Investigating the structural and functional consequences of circular permutation on lipase B from Candida antarctica.
The engineering of lipase B from Candida antarctica (CALB) by circular permutation has yielded over sixty hydrolase variants, and several show significantly improved catalytic performance. Here we report a detailed characterization of ten selected enzyme variants by kinetic and spectroscopic methods to further elucidate the impact of circular permutation on the structure and function of CALB. Our experiments identify lipase variants with up to 175-fold enhanced k(cat)/K(M) values over wild-type. In addition, circular permutation does not change the enzymes' enantiopreference and preserves or even improves their enantioselectivity compared to that of the wild-type enzyme. Finally, our spectroscopic analyses suggest that the structural effects of circular permutation on CALB are mostly local, concentrating on regions near the native and new protein termini. The observed changes in secondary structure and protein thermostability vary among enzyme variants but directly correlate with the locations of the new termini, a first step towards a predictive framework.
Qian
Zhen
Z
Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA.
Fields
Christina J
CJ
Lutz
Stefan
S
D000438:Alcohols / Q000737:chemistry; D001665:Binding Sites; D002175:Candida / Q000201:enzymology*; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism / Q000379:methods; D005656:Fungal Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D015394:Molecular Structure; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D000438:Alcohols; D002264:Carboxylic Acids; D005656:Fungal Proteins; D007527:Isoenzymes; D008049:Lipase; C431349:lipase B, Candida antarctica