ml À1 protein, 4% PEG 4000, 0.33 M NaCl, 1.5 mM CaCl 2 , 18 mM citrate buffer pH 6.0.…”

Section: Controlled Growth Of Single Crystals In the 1ndq-like Crystamentioning

confidence: 99%

Analysis of an industrial production suspension ofBacillus lentussubtilisin crystals by powder diffraction: a powerful quality-control tool

Frankær

Moroz

Turkenburg

et al. 2014

Acta Cryst D Biol Crystallogr

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A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Å and a = 52.72, b = 57.13, c = 65.86 Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Å with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.

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“…Each pair of kinetic parameters was calculated from at least 1 4 initial velocities measured at 7 substrate concen- With dimethyl casein as a substrate the proteolytic activity was determined as previously described [13]. The method is based on the digestion of a dimethyl casein solution by the enzyme at 50 "C, pH 8.0. The content of primary amino groups formed is determined, after reaction with the color reagent 2,4,6-trinitrobenzene sulfonic acid, at 410 nm on a Technicon AutoAnalyzer 11.…”

Section: Determination Of Enzyme Activity and Kinetic Constantsmentioning

confidence: 99%

“…We show here how the Met222 of a subtilisin from Bacillus lentus, i.e. sub-tilisin 309 [8], can be replaced with a group stable towards oxidation, without substantially reducing the activity. The approach is to combine site-directed mutagenesis with chemical modification as previously described for carboxypeptidase y 191.…”

mentioning

confidence: 99%

A highly active and oxidation‐resistant subtilisin‐like enzyme produced by a combination of site‐directed mutagenesis and chemical modification

Grøn

Bech

Branner

et al. 1990

European Journal of Biochemistry

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The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1 -10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222.It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H 2 0 z revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cy~~~~lsubtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cy~~~~]subtilisin, exhibited a kca,/Km value of 56% ofthat obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-N02 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cy~~~~]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kca,lKm for the hydrolysis of Suc-Ala-AlaPro-Phe-NH-Ph-NO,. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.The subtilisin family is currently being studied in several laboratories by in vitro mutagenesis. Single amino acid substitutions have resulted in enzyme derivatives with altered specificity [l], increased activity [2], altered pH dependence [3], improved alkaline stability [4] and altered stability towards oxidation [5].Sensitivity of subtilisins to oxidation has been reported and chemical analysis as well as X-ray crystallography has revealed that the apparent inactivation can be ascribed to the conversion of the methionyl residue, neighboring the cataCorrespondence to K. Breddam,

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Crystallization and preliminary X-ray diffraction studies of an alkaline protease from Bacillus lentus

Cited by 24 publications

References 13 publications

Recent advances in theCRANKsoftware suite for experimental phasing

Recent advances in theCRANKsoftware suite for experimental phasing

Analysis of an industrial production suspension ofBacillus lentussubtilisin crystals by powder diffraction: a powerful quality-control tool

A highly active and oxidation‐resistant subtilisin‐like enzyme produced by a combination of site‐directed mutagenesis and chemical modification

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