Structure-Function of Novel Carboxyl Proteinases from Microorganisms

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In this study, we aimed to identify the catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryote cells. We focussed our studies on carboxyl proteinases from Pseudomonas sp. 101 (PCP), Xanthomonas sp. T-22 (XCP). Bacillus coagulans J-4 (J-4), and Bacillus novosp. MN-32 (kumamolysin). The primary structures of them does not have any similarities to those of aspartic proteinases (pepstatin-insensitive carboxyl proteinase) reported so far. Moreover, the well-conserved structure. -Asp*-Thr-Gly-(Asp* : catalytic residue) in the active center of aspartic proteinases was not observed. The following results were obtained.1. Substrate Specificity and Subsite Structure of KumamolysinSubstrate specificity of Kumamolysin was investigated by using two sets of synthetic substrates. The subsite structure of Kumamolysin was found to be different with those of aspartic proteinases. These data will be published in J.Biochem.2. Identification of Catalytic Residues by Using [^<14>C] … More Stylene OxideIn order to identify the catalytic residue(s) of XCP, [^<14>C] stylene oxide was used. It was found that the [^<14>C]stylene oxide was bound to E75 and D110 residues of XCP, respectively. Of them, E75 residue (corresponding to E80 residue of PCP) and its vicinities were conserved in PCP.Based on these data, E80 residue of PCP and E75 residue of XCP were thought to be involved in their catalytic function, as a substrate binding site, respectively.3. Identification of Catalytic Residues by Using [^<14>C] DCCDIn order to identify the catalytic residue(s) of PCP, chemical modification was carried out by using N.N'-dicyclohexylcafrbodiimide (DCCD) and specific inhibitor. tyrostatin, It was found that [^<14>C] DCCD was bound to D140 and E222 residues of PCP, respectively. Of them, E222 residue (corresponding to E235 residue of XCP) and its vicinities were found out to be conserved in XCP.Furthermore, E222A mutant had no any activity, whereas XE235A (corresponding to E222A for PCP) had proteinase activity. Based on these data. E222 residue of PCP and E235 of XCP were thought to be involved in their catalytic function. probably as a substrate binding site, respectively.4. Cloning of Carboxyl Proteinase J-4 Gene from Bacillus coagulansBacillus coagulans J-4 carboxyl proteinase. designated as J-4. is characterized as alcohol resistant and insensitive to pepstatin. Most of the gene has been cloned, sequenced. After getting the whole gene, we will try to construct a high expression system and determine the catalytic residues by site-directed mutagenesis. Less