PDBsum entry 1wme

Hydrolase

PDB id: 1wme

Contents

Protein chain

434 a.a. *

Metals

_CA ×3

Waters ×200

* Residue conservation analysis

References listed in PDB file

Key reference

Title

The crystal structure of an oxidatively stable subtilisin-Like alkaline serine protease, Kp-43, With a c-Terminal beta-Barrel domain.

Authors

T.Nonaka, M.Fujihashi, A.Kita, K.Saeki, S.Ito, K.Horikoshi, K.Miki.

Ref.

J Biol Chem, 2004, 279, 47344-47351. [DOI no: 10.1074/jbc.M409089200]

PubMed id

15342641

Abstract

The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43 from Bacillus sp. KSM-KP43, with a C-terminal extension domain, was determined by the multiple isomorphous replacements method with anomalous scattering. The native form was refined to a crystallographic R factor of 0.134 (Rfree of 0.169) at 1.30-A resolution. KP-43 consists of two domains, a subtilisin-like alpha/beta domain and a C-terminal jelly roll beta-barrel domain. The topological architecture of the molecule is similar to that of kexin and furin, which belong to the subtilisin-like proprotein convertases, whereas the amino acid sequence and the binding orientation of the C-terminal beta-barrel domain both differ in each case. Since the C-terminal domains of subtilisin-like proprotein convertases are essential for folding themselves, the domain of KP-43 is also thought to play such a role. KP-43 is known to be an oxidation-resistant protease among the general subtilisin-like proteases. To investigate how KP-43 resists oxidizing reagents, the structure of oxidized KP-43 was also determined and refined to a crystallographic R factor of 0.142 (Rfree of 0.212) at 1.73-A resolution. The structure analysis revealed that Met-256, adjacent to catalytic Ser-255, was oxidized similarly to an equivalent residue in subtilisin BPN'. Although KP-43, as well as proteinase K and subtilisin Carlsberg, lose their hydrolyzing activity against synthetic peptides after oxidation treatment, all of them retain 70-80% activity against proteinaceous substrates. These results, as well as the beta-casein digestion pattern analysis, have indicated that the oxidation of the methionine adjacent to the catalytic serine is not a dominant modification but might alter the substrate specificities.

Figure 2.
FIG. 2. A comparison of the structures of KP-43 (blue, Nat1), subtilisin BPN' complexed with the Streptomyces subtilisin inhibitor (red, PDB accession code, 2sic [PDB] (34)), and kexin (yellow, PDB accession code, 1ot5 [PDB] (25)) by stereo drawings of C -trace models. Calcium ions of KP-43, subtilisin BPN', and kexin are drawn as cyan, orange, and yellow spheres, respectively. Streptomyces subtilisin inhibitor complexed with subtilisin BPN' was partially drawn as a stick model, and its carbon atom is colored in orange. These figures were prepared by MOLSCRIPT and Raster3D (36, 37). a, superimposition of KP-43 and subtilisin BPN'. b, superimposition of KP-43 and kexin.

Figure 5.
FIG. 5. Stereo views of the stick models around the catalytic center of KP-43 and subtilisin BPN'. Subtilisin BPN' is drawn transparently and is superimposed on KP-43. a, the native forms of KP-43 (Nat1) and subtilisin BPN' (PDB accession code, 2st1 [PDB] (8)). WAT, water. b, the oxidized forms of KP-43 (Oxi) and subtilisin BPN' (PDB accession code, 1st2 [PDB] (8)). These figures were drawn by MOLSCRIPT and Raster3D (36, 37).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 47344-47351) copyright 2004.

Secondary reference #1

Title

Crystallization and preliminary X-Ray diffraction studies of a novel alkaline serine protease (kp-43) from alkaliphilic bacillus sp. Strain ksm-Kp43.

Authors

T.Nonaka, M.Fujihashi, A.Kita, K.Saeki, S.Ito, K.Miki.

Ref.

Acta Crystallogr D Biol Crystallogr, 2001, 57, 717-718. [DOI no: 10.1107/S0907444901002566]

PubMed id

11320315

Figure 2.
Figure 2 X-ray diffraction patterns from a crystal of KP-43 taken with Cu K radiation. The crystal-to-detector distance and the oscillation range are 150 mm and 1.0°, respectively.

The above figure is reproduced from the cited reference with permission from the IUCr