title: Construction and characterization of a bifunctional enzyme with deoxyribonuclease I and thioredoxin-like activities.
abstract: One large essential (C173-C209) and one small nonessential (C101-C104) disulfide loops occur in bovine pancreatic deoxyribonuclease I (bpDNase I). In our recent study, the reduced nonessential disulfide (-CESC-), which is structurally homologous to the active-site motif (-CGPC-) of thioredoxin, was shown to have thioredoxin-like activity. In order to gain further insight into the potential redox activity of the nonessential disulfide in bpDNase I, four double (GP, PG, WK, and KW) and two quadruple (WGPK, KPGW) mutants were constructed. Most of the mutant enzymes possess similar specific DNase activities as that of WT bpDNase I, while KPGW exhibited only half of the activity, possibly due to gross structural alteration, as revealed by CD analysis. All these mutants were able to accelerate the rate of insulin precipitation. The highest thioredoxin-like activity (66%) measured for WGPK indicated that the conserved sequence (-WCGPCK-) of thioredoxin is crucial for its redox activity. Our results suggested that engineering of the nonessential disulfide in bpDNase I was able to generate a novel bifunctional enzyme with enhanced disulfide/dithiol exchange reactivity, while retaining its full DNA-hydrolyzing activity.
authors: Institute of Biotechnology, College of Bioresources, National Ilan University, Ilan 26047, Taiwan. wjchen@niu.edu.tw
authors: Wei-Jung
authors: WJ
authors: Chen
authors: Ting
authors: T
authors: Lo
authors: Yu-Shen
authors: YS
authors: Lai
authors: Po-Tsang
authors: PT
authors: Huang
authors: Chai-Ching
authors: CC
authors: Lin
authors: Ta-Hsiu
authors: TH
authors: Liao
mesh_terms: D000818:Animals; D002417:Cattle; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004220:Disulfides; D013879:Thioredoxins; D003850:Deoxyribonuclease I