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Papain is a plant cysteine protease of industrial importance having a two-domain structure with its catalytic cleft located at the domain interface. A structure-based rational design approach has been used to improve the thermostability of papain, without perturbing its enzymatic activity, by introducing three mutations at its interdomain region. A thermostable homologue in papain family, Ervatamin C, has been used as a template for this purpose. A single (K174R), a double (K174RV32S) and a triple (K174RV32SG36S) mutant of papain have been generated, of which the triple mutant shows maximum thermostability with the half-life (t1/2) extended by 94 min at 60°C and 45 min at 65°C compared to the wild type (WT). The temperature of maximum enzymatic activity (Tmax) and 50% maximal activity (T50) for the triple mutant increased by 15 and 4°C, respectively. Moreover, the triple mutant exhibits a faster inactivation rate beyond Tmax which may be a desirable feature for an industrial enzyme. The values of t1/2 and Tmax for the double mutant lie between those of the WT and the triple mutant. The single mutant however turns out to be unstable for biochemical characterization. These results have been substantiated by molecular modeling studies which also indicate highest stability for the triple mutant based on higher number of interdomain H-bonds/salt-bridges, less interdomain flexibility and lower stability free-energy compared to the WT. In silico studies also explain the unstable behavior of the single mutant. [ABSTRACT FROM PUBLISHER]

Hen serum transferring in its diferric form (hST) has been isolated, purified and the three-dimensional structure determined by X-ray crystallography at 2.8 Å resolution. The final refined structure of hST, comprising 5232 protein atoms, two Fe[SUP3+] cations, two CO[SUB3,SUP2-] anions, 54 water molecules and one fucose moiety, has an R factor of 21.5% ad an R[SUBfree} of 26.9% for all data. The structure has been compared with the three-dimensional structure of hen ovotransferrin (hOT) and also with structures of some other transferrins, viz. rabbit serum transferring (rST) and human lactoferin (hLF). The overall conformation of the hST molecule is essentially the same as that of other transferrins. However the relative orientation of the two lobes, which is related to the species-specific receptor-recognition property of transferrins, has been found to be different in hST from that in hOT, rST and hLF. On the basis of superposition of the N lobes, rotations of 5.8, 16.9 and 11.3° are required to bring the C lobes of hOT, rST and hLF, respectively, into coincidence with that of hST. A number of additional hydrogen bonds between the two domains in the N and C lobes have been identified in the structure of hST compare with that of hOT, which indicate a greater compactness of the lobes of hST than those of hOT. Being products of the same gene hST than those of hOT. Being products of the same gene. HST and hOT have 100% sequence identity and differ only in the attached carbohydrate moiety. On the other hand, despite having similar functions, hST and rST have only 51% sequence similarity. However, the nature of the interdomain interactions of hST are closer to rST than to hOT. A putative carbohydrate-binding site has been identified in the N lobe of hST at Asn52 and a fucose molecule could be modeled at the site. The variations in interdomain and itnerlobe interactions in hST, together with altered lobe orientation with respect to hOT, rST and hLF, which are the representatives of the other subfamily of transferrins, are discussed. [ABSTRACT FROM AUTHOR]