Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case.
Thermostability is a key feature for commercially attractive variants of the fungal enzyme phytase. In an initial set of experiments, we restored ionic interactions and hydrogen bonds on the surface of Aspergillus terreus phytase, which are present in the homologous but more thermostable enzyme from A. niger. Since these mutations turned out to be neutral, we replaced-in the same region and based on the crystal structure of A. niger phytase-entire secondary structure elements. The replacement of one alpha-helix on the surface of A. terreus phytase by the corresponding stretch of A. niger phytase resulted in an enzyme with improved thermostability and unaltered enzymatic activity. Surprisingly, the thermostability of this hybrid protein was very similar to that of A. niger phytase, although the fusion protein contained only a 31 amino acid stretch of the more stable parent enzyme. This report provides evidence that structure-based chimeric enzymes can be used to exploit the evolutionary information within a sequence alignment. We propose this method as an alternative to directed enzyme evolution if due to expression constraints the screening of large mutant populations is not feasible.
Jermutus
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F. Hoffmann-La Roche, Vitamins and Fine Chemicals Division, 4070, Basel, Switzerland.
Tessier
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Pasamontes
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van Loon
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AP
Lehmann
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D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D006860:Hydrogen Bonding; D007477:Ions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013499:Surface Properties
D005656:Fungal Proteins; D007477:Ions; D011993:Recombinant Fusion Proteins; D010744:Phosphoric Monoester Hydrolases; C400321:3-phytase