SCHEMA-designed variants of human Arginase I and II reveal sequence elements important to stability and catalysis.
Arginases catalyze the divalent cation-dependent hydrolysis of L-arginine to urea and L-ornithine. There is significant interest in using arginase as a therapeutic antineogenic agent against L-arginine auxotrophic tumors and in enzyme replacement therapy for treating hyperargininemia. Both therapeutic applications require enzymes with sufficient stability under physiological conditions. To explore sequence elements that contribute to arginase stability we used SCHEMA-guided recombination to design a library of chimeric enzymes composed of sequence fragments from the two human isozymes Arginase I and II. We then developed a novel active learning algorithm that selects sequences from this library that are both highly informative and functional. Using high-throughput gene synthesis and our two-step active learning algorithm, we were able to rapidly create a small but highly informative set of seven enzymatically active chimeras that had an average variant distance of 40 mutations from the closest parent arginase. Within this set of sequences, linear regression was used to identify the sequence elements that contribute to the long-term stability of human arginase under physiological conditions. This approach revealed a striking correlation between the isoelectric point and the long-term stability of the enzyme to deactivation under physiological conditions.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Philip A
PA
Romero
Everett
E
Stone
Candice
C
Lamb
Lynne
L
Chantranupong
Andreas
A
Krause
Aleksandr E
AE
Miklos
Randall A
RA
Hughes
Blake
B
Fechtel
Andrew D
AD
Ellington
Frances H
FH
Arnold
George
G
Georgiou
D000465:Algorithms; D001119:Arginase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D017076:Computer-Aided Design; D004795:Enzyme Stability; D006801:Humans; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology
D011993:Recombinant Fusion Proteins; C574619:ARG1 protein, human; C477631:ARG2 protein, human; D001119:Arginase