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Decoding Essential Amino Acid Residues in the Substrate Groove of a Non-Specific Nuclease from Pseudomonas syringae

Miltenyi Biotec B.V. & Co. KG, Friedrich-Ebert-Straße 68, 51429 Bergisch Gladbach, Germany
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Catalysts 2019, 9(11), 941; https://doi.org/10.3390/catal9110941
Received: 17 October 2019 / Revised: 4 November 2019 / Accepted: 6 November 2019 / Published: 9 November 2019
(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)
Non-specific nucleases (NSN) are of interest for biotechnological applications, including industrial downstream processing of crude protein extracts or cell-sorting approaches in microfabricated channels. Bacterial nucleases belonging to the superfamily of phospholipase D (PLD) are featured for their ability to catalyze the hydrolysis of nucleic acids in a metal-ion-independent manner. In order to gain a deeper insight into the composition of the substrate groove of a NSN from Pseudomonas syringae, semi-rational mutagenesis based on a structure homology model was applied to identify amino acid residues on the protein’s surface adjacent to the catalytic region. A collection of 12 mutant enzymes each with a substitution to a positively charged amino acid (arginine or lysine) was produced in recombinant form and biochemically characterized. Mutations in close proximity to the catalytic region (inner ring) either dramatically impaired or completely abolished the enzymatic performance, while amino acid residues located at the border of the substrate groove (outer ring) only had limited or no effects. A K119R substitution mutant displayed a relative turnover rate of 112% compared to the original nuclease. In conclusion, the well-defined outer ring of the substrate groove is a potential target for modulation of the enzymatic performance of NSNs belonging to the PLD superfamily. View Full-Text
Keywords: DNase; kinetic profiles; RNase; semi-rational mutagenesis; substrate specificity DNase; kinetic profiles; RNase; semi-rational mutagenesis; substrate specificity
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Schwardmann, L.S.; Schmitz, S.; Nölle, V.; Elleuche, S. Decoding Essential Amino Acid Residues in the Substrate Groove of a Non-Specific Nuclease from Pseudomonas syringae. Catalysts 2019, 9, 941.

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