TY - JOUR
T1 - Novel Monomeric Fungal Subtilisin Inhibitor from a Plant-Pathogenic Fungus, Choanephora cucurbitarum
T2 - Isolation and Molecular Characterization
AU - Pathiraja, Duleepa
AU - Chun, Youngeun
AU - Cho, Junghwan
AU - Min, Byoungnam
AU - Lee, Saeyoung
AU - Park, Hongjae
AU - Byun, Juan
AU - Choia, In Geol
N1 - Funding Information:
This study was supported by the School of Life Sciences and Biotechnology for BK21 PLUS, Korea University. We are grateful to Kwang Yeon Hwang, Department of Biotechnology, Korea University, for providing the resources and assisting with the microscale thermophoresis experiments. A patent application for the fungal protease inhibitor is pending approval.
Funding Information:
This study was funded by the Cooperative Research Program for the National Agricultural Genome Program, Rural Development Administration, Republic of Korea (project no. PJ01337602), a grant from the New and Renewable Energy Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP), Ministry of Trade, Industry and Energy (grant 20173010092460), and a National Research Foundation of Korea (NRF) grant funded by the government of the Republic of Korea (MEST) (grant NRF-2019R1A2C1089704).
Funding Information:
This study was supported by the School of Life Sciences and Biotechnology for BK21 PLUS, Korea University. We are grateful to Kwang Yeon Hwang, Department of Bio-technology, Korea University, for providing the resources and assisting with the mi-croscale thermophoresis experiments. A patent application for the fungal protease inhibitor is pending approval. This study was funded by the Cooperative Research Program for the National Agricultural Genome Program, Rural Development Administration, Republic of Korea (project no. PJ01337602), a grant from the New and Renewable Energy Core Technol-ogy Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP), Ministry of Trade, Industry and Energy (grant 20173010092460), and a National Research Foundation of Korea (NRF) grant funded by the government of the Republic of Korea (MEST) (grant NRF-2019R1A2C1089704).
Publisher Copyright:
© 2020 American Society for Microbiology.
PY - 2020/11
Y1 - 2020/11
N2 - sin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are hori-zontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated “cho-anepins.” Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [Kd]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a mono-meric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in in-hibitory reactivity (reactive sites) and binding affinity (structural integrity). The func-tion of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel ser-ine protease inhibitors. IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not re-active sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional char-acterization of fungal SSI-like domains will be useful in understanding their biologi-cal role and future biotechnological applications. The bacterial protease inhibitor domains known as Streptomyces subtili-
AB - sin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are hori-zontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum, designated “cho-anepins.” Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [Kd]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro, whereas bacterial SSI is a homodimer. Based on these observations, we constructed a mono-meric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in in-hibitory reactivity (reactive sites) and binding affinity (structural integrity). The func-tion of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel ser-ine protease inhibitors. IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not re-active sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional char-acterization of fungal SSI-like domains will be useful in understanding their biologi-cal role and future biotechnological applications. The bacterial protease inhibitor domains known as Streptomyces subtili-
KW - Choanephora cucurbitarum
KW - Streptomyces subtilisin inhibitor
KW - fungal protease inhibitor
UR - http://www.scopus.com/inward/record.url?scp=85094932851&partnerID=8YFLogxK
U2 - 10.1128/AEM.01818-20
DO - 10.1128/AEM.01818-20
M3 - Article
C2 - 32887713
AN - SCOPUS:85094932851
VL - 86
SP - 1
EP - 16
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 22
ER -