Insights into the Maturation of Hyperthermophilic Pyrolysin and the Roles of Its N-Terminal Propeptide and Long C-Terminal Extension

Dai, Zheng; Fu, Han; Zhang, Yufeng; Zeng, Jing; Tang, Bing; Tang, Xiaofeng

doi:10.1128/aem.00548-12

Cited by 13 publications

(21 citation statements)

References 38 publications

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“…The stabilizing effect of IS8 on pyrolysin is closely related to its involvement in electrostatic interactions important for structural integrity of the enzyme. Our previous study demonstrated that the CTE of pyrolysin confers additional enzyme stability (12). Because IS8 is located near the C terminus of the catalytic domain and resides within a surface loop (11), it could be involved in the interaction between the catalytic domain and the CTE, conferring additional enzyme stability.…”

Section: Discussionmentioning

confidence: 99%

“…4E). We showed previously that only correctly folded proforms of pyrolysin matured efficiently and that the proform is less stable than the mature form with respect to thermogenic hydrolysis resistance (12). Given this information, the low maturation efficiencies of the Ca4 site variants (e.g., D163A and D161N/D163N/D165N) could be due to slower folding and lower maturation rates compared to those of the WT.…”

Section: Figmentioning

confidence: 92%

“…Expression plasmids used for the proforms of wild-type pyrolysin (Pls) (pET26b-pls) and the active-site variant PlsS441A (pET26-plsS441A) were constructed previously (12). Table S1 in the supplemental material lists the primer sequences used in this study.…”

Section: Methodsmentioning

confidence: 99%

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Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

Gao

Zeng

et al. 2017

Appl Environ Microbiol

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Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is 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 (IS147 and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29 is dispensable. The large insert IS147 contains 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 IS29 and IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS29 and the other in IS27. Disrupting the ion pairs in IS27 and IS8 induces 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 IS29 increases 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. IMPORTANCE Pyrolysin-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.

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Section: Discussionmentioning

confidence: 99%

Section: Figmentioning

confidence: 92%

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Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

Gao

Zeng

et al. 2017

Appl Environ Microbiol

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“…Multiple mature forms have been described for other CTEcontaining subtilases, such as Vpr (31,32) and Epr (33,34) from B. subtilis and pyrolysin from Pyrococcus furiosus (35,36). Maturation of these enzymes also involves differential proteolytic processing of the CTE.…”

Section: Discussionmentioning

confidence: 99%

Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes

Meng

Dai

Bin

et al. 2016

Appl Environ Microbiol

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Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis. Its precursor is composed of a signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE). Several active forms of Bpr have been previously reported, but little is known about the maturation of this enzyme. Here, a gene encoding a Bpr (BprL) was cloned from B. subtilis LZW and expressed in B. subtilis WB700, and three fibrinolytic mature forms with apparent molecular masses of 45, 75, and 85 kDa were identified in the culture supernatant. After treatment with urea, the 75-kDa mature form had the same molecular mass as the 85-kDa mature form, from which we infer that they adopt different conformations. Mutational analysis revealed that while the 85-kDa mature form is generated via heterocatalytic processing of a BprL proform by an unidentified protease of B. subtilis, the production of the 75-and 45-kDa mature forms involves both hetero-and autocatalytic events. From in vitro analysis of BprL and its sequential C-terminal truncation variants, it appears that partial removal of the CTE is required for the initiation of autoprocessing of the N-terminal propeptide, which is composed of a core domain (N*) and a 15-residue linker peptide, thereby yielding the 45-kDa mature form. These data suggest that the differential processing of BprL, either heterocatalytically or autocatalytically, leads to the formation of multiple mature forms with different molecular masses or conformations.

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“…The Tk-subtilisin and alkaline serine protease exhibited optimum enzymatic activity at 90°C and 100°C and halflives at 100°C of 50 min and 100 min, respectively. Pyrolysin from Pyrococcus furiosus had a half-life value of 4 h at the boiling point of water (Voorhorst et al, 1996;Dai et al, 2012). An aeropyrolysin (a metalloproteinase) from Aeropyrum pernix K1 (an aerobic marine hyperthermophilic archaeon) had a maximum activity at 110°C.…”

Section: Hyperthermophilic Archaeamentioning

confidence: 99%

A Review: Biodegradation and Applications of Keratin Degrading Microorganisms and Keratinolytic Enzymes, Focusing on Thermophiles and Thermostable Serine Proteases

Kanoksilapatham¹,

Intagun²

2017

American Journal of Applied Sciences

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Keratins are hard-degrading fibrous proteins, insoluble in water and organic solvents, often accumulated in nature and major components in feathers, skins, hair, horn, nail, hoof etc., Keratin-degrading microorganisms such as bacteria, archaea, actinomycetes and fungi employ keratinases to attack keratin. Keratinases belonging to subtilisin-like serine proteases were classified based on similarity of amino acid sequences. Keratinolytic thermophilic or hyperthermophilic bacteria and archaea have been known to degrade keratin at ≥70°C. General properties of thermozymes such as stability to heat and resistance to denaturing conditions; e.g., solvents and detergents have drawn attention to various biotechnological industries. Some bacterial and archaeal keratinases degrade not only fibrous keratin but also digest recalcitrant prion proteins, an etiologic agent of spongiform encephalopathies of brain and nervous system. Keratin and keratinase have a number of applications in various sectors, i.e., biotechnology, cosmetic and pharmaceutical industries, medical therapy and waste management. On the other hand, accumulation of excess amount of keratin is recognized as solid waste and troublesome environmental pollutant. Biodegradation involving either keratinolytic thermophiles or thermostable keratinases not only improve digestibility and nutritive values of keratinous meals (for mixers in animal feed stuffs), but also minimize risk from infection and microbial toxin transmitted to livestock.

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Insights into the Maturation of Hyperthermophilic Pyrolysin and the Roles of Its N-Terminal Propeptide and Long C-Terminal Extension

Cited by 13 publications

References 38 publications

Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes

A Review: Biodegradation and Applications of Keratin Degrading Microorganisms and Keratinolytic Enzymes, Focusing on Thermophiles and Thermostable Serine Proteases

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