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

Abstract

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.

Keywords: Ca2+-binding; hyperthermophilic archaeon; insertion sequence; serine protease; subtilisin; thermostability.

FIG 1

Maturation of pyrolysin and its variants. (A) A schematic representation of the primary structure of pyrolysin proform (Pls) and its insert-deletion variants. The locations of the active-site residues (Asp³⁰, His²¹⁶, and Ser⁴⁴¹) and four inserts (IS147, IS29, IS27, and IS8) are shown. The core of the catalytic domain is shown in black. The N-terminal propeptide (N), C-terminal extension (CTEm), C-terminal propeptide (C), and putative prepeptidase C-terminal (PPC) domain are indicated. (B) SDS-PAGE analysis of the Pls and variant maturation. Crude samples containing approximately 25 to 30 μg/ml of Pls and its variants in buffer A were incubated at 95°C for the time intervals indicated and electrophoresed using SDS-PAGE. The positions of the proform (P) and the mature form (M) are indicated on the gels.

FIG 2

Alignment of amino acid sequences of pyrolysin and its homologs around two potential Ca²⁺-binding sites (Ca3 and Ca4) within the large insert IS147. The target sequence of pyrolysin from Pyrococcus furiosus (Pfu) was aligned with that from Pyrococcus woesei (Pwo), Thermococcus sp. strain PK (Tsp), Palaeococcus pacificus (Ppa), Thermincola potens (Tpo), Kineosphaera limosa (Kli), Streptomyces sp. strain LaPpAH-108 (Ssp), Verrucosispora maris (Vma), Salinispora pacifica (Spa), Amycolatopsis rifamycinica (Ari), Kutzneria albida (Kal), Saccharomonospora glauca (Sgl), and Tepidanaerobacter acetatoxydans (Tac). GenBank accession numbers of the proteins are shown in parentheses. The amino acid residues are numbered starting from the N terminus of the precursor or the mature enzyme (Pfu). The two Dx[DN]xDG Ca²⁺-binding motifs are indicated. Arrowheads indicate residues that were mutated. A schematic representation of the primary structure of mature Pls (mPls) and the residues of Ca1 and Ca2 sites (indicated by filled circles) are also shown.

FIG 3

Mutating residues of the Ca3 site affects pyrolysin activity and stability. (A) SDS-PAGE analysis (lower) and activity assays (upper) of purified samples of mature enzymes. Azocaseinolytic activities of the enzymes were carried out at 95°C in buffer A, and relative activity was calculated by defining the activity of the WT as 100%. (B and C) Heat inactivation profiles. Enzymes (8.0 μg/ml) in buffer A were incubated at 95°C in the absence (B) or presence (C) of 2 mM EGTA. At the time intervals indicated, samples were removed and an azocaseinolytic activity assay (B and C) and SDS-PAGE (C) were performed. The residual activity is expressed as a percentage of the original activity for each enzyme sample. The values are expressed as means ± standard deviations (SD) from three independent experiments.

FIG 4

Properties of pyrolysin Ca4 site variants. (A) SDS-PAGE analysis of variant maturation. Crude samples of the variants in buffer A were incubated at 95°C for the time intervals indicated and electrophoresed using SDS-PAGE. The positions of the proform (P) and the mature form (M) are indicated on the gels. (B) SDS-PAGE analysis of purified samples of the variants. (C) Thermogenic hydrolysis of active-site variants. Purified samples of the variant proforms (8.0 μg/ml) in buffer A were incubated at 95°C for the time intervals indicated and electrophoresed using SDS-PAGE. (D) Heat inactivation profiles. The enzymes (8.0 μg/ml) in buffer A were incubated at 95°C in the absence or presence of 2 mM EGTA for the time intervals indicated, and activity was tested using the azocaseinolytic activity assay. The residual activity is expressed as a percentage of the original activity of each enzyme sample. (E) Activity assay of purified mature enzymes. Azocaseinolytic activities of the enzymes were determined at 95°C in buffer A, and relative activity was calculated by defining the activity of the WT as 100%. (D and E) The values are expressed as means ± SD from three independent experiments.

FIG 5

Properties of mature PlsΔIS29 and PlsΔIS8. (A) Heat inactivation profiles. The enzymes (8.0 μg/ml) were incubated at 95°C in buffer A for the time intervals indicated, and an azocaseinolytic activity assay was performed. Residual activity is expressed as a percentage of the original activity of each enzyme sample. The inset shows the SDS-PAGE analysis of purified mature PlsΔIS29 and PlsΔIS8. (B) Temperature dependence of azocaseinolytic activity. Activity assays were performed in buffer A for 10 min at the indicated temperatures using 0.5% azocasein as the substrate. The values are expressed as means ± SD (bars) from three independent experiments. (C) Digestion patterns of β-casein cleaved by the enzymes. The reaction was carried out at 85°C in buffer A containing 0.1 mg/ml of β-casein and 0.5 nM enzyme for different time periods, and then the samples were subjected to tricine-SDS-PAGE analysis.

FIG 6

Salt-induced formation of nicked pyrolysin. (A) SDS-PAGE analysis of mPls salt-induced autocleavage. The purified sample (15.0 μg/ml) of mPls in buffer A was incubated at 95°C for 1 h in the absence (−) or presence (+) of 100 or 600 mM NaCl. The proteins were precipitated with TCA and electrophoresed using tricine–SDS-PAGE. Samples incubated in 100 or 600 mM NaCl were loaded with a 4-fold increase in protein concentration compared with samples without NaCl. The positions of mPls (M) and the autocleavage products (I, II, III, IV, and V) are indicated with closed arrowheads, and Md is indicated with an open arrowhead. Bands of products I to V were assessed using N-terminal sequencing. (B) Effects of BSA on pyrolysin autocleavage. A purified sample (15.0 μg/ml) of mPls in buffer A with (+) or without (−) 100 mM NaCl was incubated at 95°C in the absence (−) or presence (+) of BSA (150.0 μg/ml). At the time intervals indicated, aliquots were withdrawn, precipitated with TCA, and analyzed using tricine–SDS-PAGE. The positions of mPls (M) and the autocleavage products I and II are indicated with closed arrowheads. (C) Urea–SDS-PAGE and gelatin overlay assays of nicked pyrolysin. Enzyme samples were electrophoresed using urea–SDS-PAGE (s), and a gelatin overlay assay was performed at 90°C (g). (D) Stability and activity of nicked enzyme. The enzymes (8.0 μg/ml) were incubated at 95°C in buffer A for the time intervals indicated and analyzed using an azocaseinolytic activity assay. Residual activity is expressed as a percentage of the original activity of each enzyme. The inset shows the specific activities of the enzymes against azocasein (0.5%) at 95°C. (E) Schematic representation of the primary structure of mPls and the identified autocleavage sites. The amino acid sequences of inserts IS29, IS27, and IS8 are indicated in boldface. The first four or five residues of products I to V, identified by N-terminal sequencing, are underlined. The locations of the two autocleavage sites a and b (Tyr²⁵¹-Gly²⁵² and Ala³⁹²-Tyr³⁹³ bonds) are indicated with vertical arrows. Double-headed arrows show the salt-induced autocleavage products of mPls. Residues predicted to be involved in ionic interactions and subsequently mutated are indicated with filled circles.

FIG 7

Mutation of residues involved in ionic interactions affects pyrolysin stability. (A) SDS-PAGE analysis of variant autocleavage. Purified samples (8.0 μg/ml) of mPls (WT) and its variants in buffer A were incubated at 95°C for the time intervals indicated and analyzed using SDS-PAGE. The positions of the mature form (M) and autocleavage products (I, II, III, and IV) are indicated. (B) Thermostabilities of the variants. The purified samples (8.0 μg/ml) of the WT and its variants in buffer A were incubated at 95°C for 12 h and analyzed using an azocaseinolytic activity assay. Residual activity is expressed as a percentage of the original activity of each enzyme sample. The values are expressed as means ± SD (bars) from three independent experiments.

FIG 8

Mutation of Arg²⁴⁹ in IS29 affects pyrolysin stability. (A and C) Heat inactivation of enzymes. Enzyme samples (8.0 μg/ml) in buffer A were incubated at 95°C (A) or 100 to 115°C (C) for the time intervals indicated and analyzed using an azocaseinolytic activity assay. Residual activity is expressed as a percentage of the original activity of each enzyme sample (control). The values are expressed as means ± SD (bars) from three independent experiments. The inset shows SDS-PAGE analysis of purified mature proteins. (B) SDS-PAGE analysis of the salt-induced autocleavage of the enzymes. The enzymes (8.0 μg/ml) in buffer A containing 100 mM NaCl were incubated at 95°C for the time intervals indicated and electrophoresed using SDS-PAGE. The positions of the mature form (M) and autocleavage products I and II are indicated with closed arrowheads.