title: Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus.
abstract: Pyrolysin from the hyperthermophilic archaeonPyrococcus furiosusis the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS147, IS29, IS27, and IS8) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS147and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29is dispensable. The large insert IS147contains Ca3 and Ca4, two calcium-binding Dx[DN]xDG motifs that are conserved in many pyrolysin-like proteases. Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation. Mature insert-deletion variants were characterized and showed that IS29and IS8contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS29and the other in IS27Disrupting the ion pairs in IS27and IS8induces autocleavage in the absence of salts. Interestingly, autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation. Additionally, a single mutation in IS29increases resistance to salt-induced autocleavage and further increases enzyme thermostability. Our results suggest that these extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments.IMPORTANCEPyrolysin-like proteases belong to the subtilase superfamily and are characterized by large inserts and long C-terminal extensions; however, the role of the inserts in enzyme function is unclear. Our results demonstrate that four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zeng
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Huawei
authors.initials: H
authors.lastname: Yi
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China tangb@whu.edu.cn tangxf@whu.edu.cn.|Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China tangb@whu.edu.cn tangxf@whu.edu.cn.|Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan, China.
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics*; D004251:DNA Transposable Elements; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D019766:Genes, Archaeal; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics*; D057057:Serine Proteases / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D020860:Subtilisin / Q000737:chemistry
chemical_list: D019843:Archaeal Proteins; D004251:DNA Transposable Elements; D012965:Sodium Chloride; D057057:Serine Proteases; D012697:Serine Endopeptidases; C101133:pls protein, Pyrococcus furiosus; D020860:Subtilisin; D002118:Calcium
keywords: Ca2+-binding; hyperthermophilic archaeon; insertion sequence; serine protease; subtilisin; thermostability