Importance of Conformational Variability in Protein Engineering of Subtilisin

Bott, R.; Ultsch, Mark; Wells, J.A.; Powers, David B.; Burdick, Daniel J.; Struble, Martin; Burnier, John; Estell, David A.; Miller, Jeffrey H; Graycar, Thomas P.; Adams, Rhys M.; Power, Scott D.

doi:10.1021/bk-1987-0334.ch011

Cited by 34 publications

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“…In 1969 Stauffer and Etson showed that the drop in activity of subtilisin Carlsberg upon oxidation was due to the oxidation of Met222 to a methionine sulfoxide [6]. This methionyl residue is positioned next to Ser221 which is part of the catalytic triad [7,141. In 1988 Bott et al solved the three-dimensional structure of a subtilisin from Bacillus amyloliguefaciens and analyzed the structural consequences of peroxide inactivation [7].…”

Section: Discussionmentioning

confidence: 99%

“…This methionyl residue is positioned next to Ser221 which is part of the catalytic triad [7,141. In 1988 Bott et al solved the three-dimensional structure of a subtilisin from Bacillus amyloliguefaciens and analyzed the structural consequences of peroxide inactivation [7]. Of the five methionines, only Met222 and Met124 were fully oxidized.…”

Section: Discussionmentioning

confidence: 99%

“…Prolonged exposure to high concentrations of H 2 0 2 affects all the enzymes described. However, this may be attributed to oxidative damage at positions other than 222, such as oxidation of tyrosyl and other methionyl residues [7].…”

Section: Discussionmentioning

confidence: 99%

“…It has been suggested that the decrease in catalytic efficiency is due to unfavorable interactions at the oxyanion hole between the sulfoxide group of the oxidized methionyl residue and the carbonyl oxygen of the scissile bond of the bound peptide substrate [7].…”

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…amyloliquefaciens have been determined previously (subtilisin BPN' [8], subtilisin Novo [9], subtilisin BAS [10]). For subtilisin Carlsberg from B. subtilis the crystal structure of the native enzyme [11] and the complex formed with the inhibitor eglin C [12] were reported.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of an alkaline protease from Bacillus alcalophilus at 2.4Åresolution

et al. 1990

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The crystal structure of an alkaline protease from Bacillus alcalophilus has been determined by X-ray diffraction at 2.4 ,~ resolution. The enzyme crystallizes in space group P212~21 with lattice constants a= 53.7, b= 61.6, c=75.9 .&. The structure was solved by molecular replacement using the structure of subtilisin Carlsberg as search model. Refinement using molecular dynamics and restrained least squares methods results in a crystallographic R-factor of 0.185. The tertiary structure is very similar to that of subtilisin Carlsberg. The greatest structural differences occur in loops at the surface of the protein.

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Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering

2017

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Generally, the catalytic activity of subtilisin Carlsberg (SC) for transacylation reactions with secondary alcohols in organic solvent is low. Enzyme immobilization and protein engineering was performed to improve the enantioselectivity of SC towards secondary alcohols. Possible amino-acid residues for mutagenesis were found by combining available literature data with molecular modeling. SC variants were created by site-directed mutagenesis and were evaluated for a model transacylation reaction containing 1-phenylethanol in THF. Variants showing high E values (>100) were found. However, the conversions were still low. A second mutation was made, and both the E values and conversions were increased. Relative to that shown by the wild type, the most successful variant, G165L/M221F, showed increased conversion (up to 36 %), enantioselectivity (E values up to 400), substrate scope, and stability in THF.

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Importance of Conformational Variability in Protein Engineering of Subtilisin

Cited by 34 publications

References 0 publications

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

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

Crystal structure of an alkaline protease from Bacillus alcalophilus at 2.4Åresolution

Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering

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