High selectivity of the hyperthermophilic subtilase propeptide domain toward inhibition of its cognate protease

Bahun, Miha (author), Poklar Ulrih, Nataša (author)

Microbial extracellular subtilases are highly active proteolytic enzymes commonly used in commercial applications. These subtilases are synthesized in their inactive proform, which matures into the active protease under the control of the propeptide domain. In mesophilic bacterial prosubtilases, the propeptide functions as both an obligatory chaperone and an inhibitor of the subtilase catalytic domain. In contrast, the propeptides of hyperthermophilic archaeal prosubtilases act mainly as tight inhibitors and are not essential for subtilase folding. It is unclear whether this stronger inhibitory activity of hyperthermophilic propeptides results in their higher selectivity toward their cognate subtilases, in contrast to promiscuous mesophilic propeptides. Here, we showed that the propeptide of pernisine, a hyperthermostable archaeal subtilase, strongly interacts with and inhibits pernisine, but not the homol­ogous subtilisin Carlsberg and proteinase K. Instead, the pernisine propeptide was readily degraded by subtilisin Carlsberg and proteinase K. In addition, the catalytic domain of unprocessed propernisine was also susceptible to degradation but became proteolytically stable after autoprocessing of propernisine into the inactive, noncovalent complex propeptide:pernisine. This allowed efficient transactivation of the autopro­cessed complex propeptide:pernisine through degradation of pernisine propeptide by subtilisin Carlsberg and proteinase K at mesophilic temperature. Moreover, we demon­strated that active pernisine molecules are inhibited by the propeptide that is released after pernisine-catalyzed degradation of the unprocessed propernisine catalytic domain. This highlights the high inhibitory potency of the hyperthermophilic propeptide toward its cognate subtilase and its importance in regulating subtilase maturation, to prevent the degradation of the unprocessed subtilase precursors by the prematurely activated molecules.

• Language: English
• Keywords: hyperthermophile, subtilase, propeptide, enzyme activation, enzyme inhibition, protein stability
• Work type: Scientific work
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2023
• Number of pages: 24 str.
• Numbering: Vol. 11, iss. 5, art. 01487-23
• PID: 20.500.12556/RUL-155313
• UDC: 577.15
• ISSN on article: 2165-0497
• DOI: 10.1128/spectrum.01487-23
• COBISS.SI-ID: 163687939
• Publication date in RUL: 26.03.2024

Record is a part of a journal

• Title: Microbiology spectrum
• Shortened title: Microbiol. spectr.
• Publisher: ASM Press
• ISSN: 2165-0497
• COBISS.SI-ID: 4526968

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: hipertermofili, encimi, proteaze

Projects

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: P4-0121
• Name: Biokemijska in biofizikalno-kemijska karakterizacija naravnih snovi