Formation of the High-Affinity Calcium Binding Site in Pro-subtilisin E with the Insertion Sequence IS1 of Pro-Tk-subtilisin

Uehara, Ryo; Angkawidjaja, Clement; Koga, Yuichi; Kanaya, Shigenori

doi:10.1021/bi401342k

Cited by 16 publications

(17 citation statements)

References 46 publications

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“…While the signal that activates CspB has yet to be elucidated, a recent study showed that calcium is required for CspB-mediated cleavage of SleC (66). Since subtilisin-like proteases are calcium sensitive, one hypothesis is that CspB binds calcium and that this binding is required for CspB activity (66,(81)(82)(83)(84)(85). However, another study did not identify any calcium ions associated with the C. perfringens CspB (70).…”

Section: Initiation Of Cortex Hydrolysis: Cspbac Operonmentioning

confidence: 99%

Updates to Clostridium difficile Spore Germination

et al. 2018

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Germination of spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances in our understanding of spore germination mechanisms indicate that this process is both complex and unique. This review defines unique aspects of the germination pathways of and compares them to those of two other well-studied organisms, and germination is unique, as does not contain any orthologs of the traditional GerA-type germinant receptor complexes and is the only known sporeformer to require bile salts in order to germinate. While recent advances describing germination mechanisms have been made on several fronts, major gaps in our understanding of germination signaling remain. This review provides an updated, in-depth summary of advances in understanding of germination and potential avenues for the development of therapeutics, and discusses the major discrepancies between current models of germination and areas of ongoing investigation.

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Section: Initiation Of Cortex Hydrolysis: Cspbac Operonmentioning

confidence: 99%

Updates to Clostridium difficile Spore Germination

et al. 2018

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“…For M179, the hydroxyl group of Thr176 is located closely (2.9 Å) to the calcium ion, whereas Ala176 of AprE176 does not show any interaction with calcium ions. Bacillus fibrinolytic enzymes (subtilisin E [34], Carlsberg [25], and BPN [3]) have two calcium-binding sites, Ca A and Ca B sites. The Ca A site is located near the N-terminus and has higher calcium-binding affinity, whereas Ca B site has weaker binding affinity.…”

Section: Thermostability Of M179mentioning

confidence: 99%

Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176

Jeong¹,

Heo²,

Park³

et al. 2015

Journal of Microbiology and Biotechnology

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Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40°C, respectively. Error-prone PCR was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (kcat/Km) of M179 was not different from the wild-type enzyme owing to reduced substrate affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45°C, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176(th) residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca(2+), which increased the thermostability of M179.

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“…IS3-deleted mutant reveals the importance of IS3 in TKS folding Three insertion sequences (IS1-IS3) in the subtilisin domain of Tk-subtilisin form short surface loops in the tertiary structure [7,8]. We previously showed that IS1 and IS2 allow Tk-subtilisin to bind Ca 2+ ions, which facilitate the folding and maturation at high temperatures [10,13,14]. IS3, in contrast, is not responsible for Ca 2+ ion binding, yet this insertion loop seems necessary for the molecular architecture.…”

Section: Resultsmentioning

confidence: 99%

“…In this study, we found that IS3 further enhances the folding rate and, thereby, the maturation efficiency of proTKS by 10‐fold. Hence, all three insertions in TKS (IS1‐IS3) are eventually required for maturation at a high temperature, although the underlying mechanisms are different [10,13,14]. Such insertions can also be found in other groups of hyperthermophilic subtilases [38].…”

Section: Resultsmentioning

confidence: 99%

“…The highly conserved Ca1 site formation is the final step in the folding of many subtilisin-like proteases (subtilases). Mesophilic subtilisins lacking IS1 are partially unfolded and thermally unstable until autoprocessing due to the absence of Ca 2+ ions [11][12][13]. Tk-subtilisin bypasses such an unstable intermediate to enhance the folding efficiency at high temperatures.…”

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confidence: 99%

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Insertion loop‐mediated folding propagation governs efficient maturation of hyperthermophilic Tk‐subtilisin at high temperatures

et al. 2020

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The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments.

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Formation of the High-Affinity Calcium Binding Site in Pro-subtilisin E with the Insertion Sequence IS1 of Pro-Tk-subtilisin

Cited by 16 publications

References 46 publications

Updates to Clostridium difficile Spore Germination

Updates to Clostridium difficile Spore Germination

Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176

Insertion loop‐mediated folding propagation governs efficient maturation of hyperthermophilic Tk‐subtilisin at high temperatures

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