title: Effects of hydrophobic amino acid substitution in Pleurotus ostreatus proteinase A inhibitor 1 on its structure and functions as protease inhibitor and intramolecular chaperone.
abstract: We previously demonstrated that Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) could function as an intramolecular chaperone of subtilisin BPN', as in the case of the propeptide of subtilisin BPN', and that its Phe44 --> Ala mutant, which lost its tertiary structure, could not assist the refolding of subtilisin BPN'. In this study, we examined the effects of hydrophobic amino acid substitutions at other sites and substitutions of Phe44 with other hydrophobic residues on the structure and functions of POIA1. These mutations were introduced into POIA1cm that had been obtained by the substitution of the C-terminal six residues of POIA1 with those of the propeptide of subtilisin BPN'. When Ile32 or Ile64 was substituted with Ala, the tertiary structure of the resultant mutant was markedly destroyed, and the activities as a protease inhibitor and an intramolecular chaperone were significantly lowered. Among the position 44 mutants, the Phe44 --> Val mutant was a much less effective intramolecular chaperone with conversion to a digestible inhibitor, possibly owing to destruction of the tertiary structure. On the other hand, the Phe44 --> Leu or Ile mutant maintained its tertiary structure, and hence could function as a more effective intramolecular chaperone than the Phe44 --> Val mutant. Furthermore, since the Phe44 --> Leu mutant was a more susceptible inhibitor than POIA1cm, the halo formed around a colony of Bacillus cells transformed with a plasmid encoding this mutant was larger than others. These results clearly show the close relationship between the tertiary structure and functions of POIA1 as a protease inhibitor and an intramolecular chaperone, and that a combination of such inhibitory properties and intramolecular chaperone activity of POIA1 might affect the diameter of the halo formed around Bacillus colonies in vivo.
authors: Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588, Japan. shuichi.kojima@gakushuin.ac.jp
authors: Shuichi
authors: S
authors: Kojima
authors: Akane
authors: A
authors: Iwahara
authors: Yuri
authors: Y
authors: Hisano
authors: Hideyuki
authors: H
authors: Yanai
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000597:Amino Acids, Branched-Chain / Q000737:chemistry / Q000235:genetics; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors*; D002942:Circular Dichroism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D057927:Hydrophobic and Hydrophilic Interactions; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology / Q000378:metabolism*; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D017434:Protein Structure, Tertiary / Q000235:genetics
chemical_list: D000597:Amino Acids, Branched-Chain; D005656:Fungal Proteins; D018832:Molecular Chaperones; D011480:Protease Inhibitors; D010649:Phenylalanine; D016282:Aspartic Acid Endopeptidases; C022442:aspergillopepsin II