title: Mutants of Micromonospora viridifaciens sialidase have highly variable activities on natural and non-natural substrates.
abstract: This study aimed to improve the hydrolase activity of the well-characterised bacterial sialidase from Micromonospora viridifaciens. The enzyme and its mutated versions were produced in Bacillus subtilis and secreted to the growth medium. Twenty amino acid positions in or near the active site were subjected to site-saturation mutagenesis and evaluated on the artificial sialidase substrate 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid and on the natural substrate casein glycomacropeptide. A considerably higher fraction of the mutants exhibited increased activity on the artificial substrate compared with the natural one, with the most proficient mutant showing a 13-fold improvement in kcat/Km. In contrast, no mutants displayed more than a 2-fold increase in activity on the natural substrate. To gain further insight into this important discrepancy, we analysed the stability of mutants using the PoPMuSiC software, a property that also correlates with the potential for introducing chemical variation, after validating the method with a set of experimental stability estimates. We found a significant correlation between improved hydrolase activity on the artificial substrate and reduced apparent stability. Together with the minor improvement on the natural substrate this shows an important difference between naturally evolved functionality and new laboratory functionality. Our results suggest that when engineering sialidases and potentially other proteins towards non-natural substrates that are not optimized by natural evolution, major changes in chemical properties are advantageous, and these changes tend to correlate with decreased stability, partly explaining commonly observed trade-offs between stability and proficiency.
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800 Kgs. Lyngby, Denmark cj@kt.dtu.dk.
authors.forename: C
authors.initials: C
authors.lastname: Jers
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800 Kgs. Lyngby, Denmark.
authors.forename: Y
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Chemistry, Technical University of Denmark, Building 206, 2800 Kgs. Lyngby, Denmark.
authors.forename: K P
authors.initials: KP
authors.lastname: Kepp
authors.affiliation: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800 Kgs. Lyngby, Denmark.
authors.forename: J D
authors.initials: JD
authors.lastname: Mikkelsen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D008847:Micromonospora / Q000201:enzymology* / Q000235:genetics; D009112:Muramic Acids / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D009439:Neuraminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009112:Muramic Acids; C031651:N-acetylmuramic acid; D009439:Neuraminidase
keywords: Micromospora viridifaciens; mutagenesis; protein evolution; sialidase; stability–activity trade-off