Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.

The PH1704 protease from hyperthermophilic archaean Pyrococcus horikoshii OT3 is a member of DJ-1/ThiJ/PfpI superfamily with diverse functional subclasses. The recombinant PH1704 was efficiently purified and was systematically characterized by a combination of substrate specificity analysis, steady-...

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Autores principales:	Dongling Zhan, Aixi Bai, Lei Yu, Weiwei Han, Yan Feng
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5d8cb75ed7e24bc78f41c3ee5fdf0f3e
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spelling	oai:doaj.org-article:5d8cb75ed7e24bc78f41c3ee5fdf0f3e2021-11-25T06:01:39ZCharacterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.1932-620310.1371/journal.pone.0103902https://doaj.org/article/5d8cb75ed7e24bc78f41c3ee5fdf0f3e2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25192005/?tool=EBIhttps://doaj.org/toc/1932-6203The PH1704 protease from hyperthermophilic archaean Pyrococcus horikoshii OT3 is a member of DJ-1/ThiJ/PfpI superfamily with diverse functional subclasses. The recombinant PH1704 was efficiently purified and was systematically characterized by a combination of substrate specificity analysis, steady-state kinetics study and molecular docking research. The homogeneous protease was obtained as a presumed dodecamer with molecular weight of ∼240 kDa. Iodoacetamide strongly inhibited the peptidase activity, confirming that Cys100 is a nucleophilic residue. The recombinant protein was identified as both an aminopeptidase and an endopeptidase. Experimental data showed that L-R-amc was the best substrate of PH1704. Structural interaction fingerprint analysis (SIFt) indicated the binding pose of PH1704 and showed that Tyr120 is important in substrate binding. Kinetic parameters Kcat and Kcat/Km of the Y120P mutant with L-R-amc was about 7 and 7.8 times higher than that of the wild type (WT). For the endopeptidase Y120P with AAFR-amc, Kcat and Kcat/Km is 10- and 21-fold higher than that of WT. Experimental data indicate the important functions of Tyr120: involvement in enzyme activity to form a hydrogen bond with Cys100 and as an entrance gate of the substrate with Lys43. The results of this study can be used to investigate the DJ-1/ThiJ/PfpI superfamily.Dongling ZhanAixi BaiLei YuWeiwei HanYan FengPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 9, p e103902 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Dongling Zhan Aixi Bai Lei Yu Weiwei Han Yan Feng Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.
description	The PH1704 protease from hyperthermophilic archaean Pyrococcus horikoshii OT3 is a member of DJ-1/ThiJ/PfpI superfamily with diverse functional subclasses. The recombinant PH1704 was efficiently purified and was systematically characterized by a combination of substrate specificity analysis, steady-state kinetics study and molecular docking research. The homogeneous protease was obtained as a presumed dodecamer with molecular weight of ∼240 kDa. Iodoacetamide strongly inhibited the peptidase activity, confirming that Cys100 is a nucleophilic residue. The recombinant protein was identified as both an aminopeptidase and an endopeptidase. Experimental data showed that L-R-amc was the best substrate of PH1704. Structural interaction fingerprint analysis (SIFt) indicated the binding pose of PH1704 and showed that Tyr120 is important in substrate binding. Kinetic parameters Kcat and Kcat/Km of the Y120P mutant with L-R-amc was about 7 and 7.8 times higher than that of the wild type (WT). For the endopeptidase Y120P with AAFR-amc, Kcat and Kcat/Km is 10- and 21-fold higher than that of WT. Experimental data indicate the important functions of Tyr120: involvement in enzyme activity to form a hydrogen bond with Cys100 and as an entrance gate of the substrate with Lys43. The results of this study can be used to investigate the DJ-1/ThiJ/PfpI superfamily.
format	article
author	Dongling Zhan Aixi Bai Lei Yu Weiwei Han Yan Feng
author_facet	Dongling Zhan Aixi Bai Lei Yu Weiwei Han Yan Feng
author_sort	Dongling Zhan
title	Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.
title_short	Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.
title_full	Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.
title_fullStr	Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.
title_full_unstemmed	Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.
title_sort	characterization of the ph1704 protease from pyrococcus horikoshii ot3 and the critical functions of tyr120.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/5d8cb75ed7e24bc78f41c3ee5fdf0f3e
work_keys_str_mv	AT donglingzhan characterizationoftheph1704proteasefrompyrococcushorikoshiiot3andthecriticalfunctionsoftyr120 AT aixibai characterizationoftheph1704proteasefrompyrococcushorikoshiiot3andthecriticalfunctionsoftyr120 AT leiyu characterizationoftheph1704proteasefrompyrococcushorikoshiiot3andthecriticalfunctionsoftyr120 AT weiweihan characterizationoftheph1704proteasefrompyrococcushorikoshiiot3andthecriticalfunctionsoftyr120 AT yanfeng characterizationoftheph1704proteasefrompyrococcushorikoshiiot3andthecriticalfunctionsoftyr120
_version_	1718414283779342336

Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.

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