Peptidase S8, subtilisin-related | |||||||||
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Identifiers | |||||||||
Symbol | Peptidase_S8 | ||||||||
Pfam | PF00082 | ||||||||
InterPro | IPR015500 | ||||||||
PROSITE | PDOC00125 | ||||||||
CATH | 1cse | ||||||||
SCOP2 | 1cse / SCOPe / SUPFAM | ||||||||
CDD | cd07477 | ||||||||
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Subtilisin BPN' | |||||||
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Identifiers | |||||||
Organism | |||||||
Symbol | apr | ||||||
CAS number | 9014-01-1 | ||||||
Entrez | 5712479 | ||||||
PDB | 1st2 More structures | ||||||
UniProt | P00782 | ||||||
Other data | |||||||
EC number | 3.4.21.62 | ||||||
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GO:0004252 |
Subtilisin is a protease (a protein-digesting enzyme) initially obtained from Bacillus subtilis . [2] [3] [4] [5] [6] [7] [8]
Subtilisins belong to subtilases, a group of serine proteases that – like all serine proteases – initiate the nucleophilic attack on the peptide (amide) bond through a serine residue at the active site. Subtilisins typically have molecular weights 27kDa. They can be obtained from certain types of soil bacteria, for example, Bacillus amyloliquefaciens from which they are secreted in large amounts.
"Subtilisin" does not refer to a single protein, but to an entire clade under subtilases containing the classical subtilisins. The clade can be further divided into four groups: "true subtilisins" (containing the classical members), "high-alkaline subtilisins", "intracellular subtilisins", and "phylogenetically intermediate subtilisins" (PIS). [9] [10] Notable subtilisins include:
Family | Organism | Uniprot | Names | Notes |
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True | B. licheniformis | P00780 | Subtilisin Carlsberg, Alcalase (Novozymes), Maxatase (?) "subtilisin DY" (X-ray mutant) [11] | Type serine endopeptidase of MEROPS family S8. |
? | B. licheniformis | ? | Endocut-02L (Tailorzyme ApS) | |
? | ? | ? | bioprase, bioprase AL | |
? | Lederbergia lenta | Esperase (Novozymes) | Structure determined, but not found on PDB. [12] | |
High-alkaline | Lederbergia lenta | P29600 | Subtilisin Savinase, Savinase (Novozymes) | PDB: 1SVN [13] |
True | B. amyloliquefaciens | P00782 | Subtilisin BPN’, Alcalase (Novozymes) | |
? | Geobacillus stearothermophilus | P29142 | Subtilisin J, Thermoase (Amano) | [14] |
Other non-commercial names include ALK-enzyme, bacillopeptidase, Bacillus subtilis alkaline proteinase, colistinase, genenase I, protease XXVII, subtilopeptidase, kazusase, protease VIII, protin A 3L, protease S.
Other commercial names with unidentified molecular identities include SP 266, orientase 10B (HBI Enzymes), Progress (Novozyme), Liquanase (Novozyme).
The structure of subtilisin has been determined by X-ray crystallography. The mature form is a 275-residue globular protein with several alpha-helices, and a large beta-sheet. The N-terminal contains an I9 propeptide domain (InterPro : IPR010259 ) that assists the folding of subtilisin. Proteolytic removal of the domain activates the enzyme. It is structurally unrelated to the chymotrypsin-clan of serine proteases, but uses the same type of catalytic triad in the active site. This makes it a classic example of convergent evolution.
The active site features a charge-relay network involving Asp-32, His-64, and active site Ser-221 arranged in a catalytic triad. The charge-relay network functions as follows: The carboxylate side-chain of Asp-32 hydrogen-bonds to a nitrogen-bonded proton on His-64's imidazole ring. This is possible because Asp is negatively charged at physiological pH. The other nitrogen on His-64 hydrogen-bonds to the O-H proton of Ser-221. This last interaction results in charge-separation of O-H, with the oxygen atom being more nucleophilic. This allows the oxygen atom of Ser-221 to attack incoming substrates (i.e., peptide bonds), assisted by a neighboring carboxyamide side-chain of Asn-155.
Even though Asp-32, His-64, and Ser-221 are sequentially far apart, they converge in the 3D structure to form the active site.
To summarize the interactions described above, Ser-221 acts as a nucleophile and cleaves peptide bonds with its partially negative oxygen atom. This is possible due to the nature of the charge-relay site of subtilisin.
In molecular biology using B. subtilis as a model organism, the gene encoding subtilisin (aprE) is often the second gene of choice after amyE for integrating reporter constructs into, due to its dispensability.
Protein-engineered subtilisins are widely used in commercial products (the native enzyme is easily inactivated by detergents and high temperatures) and is also called a stain cutter, for example, in laundry [15] and dishwashing detergents, cosmetics, food processing, [16] skin care products, contact lens cleaners, and for research in synthetic organic chemistry.
People can be exposed to subtilisin in the workplace by breathing it in, swallowing it, skin contact, and eye contact. The National Institute for Occupational Safety and Health (NIOSH) has set a recommended exposure limit (REL) of 60 ng/m3 over a 60-minute period. [17]
Subtilisin can cause "enzymatic detergent asthma". People who are sensitive to Subtilisin (Alcalase) usually are also allergic to the bacterium Bacillus subtilis. [18]
Bacillus is a genus of Gram-positive, rod-shaped bacteria, a member of the phylum Bacillota, with 266 named species. The term is also used to describe the shape (rod) of other so-shaped bacteria; and the plural Bacilli is the name of the class of bacteria to which this genus belongs. Bacillus species can be either obligate aerobes which are dependent on oxygen, or facultative anaerobes which can survive in the absence of oxygen. Cultured Bacillus species test positive for the enzyme catalase if oxygen has been used or is present.
Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called proteases, but may also occur by intra-molecular digestion.
A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in numerous biological pathways, including digestion of ingested proteins, protein catabolism, and cell signaling.
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the binding site, and residues that catalyse a reaction of that substrate, the catalytic site. Although the active site occupies only ~10–20% of the volume of an enzyme, it is the most important part as it directly catalyzes the chemical reaction. It usually consists of three to four amino acids, while other amino acids within the protein are required to maintain the tertiary structure of the enzymes.
Serine proteases are enzymes that cleave peptide bonds in proteins. Serine serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both eukaryotes and prokaryotes. Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.
A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes. An acid-base-nucleophile triad is a common motif for generating a nucleophilic residue for covalent catalysis. The residues form a charge-relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine amino acid, but occasionally threonine or even selenocysteine. The 3D structure of the enzyme brings together the triad residues in a precise orientation, even though they may be far apart in the sequence.
Bacillus licheniformis is a bacterium commonly found in the soil. It is found on bird feathers, especially chest and back plumage, and most often in ground-dwelling birds and aquatic species.
Aspartic proteases are a catalytic type of protease enzymes that use an activated water molecule bound to one or more aspartate residues for catalysis of their peptide substrates. In general, they have two highly conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin.
TEV protease is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). It is a member of the PA clan of chymotrypsin-like proteases. Due to its high sequence specificity, TEV protease is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo. The consensus sequence recognized by TEV protease is Glu-Asn-Leu-Tyr-Phe-Gln-|-Ser, where "|" denotes cleaved peptide bond.
Nattokinase is an enzyme extracted and purified from a Japanese food called nattō. Nattō is produced by fermentation by adding the bacterium Bacillus subtilisvar natto, which also produces the enzyme, to boiled soybeans. While other soy foods contain enzymes, it is only the nattō preparation that contains the specific nattokinase enzyme.
In molecular biology, Proteinase K is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Parengyodontium album. Proteinase K is able to digest hair (keratin), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8 (subtilisin). The molecular weight of Proteinase K is 28,900 daltons.
Subtilases are a family of subtilisin-like serine proteases. They appear to have independently and convergently evolved an Asp/Ser/His catalytic triad, like in the trypsin serine proteases. The structure of proteins in this family shows that they have an alpha/beta fold containing a 7-stranded parallel beta sheet.
Keratinases are proteolytic enzymes that digest keratin.
In molecular biology the protein SSI is a Subtilisin inhibitor-like which stands for Streptomyces subtilisin inhibitor. This is a protease inhibitor. These are often synthesised as part of a larger precursor protein, either as a prepropeptide. The function of this protein domain is to prevent access of the substrate to the active site. It is found only in bacteria.
Cyanophycinase (EC 3.4.15.6, cyanophycin degrading enzyme, beta-Asp-Arg hydrolysing enzyme, CGPase, CphB, CphE, cyanophycin granule polypeptidase, extracellular CGPase) is an enzyme. It catalyses the following chemical reaction
Oryzin is an enzyme. This enzyme catalyses the following chemical reaction
Thermitase is an enzyme. This enzyme catalyses the following chemical reaction
Bacillolysin is an enzyme. This enzyme catalyses the following chemical reaction
Ribosomally synthesized and post-translationally modified peptides (RiPPs), also known as ribosomal natural products, are a diverse class of natural products of ribosomal origin. Consisting of more than 20 sub-classes, RiPPs are produced by a variety of organisms, including prokaryotes, eukaryotes, and archaea, and they possess a wide range of biological functions.
The sedolisin family of peptidases are a family of serine proteases structurally related to the subtilisin (S8) family. Well-known members of this family include sedolisin ("pseudomonalisin") found in Pseudomonas bacteria, xanthomonalisin ("sedolisin-B"), physarolisin as well as animal tripeptidyl peptidase I. It is also known as sedolysin or serine-carboxyl peptidase. This group of enzymes contains a variation on the catalytic triad: unlike S8 which uses Ser-His-Asp, this group runs on Ser-Glu-Asp, with an additional acidic residue Asp in the oxyanion hole.