1sua
From Proteopedia
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SUBTILISIN BPN'
Overview
The three-dimensional structure of a subtilisin BPN' construct that was, produced and folded without its prodomain shows the tertiary structure is, nearly identical to the wild-type enzyme and not a folding intermediate., The subtilisin BPN' variant, Sbt70, was cloned and expressed in, Escherichia coli without the prodomain, the 77-residue N-terminal domain, that catalyzes the folding of the enzyme into its native tertiary, structure. Sbt70 has the high-affinity calcium-binding loop, residues 75, to 83, deleted. Such calcium-independent forms of subtilisin BPN' refold, independently while retaining high levels of activity [Bryan et al., Biochemistry, 31:4937-4945, 1992]. Sbt70 has, in addition, seven, stabilizing mutations, K43N, M50F, A73L, Q206V, Y217K, N218S, Q271E, and, the active site serine has been replaced with alanine to prevent, autolysis. The purified Sbt70 folded spontaneously without the prodomain, and crystallized at room temperature. Crystals of Sbt70 belong to space, group P2(1)2(1)2(1) with unit cell parameters a = 53.5 A, b = 60.3 A, and, c = 83.4 A. Comparison of the refined structure with other high-resolution, structures of subtilisin BPN' establishes that the conformation of Sbt70, is essentially the same as that previously determined for other, calcium-independent forms and that of other wild-type subtilisin BPN', structures, all folded in the presence of the prodomain. These findings, confirm the results of previous solution studies that showed subtilisin, BPN' can be refolded into a native conformation without the presence of, the prodomain [Bryan et al., Biochemistry 31:4937-4945, 1992]. The, structure analysis also provides the first descriptions of four, stabilizing mutations, K43N, A73L, Q206V, and Q271E, and provides details, of the interaction between the enzyme and the Ala-Leu-Ala-Leu tetrapeptide, found in the active-site cleft.
About this Structure
1SUA is a Single protein structure of sequence from Bacillus amyloliquefaciens. Active as Subtilisin, with EC number 3.4.21.62 Full crystallographic information is available from OCA.
Reference
Crystal structure of calcium-independent subtilisin BPN' with restored thermal stability folded without the prodomain., Almog O, Gallagher T, Tordova M, Hoskins J, Bryan P, Gilliland GL, Proteins. 1998 Apr 1;31(1):21-32. PMID:9552156
Page seeded by OCA on Wed Nov 21 02:40:28 2007
