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

Abstract

The crystal structure of an oxidatively stable subtilisinlike 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.

Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus

“…These results exclude the possibility that RaCE is a thiol enzyme, and the sensitivity to heavy metal ions might be the result of interaction with Trp residues required for catalysis (Pettersson 1968;Hurst et al 1977). RaCE is very sensitive to H 2 O 2 at a concentration lower than 1 mM, suggesting that certain Met residues are readily inactivated by oxidants and air (Saeki et al 2000;Hagihara et al 2001;Hagihara et al 2003;Nonaka et al 2004). Besides the two Met residues at the N-terminus, RaCE has 10 Met residues at positions 62, 95, 96, 106, 179, 212, 251, 270, 378, and 380 (Fig.…”

Characterization of a Novel Subtilisin-like Protease Myroicolsin from Deep Sea Bacterium Myroides profundi D25 and Molecular Insight into Its Collagenolytic Mechanism

“…Myroicolsin exhibits the highest identity (46%) with a putative subtilisin-like protease (NCBI reference sequence: WP_002990379.1). Among characterized proteases, myroicolsin exhibits the highest identity (28%) with protease Kp43, an oxidatively stable alkaline protease of the S8 family (43). Moreover, myroicolsin has a domain architecture that is different from other reported subtilisin-like collagenolytic proteases (Fig.…”

Structural Comparison of ColH and ColG Collagen-Binding Domains from Clostridium histolyticum

“…No structure of PPC-M9A has been reported. The crystal structure of PPC from serine protease S8 superfamily with a sequence identity of 15% (PDB accession code 1WME [46]) cannot offer additional insights either. The function of the PPC is not known either, but PPC-M9A may adopt a tertiary fold similar to that of CBD.…”