Directed molecular evolution of cytochrome c peroxidase.
Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed molecular evolution to generate mutants with increased activity against the classical peroxidase substrate guaiacol, thus changing the substrate specificity of CCP from the protein cytochrome c to a small organic molecule. After three rounds of DNA shuffling and screening, mutants were isolated which possessed a 300-fold increased activity against guaiacol and an up to 1000-fold increased specificity for this substrate relative to that for the natural substrate. In all of the selected mutants, the distal arginine (Arg48), which is fully conserved in the superfamily of peroxidases, was mutated to histidine, showing that this mutation plays a key role in the significant increase in activity against phenolic substrates. The results suggest that, in addition to stabilizing the reactive intermediate compound I, the distal arginine plays an important role as a gatekeeper in the active site of CCP, controlling the access to the ferryl oxygen and the distal histidine. Other isolated mutations increase the general reactivity of the peroxidase or increase the intracellular concentration of the active holo form, allowing their selection under the employed screening conditions. The results illustrate the ability of directed molecular evolution technologies to deliver solutions to biochemical problems that would not be readily predicted by rational design.
Iffland
A
A
Institut de Chimie Organique and Institut de Chimie Minerale et Analytique, Université de Lausanne, CH-1015 Lausanne, Switzerland.
Tafelmeyer
P
P
Saudan
C
C
Johnsson
K
K
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D003578:Cytochrome-c Peroxidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods*; D004789:Enzyme Activation / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors / Q000138:chemical synthesis; D006139:Guaiacol / Q000737:chemistry; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D013912:Threonine; D001224:Aspartic Acid; D014443:Tyrosine; D006139:Guaiacol; D001216:Asparagine; D003578:Cytochrome-c Peroxidase; D000409:Alanine