title: Kinetic Characterization of 100 Glycoside Hydrolase Mutants Enables the Discovery of Structural Features Correlated with Kinetic Constants.
abstract: The use of computational modeling algorithms to guide the design of novel enzyme catalysts is a rapidly growing field. Force-field based methods have now been used to engineer both enzyme specificity and activity. However, the proportion of designed mutants with the intended function is often less than ten percent. One potential reason for this is that current force-field based approaches are trained on indirect measures of function rather than direct correlation to experimentally-determined functional effects of mutations. We hypothesize that this is partially due to the lack of data sets for which a large panel of enzyme variants has been produced, purified, and kinetically characterized. Here we report the kcat and KM values of 100 purified mutants of a glycoside hydrolase enzyme. We demonstrate the utility of this data set by using machine learning to train a new algorithm that enables prediction of each kinetic parameter based on readily-modeled structural features. The generated dataset and analyses carried out in this study not only provide insight into how this enzyme functions, they also provide a clear path forward for the improvement of computational enzyme redesign algorithms.
authors.affiliation: Biophysics Graduate Group, University of California Davis, California, United States of America.
authors.forename: Dylan Alexander
authors.initials: DA
authors.lastname: Carlin
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Ryan W
authors.initials: RW
authors.lastname: Caster
authors.affiliation: Department of Biomedical Engineering, University of California Davis, Davis, California, United States of America.
authors.forename: Xiaokang
authors.initials: X
authors.lastname: Wang
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Betzenderfer
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Claire X
authors.initials: CX
authors.lastname: Chen
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Veasna M
authors.initials: VM
authors.lastname: Duong
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Carolina V
authors.initials: CV
authors.lastname: Ryklansky
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Alp
authors.initials: A
authors.lastname: Alpekin
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Nathan
authors.initials: N
authors.lastname: Beaumont
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Harshul
authors.initials: H
authors.lastname: Kapoor
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Nicole
authors.initials: N
authors.lastname: Kim
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Hosna
authors.initials: H
authors.lastname: Mohabbot
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Boyu
authors.initials: B
authors.lastname: Pang
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Rachel
authors.initials: R
authors.lastname: Teel
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.
authors.forename: Lillian
authors.initials: L
authors.lastname: Whithaus
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.|Department of Computer Science, University of California Davis, Davis, California, United States of America.
authors.forename: Ilias
authors.initials: I
authors.lastname: Tagkopoulos
authors.affiliation: Genome Center, University of California Davis, Davis, California, United States of America.|Department of Chemistry, University of California Davis, Davis, California, United States of America.|Department of Biochemistry & Molecular Medicine, University of California Davis, Davis, California, United States of America.
authors.forename: Justin B
authors.initials: JB
authors.lastname: Siegel
mesh_terms: D003198:Computer Simulation; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D006026:Glycoside Hydrolases
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