Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold.
In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the alpha/beta-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the alpha/beta-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.
Altamirano
M M
MM
Cambridge Centre for Protein Engineering, and Cambridge University Chemical Laboratory, MRC Centre, UK.
Blackburn
J M
JM
Aguayo
C
C
Fersht
A R
AR
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007209:Indole-3-Glycerol-Phosphate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017510:Protein Folding
D007209:Indole-3-Glycerol-Phosphate Synthase; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase