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J. Biol. Chem., Vol. 279, Issue 45, 47344-47351, November 5, 2004

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The Crystal Structure of an Oxidatively Stable Subtilisin-like Alkaline Serine Protease, KP-43, with a C-terminal -Barrel Domain^*

Tsuyoshi Nonaka, Masahiro Fujihashi, Akiko Kita, Katsuhisa Saeki¶, Susumu Ito||, Koki Horikoshi||, and Kunio Miki**

From the Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, the Research Reactor Institute, Kyoto University, Kumatori, Osaka 590-0494, the ^¶Biological Science Laboratories, Kao Corporation, 2606, Akabane Ichikaimachi, Haga, Tochigi 321-3497, the ^||Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, and the ^**RIKEN Harima Institute/SPring-8, Koto 1-1-1, Mikazukicho, Sayo-gun, Hyogo 679-5148, Japan

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 (R_free of 0.169) at 1.30-Å resolution. KP-43 consists of two domains, a subtilisin-like / domain and a C-terminal jelly roll -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 -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 (R_free of 0.212) at 1.73-Å 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 -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.

Received for publication, August 9, 2004 , and in revised form, August 30, 2004.

The atomic coordinates and structure factors (codes 1wmd, 1wme, and 1wmf) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was partially supported by grants from the "Research for the Future" Program (JSPS-RFTF 97L00501) of the Japan Society for the Promotion of Science and the "National Project on Protein Structural and Functional Analyses" of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-75-753-4029; Fax: 81-75-753-4032; E-mail: miki{at}kuchem.kyoto-u.ac.jp.

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