S1 Pocket of a Bacterially Derived Subtilisin-like Protease Underpins Effective Tissue Destruction

Wong, Wilson; Wijeyewickrema, Lakshmi C.; Kennan, Ruth M.; Reeve, Shane; Steer, David; Reboul, Cyril; Smith, A. Ian; Pike, Robert N.; Rood, Julian I.; Whisstock, James C.; Porter, Corrine Joy

doi:10.1074/jbc.m111.298711

Cited by 18 publications

(22 citation statements)

References 30 publications

(29 reference statements)

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“…studies on site-directed mutagenesis at Gly166 in subtilisin, and that hydrophobic substitutions at position 166 could change catalytic efficiency towards small hydrophobic peptides. 28 In our study, the mutant S180G also showed an increase in keratinolytic activity. For example, the mutagenesis Tyr215 / Gly obtained the maximum activities to AAPF and AAPL by shortening the side chain and reducing the electrostatic effect to increase the depth of the S1 pocket (Fig.…”

Section: Structure Modeling and Analysis Of Wild Type Keratinase And supporting

confidence: 61%

“…5, which showed that constitutive factors of substrate-binding cles were important to keratinolytic activity of KerSMD. 16,28 It has been reported that the S1 pocket of subtilisin-like protease is near the catalytic center and binds to special residues in the protein substrate. 5).…”

Section: Structure Modeling and Analysis Of Wild Type Keratinase And mentioning

confidence: 99%

“…The substitutions of Gly and Ser were all short side chains, opening the pocket entrance to bind to a larger residue at the P1 site (Fig. [28][29][30] KerSMD has a subtilisin-like catalytic domain and broad specicity to hydrolyze casein, collagen, and keratin, and it may be related to the S1 pocket. We deduce that the size of the S1 pocket is the main factor on substrate selectivity.…”

Section: Structure Modeling and Analysis Of Wild Type Keratinase And mentioning

confidence: 99%

“…18 Since many keratinases belong to the subtilisin-like protease and they oen share highly conserved amino acid sequences, site-directed mutagenesis based on homology modeling can be used to modify enzyme characteristics. 28,33 Except for subtilisin BPN 0 , crystal structures and molecular modications of proteases were investigated and it stated that S1 pocket is important to identify special residues for binding and catalysis. 18 So, the site-directed mutagenesis is convenient to shi the substrate specicity by changing the size or electrostatic interactions of the substrate binding cle.…”

Section: Introductionmentioning

confidence: 99%

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Insight into the substrate specificity of keratinase KerSMD from Stenotrophomonas maltophilia by site-directed mutagenesis studies in the S1 pocket

et al. 2015

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The keratinase KerSMD from Stenotrophomonas maltophilia can hydrolyze a broad range of substrates but its keratinolytic activity needs to be improved for industrial application. From sequence alignment and homologous modeling, we deduced that four residues, Ser180, Glu208, Tyr215, and Arg216, lying at the entrance or bottom of the S1 pocket, were related to the substrate specificity. The S1 pocket was enlarged by mutating a series of amino acids. We found that mutagenesis at position 215 could shift the catalytic ability of KerSMD to hydrolyze synthetic peptides and macromolecular substrates. We improved the keratinolytic activities of five mutants (Y215G, Y215S, Y215A, S180G/Y215A, and S180G/Y215S) and obtained two thermophilic keratinases (Y215S and S180G/Y215S). Compared to the wild type, the S180G/ Y215S showed the highest keratinolytic activity (4755 U mg À1 ) and the S180G/Y215A showed the most excellent specificity to degrade feather. Our results indicate that steric hindrance and hydrophilia in the S1 pocket have a significant effect on feather keratin preference of keratinase, and the hydrogen bonds in the S1 pocket have great influence on the thermostability. These findings not only give an insight into the relationship between the S1 pocket and substrate specificity but also suggest approaches for protein engineering of keratinase.

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Section: Structure Modeling and Analysis Of Wild Type Keratinase And supporting

confidence: 61%

Section: Structure Modeling and Analysis Of Wild Type Keratinase And mentioning

confidence: 99%

Section: Structure Modeling and Analysis Of Wild Type Keratinase And mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Insight into the substrate specificity of keratinase KerSMD from Stenotrophomonas maltophilia by site-directed mutagenesis studies in the S1 pocket

et al. 2015

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“…The catalytic domain model was based on crystal structures of subtilisin-like proteases (Protein Data Bank: 3LPA, 3LPC, 3LPD, 3TI937, 3TI737, 1DBI38, 1THM39, 3AFG17), and the lowest score model was chosen. We did the energy minimization and fully reduced the steric clashes of model protein by molecular dynamics (MD) simulations with NAMD software (http://www.ks.uiuc.edu/).…”

Section: Methodsmentioning

confidence: 99%

Improved catalytic efficiency, thermophilicity, anti-salt and detergent tolerance of keratinase KerSMD by partially truncation of PPC domain

Fang

Zhang

et al. 2016

Sci Rep

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The keratinase from Stenotrophomonas maltophilia (KerSMD) is known for its high activity and pH stability in keratin degradation. However, catalytic efficiency and detergent tolerability need to be improved in order to be used for industrial application. In this work, we obtained several keratinase variants with enhanced catalytic efficiency, thermophilicity, and anti-salt and detergent tolerability by partially truncating the PPC domain of KerSMD. The variants all showed improved catalytic efficiency to synthetic substrate AAPF, with the V355 variant having the highest kcat /Km value of 143.6 s−1 mM−1. The truncation of keratinase had little effect on alkaline stability but obviously decreased collagenase activity, developing its potential application in leather treatment. The variants V380, V370, and V355 were thermophilic, with a 1.7-fold enhancement of keratinlytic activity at 60 °C when compared to the wild type. The entire truncation of PPC domain obtained the variant V355 with improved tolerance to alkalinity, salt, chaotropic agents, and detergents. The V355 variant showed more than a 40% improvement in activity under 15% (w/v) NaCl or 4% (w/v) SDS solution, showing excellent stability under harsh washing and unhairing conditions. Our work investigated how protein engineering affects the function of PPC domain of KerSMD.

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Non‐proteolytic functions of microbial proteases increase pathological complexity

2015

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Proteases are enzymes that catalyse hydrolysis of peptide bonds thereby controlling the shape, size, function, composition, turnover and degradation of other proteins. In microbes, proteases are often identified as important virulence factors and as such have been targets for novel drug design. It is emerging that some proteases possess additional non-proteolytic functions that play important roles in host epithelia adhesion, tissue invasion and in modulating immune responses. These additional "moonlighting" functions have the potential to obfuscate data interpretation and have implications for therapeutic design. Moonlighting enzymes comprise a subcategory of multifunctional proteins that possess at least two distinct biological functions on a single polypeptide chain. Presently, identifying moonlighting proteins relies heavily on serendipitous empirical data with clues arising from proteins lacking signal peptides that are localised to the cell surface. Here, we describe examples of microbial proteases with additional non-proteolytic functions, including streptococcal pyrogenic exotoxin B, PepO and C5a peptidases, mycoplasmal aminopeptidases, mycobacterial chaperones and viral papain-like proteases. We explore how these non-proteolytic functions contribute to host cell adhesion, modulate the coagulation pathway, assist in non-covalent folding of proteins, participate in cell signalling, and increase substrate repertoire. We conclude by describing how proteomics has aided in moonlighting protein discovery, focusing attention on potential moonlighters in microbial exoproteomes.

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S1 Pocket of a Bacterially Derived Subtilisin-like Protease Underpins Effective Tissue Destruction

Cited by 18 publications

References 30 publications

Insight into the substrate specificity of keratinase KerSMD from Stenotrophomonas maltophilia by site-directed mutagenesis studies in the S1 pocket

Insight into the substrate specificity of keratinase KerSMD from Stenotrophomonas maltophilia by site-directed mutagenesis studies in the S1 pocket

Improved catalytic efficiency, thermophilicity, anti-salt and detergent tolerance of keratinase KerSMD by partially truncation of PPC domain

Non‐proteolytic functions of microbial proteases increase pathological complexity

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