A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure-guided recombination.
Sequence diversity within a family of functional enzymes provides a platform for elucidating structure-function relationships and for protein engineering to improve properties important for applications. Access to nature's vast sequence diversity is often limited by the fact that only a few enzymes have been characterized in a given family. Here, we recombined the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48; EC 3.2.1.176) - Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS - to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. Within this set, we found large variations in properties such as the functional temperature range, stability, and specific activity on crystalline cellulose. We showed that functional status and stability were predictable from simple linear models of the sequence-property data: recombined protein fragments contributed additively to these properties in a given chimera. Using this, we correctly predicted sequences that were as stable as any of the native Cel48 enzymes described to date. The characterization of 60 active Cel48 chimeras expands the number of characterized Cel48 enzymes from 13 to 73. Our work illustrates the role that structure-guided recombination can play in helping to identify sequence-function relationships within a family of enzymes by supplementing natural diversity with synthetic diversity.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Matthew A
MA
Smith
Andrea
A
Rentmeister
Christopher D
CD
Snow
Timothy
T
Wu
Mary F
MF
Farrow
Florence
F
Mingardon
Frances H
FH
Arnold
D020134:Catalytic Domain; D044602:Cellulases / Q000737:chemistry / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation
D044602:Cellulases; D006026:Glycoside Hydrolases