Enzyme redesign: two mutations cooperate to convert cycloartenol synthase into an accurate lanosterol synthase.
Efforts to modify the catalytic specificity of enzymes consistently show that it is easier to broaden the substrate or product specificity of an accurate enzyme than to restrict the selectivity of one that is promiscuous. Described herein are experiments in which cycloartenol synthase was redesigned to become a highly accurate lanosterol synthase. Several single mutants have been described that modify the catalytic specificity of cycloartenol to form some lanosterol. Modeling studies were undertaken to identify combinations of mutations that cooperate to decrease the formation of products other than lanosterol. A double mutant was constructed and characterized and was shown to cyclize oxidosqualene accurately to lanosterol (99%). This catalytic change entailed both relocating polarity with a His477Asn mutation and modifying steric constraints with an Ile481Val mutation.
Lodeiro
Silvia
S
Department of Chemistry and Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA.
Schulz-Gasch
Tanja
T
Matsuda
Seiichi P T
SP
D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008968:Molecular Conformation; D016296:Mutagenesis; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013185:Squalene / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry
C002821:2,3-oxidosqualene; D013185:Squalene; D019751:Intramolecular Transferases; C021924:lanosterol synthase; C021923:cycloartenol synthase