The thioredoxin-like activity and calcium binding ability of the short-range disulfide in bovine pancreatic deoxyribonuclease

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Bovine pancreatic deoxyribonuclease (bpDNase) is the best-characterized DNase. It is composed of 260 amino acids, and is a glycoprotein with a molecular weight of 31 kDa. One large (C173-C209) and one small (C101-C104) disulfide loops were found in bpDNase. Earlier studies showed that the large disulfide loop was responsible for the enzyme conformation. When the large disulfide loop was reduced, bpDNase lost its enzyme activity. In contrast, reduction of the small loop resulted in an enzyme with the full activity. However, sequence alignment of DNases from various species revealed that this small loop was still highly conserved among species. Moreover, because the structure-based sequence alignment revealed homology between the small disulfide loop and the active site motif (-CXXC-) of thioredoxin, we are interested in seeking the possible biological roles of the small loop in bpDNase. Although not related to enzyme activity, the nonessential disulfide C101-C104 might be very important for other unknown functions. The importance of this disulfide also can be found in conservative sequences of various DNases. According to our recent studies, the reduced bpDNase actually contained the thioredoxin-like activity based on the rate of insulin precipitation assay. In order to gain further insight into the biological functions of the small loop, four double (E102G/S103P、E102P/S103G、G100K/G105W、G100W/G105K) and two quadruple (G100K/E102P/S103G/G105W、G100W/E102G/S103P/G105K) mutants were constructed using site-directed mutagenesis. Recombinant proteins were expressed in E. coli strain BL21(DE3)pLysE and were purified through a SOURCE 15Q anion and a S HyperD cation-exchange columns. SDS-PAGE with silver stain confirmed the homogeneity of the purified brDNase variants. Most of the recombinant proteins possess similar specific activities as the wild type bpDNase. However, quadruple mutant KPGW exhibited only half of the activity. CD spectra analysis also revealed significant different for this mutant. In our studies, we found that all these brDNase variants were able to accelerate the rate of insulin precipitation. And the highest thioredoxin-like activity (66%) of the quadruple mutant WGPK suggests that the conserved sequence (-WCGPCK-) of thioredoxin is crucial for its activity. Previous studies also showed that these two disulfide bonds were correlated with the two calcium binding sites of bpDNase, site I and site II. It was shown that the binding of calcium of site II is responsible for the conformation of the loose loop, C101-C104. We found that among brDNase variants which were mutated around the small loop only the reversed-sequence quadruple mutant KPGW presented a weaker binding ability, probably due to the alteration of its secondary structure.