Serratia marcescens nuclease

Last updated
Serratia marcescens nuclease
Identifiers
EC no. 3.1.30.2
CAS no. 9025-65-4
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Search
PMC articles
PubMed articles
NCBI proteins
Serratia marcescens nuclease
Identifiers
Organism Serratia marcescens
SymbolnucA
UniProt P13717
Search for
Structures Swiss-model
Domains InterPro

Serratia marcescens nuclease (EC 3.1.30.2, endonuclease ( Serratia marcescens ), barley nuclease, plant nuclease I, nucleate endonuclease) is an enzyme. [1] [2] [3] [4] This enzyme catalyses the following chemical reaction

Contents

Endonucleolytic cleavage to 5'-phosphomononucleotide and 5'-phosphooligonucleotide end-products

Hydrolyses double- or single-stranded substrate DNA or RNA. It is a representative of the DNA/RNA non-specific endonuclease family.

It is commercially available.

Characteristics

Serratia nuclease was first purified from its native source in 1969. [5] It was cloned in 1987 and shown to consist of a 266 protein precursor, [6] which is further cleaved and secreted as a 245 amino acid active nuclease. [7] Its active form in solution is a homodimer. [8] It has two disulfide bonds, the first between cysteine 30 & 34 and the second between cysteine 222 & 264. [7] Reduction of these disulfides or site directed mutagenesis of their residues to serine, specifically the first one, leads to a large loss in nuclease activity, [8] and a loss of the ability to reversibly regain activity after inactivating 40-60˚C heat treatments. [7] It has a much higher catalytic efficiency than other nucleases, about 4 times greater than staphylococcal nuclease, and about 34 times greater than bovine pancreatic DNase I. [8] The enzyme cleaves single or double stranded DNA and RNA with similar rates, so long as the substrate DNA or RNA contains no fewer than 5 nucleotides (or basepairs). [8] Magnesium (II) (Mg2+) is an essential cofactor for its nuclease activity. [8] Serratia nuclease is activated by up to 4M urea. [9] At 5M urea the initial activity is decreased from its peak although still above its baseline, and the enzyme is significantly inhibited after 60 minutes. At 6M urea, the nuclease activity is below baseline and almost completely inactivated within 60 minutes. At 7M the nuclease becomes essentially completely inactivated within 15 minutes, but significant and workable degradation of nucleic acids can occur before the nuclease is inactivated. [9] 8M urea causes a complete inactivation of the enzyme within 5 minutes. [7]

Optimal conditions [9]

ConditionOptimal1Effective2
Mg2+ concentration1 - 2 mM1 - 10 mM
pH 8.2 - 9.26.0 - 10.0
Temperature 37˚C0 - 42˚C
Dithiothreitol (DTT)< 100 mM> 100 mM
β-Mercaptoethanol (BME)< 100 mM> 100 mM
Monovalent cation concentration (Na+, K+, etc.)0 - 20 mM0 - 150 mM
PO43- 0 - 10 mM0 - 100 mM
Urea < 4M> 4M

1="Optimal" is the condition in which Serratia nuclease retains >90 % of its activity.

2="Effective" is the condition in which Serratia nuclease retains >15 % of its activity.

Inhibitory conditions

Some inhibitory conditions are known: [9]

Use in biotechnology

Given its high activity, high stability & reversible inactivation to heat treatments, rate enhancement or otherwise compatibility with some denaturing reagents like urea, Serratia nuclease was recognized early on to have industrial & commercialization potential. A patent covering the recombinant expression of Serratia nuclease in E. coli was submitted by Benzon Pharma in 1986, granted in 1992, & expired in 2006. [10] This recombinant Serratia nuclease was commercialized as Benzonase, and is still available from and a registered trademark of Merck KGaA. [11] Notably, the patented sequence [10] [12] for Benzonase is slightly different (1 amino acid substitution) from the Serratia marcescens nuclease which was cloned publicly. [13]

As the benzonase patent is now expired, and in fact was never submitted nor granted in the United States, several commercial alternatives for recombinantly produced Serratia marcescens nuclease are now available:

(A current notable non-producer is New England Biolabs) [23]

See also

Related Research Articles

A restriction enzyme, restriction endonuclease, REase, ENase orrestrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

<span class="mw-page-title-main">Nuclease</span> Class of enzymes

A nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.

<span class="mw-page-title-main">Protein splicing</span> The post-translational removal of peptide sequences from within a protein sequence

Protein splicing is an intramolecular reaction of a particular protein in which an internal protein segment is removed from a precursor protein with a ligation of C-terminal and N-terminal external proteins on both sides. The splicing junction of the precursor protein is mainly a cysteine or a serine, which are amino acids containing a nucleophilic side chain. The protein splicing reactions which are known now do not require exogenous cofactors or energy sources such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP). Normally, splicing is associated only with pre-mRNA splicing. This precursor protein contains three segments—an N-extein followed by the intein followed by a C-extein. After splicing has taken place, the resulting protein contains the N-extein linked to the C-extein; this splicing product is also termed an extein.

In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically, while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. Endonucleases differ from exonucleases, which cleave the ends of recognition sequences instead of the middle (endo) portion. Some enzymes known as "exo-endonucleases", however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like. Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity.

<span class="mw-page-title-main">Exonuclease</span> Class of enzymes; type of nuclease

Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have three types of exonucleases involved in the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), which is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly(A)-specific 3′ to 5′ exonuclease.

<span class="mw-page-title-main">Micrococcal nuclease</span> Class of enzymes

Micrococcal nuclease is an endo-exonuclease that preferentially digests single-stranded nucleic acids. The rate of cleavage is 30 times greater at the 5' side of A or T than at G or C and results in the production of mononucleotides and oligonucleotides with terminal 3'-phosphates. The enzyme is also active against double-stranded DNA and RNA and all sequences will be ultimately cleaved.

Mung bean nuclease is a nuclease derived from sprouts of the mung bean that removes nucleotides in a step-wise manner from single-stranded DNA molecules (ssDNA) and is used in biotechnological applications to remove such ssDNA from a mixture also containing double-stranded DNA (dsDNA). This enzyme is useful for transcript mapping, removal of single-stranded regions in DNA hybrids or single-stranded overhangs produced by restriction enzymes, etc. It has an activity similar to Nuclease S1, but it has higher specificity for single-stranded molecules.

<span class="mw-page-title-main">Homing endonuclease</span>

The homing endonucleases are a collection of endonucleases encoded either as freestanding genes within introns, as fusions with host proteins, or as self-splicing inteins. They catalyze the hydrolysis of genomic DNA within the cells that synthesize them, but do so at very few, or even singular, locations. Repair of the hydrolyzed DNA by the host cell frequently results in the gene encoding the homing endonuclease having been copied into the cleavage site, hence the term 'homing' to describe the movement of these genes. Homing endonucleases can thereby transmit their genes horizontally within a host population, increasing their allele frequency at greater than Mendelian rates.

<span class="mw-page-title-main">Nuclease S1</span> Class of enzymes

Nuclease S1 is an endonuclease enzyme that splits single-stranded DNA (ssDNA) and RNA into oligo- or mononucleotides. This enzyme catalyses the following chemical reaction

Deoxyribonuclease IV (phage-T4-induced) is catalyzes the degradation nucleotides in DsDNA by attacking the 5'-terminal end.

<span class="mw-page-title-main">Proteinase K</span> Broad-spectrum serine protease

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.

PstI is a type II restriction endonuclease isolated from the Gram negative species, Providencia stuartii.

<span class="mw-page-title-main">FPG IleRS zinc finger</span>

The FPG IleRS zinc finger domain represents a zinc finger domain found at the C-terminal in both DNA glycosylase/AP lyase enzymes and in isoleucyl tRNA synthetase. In these two types of enzymes, the C-terminal domain forms a zinc finger.

<span class="mw-page-title-main">DNA/RNA non-specific endonuclease</span>

In molecular biology, enzymes in the DNA/RNA non-specific endonuclease family of bacterial and eukaryotic endonucleases EC 3.1.30.- share the following characteristics: they act on both DNA and RNA, cleave double-stranded and single-stranded nucleic acids and require a divalent ion such as magnesium for their activity. A histidine has been shown to be essential for the activity of the Serratia marcescens nuclease. This residue is located in a conserved region which also contains an aspartic acid residue that could be implicated in the binding of the divalent ion.

<span class="mw-page-title-main">XPG I protein domain</span>

In molecular biology, the XPG-I is a protein domain found on Xeroderma Pigmentosum Complementation Group G (XPG) protein. The XPG protein is an endonuclease which repairs DNA damage caused by ultraviolet light. The XPG protein repairs DNA by a process called, Nucleotide excision repair. Mutations in the protein commonly cause Xeroderma Pigmentosum which often lead to skin cancer.

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

<i>Eco</i>RI

EcoRI is a restriction endonuclease enzyme isolated from species E. coli. It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modification system. The Eco part of the enzyme's name originates from the species from which it was isolated - "E" denotes generic name which is "Escherichia" and "co" denotes species name, "coli" - while the R represents the particular strain, in this case RY13, and the I denotes that it was the first enzyme isolated from this strain.

<span class="mw-page-title-main">George Stark</span>

George Stark is an American chemist and biochemist. His research interests include protein and enzyme function and modification, interferons and cytokines, signal transduction, and gene expression.

References

  1. ↑ Mikulski AJ, Laskowski M (October 1970). "Mung bean nuclease I. 3. Purification procedure and (3') omega monophosphatase activity". The Journal of Biological Chemistry. 245 (19): 5026–5031. doi: 10.1016/S0021-9258(18)62813-3 . PMID   4319109.
  2. ↑ Stevens A, Hilmoe RJ (1960). "Studies on a nuclease from Azotobacter agilis. I. Isolation and mode of action". Journal of Biological Chemistry. 235 (10): 3016–3022. doi: 10.1016/S0021-9258(18)64581-8 .
  3. ↑ Stevens A, Hilmoe RJ (1960). "Studies on a nuclease from Azotobacter agilis. II. Hydrolysis of ribonucleic and deoxyribonucleic acids". Journal of Biological Chemistry. 235 (10): 3023–3027. doi: 10.1016/S0021-9258(18)64582-X .
  4. ↑ Wechter WJ, Mikulski AJ, Laskowski M (February 1968). "Gradation of specificity with regard to sugar among nucleases". Biochemical and Biophysical Research Communications. 30 (3): 318–322. doi:10.1016/0006-291x(68)90453-1. PMID   4296679.
  5. ↑ Nestle M, Roberts WK (October 1969). "An extracellular nuclease from Serratia marcescens. I. Purification and some properties of the enzyme". The Journal of Biological Chemistry. Elsevier BV. 244 (19): 5213–5218. doi: 10.1016/s0021-9258(18)63648-8 . PMID   4899013.
  6. ↑ Ball TK, Saurugger PN, Benedik MJ (1987). "The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli". Gene. Elsevier BV. 57 (2–3): 183–192. doi:10.1016/0378-1119(87)90121-1. PMID   3319779.
  7. 1 2 3 4 Biedermann K, Jepsen PK, Riise E, Svendsen I (1989). "Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli". Carlsberg Research Communications. Springer Science and Business Media LLC. 54 (1): 17–27. doi: 10.1007/bf02910469 . PMID   2665765. S2CID   12831178.
  8. 1 2 3 4 5 Benedik MJ, Strych U (August 1998). "Serratia marcescens and its extracellular nuclease". FEMS Microbiology Letters. Oxford University Press (OUP). 165 (1): 1–13. doi: 10.1111/j.1574-6968.1998.tb13120.x . PMID   9711834.
  9. 1 2 3 4 "Benzonase® Nuclease - Effective removal of nucleic acids and viscosity reduction from protein solutions" (PDF). EMD Biosciences. SigmaAldrich. Retrieved 29 April 2023.
  10. 1 2 EP 0229866A1,Molin S, Givskov M, Riise E,"Bacterial enzymes and method for their production",issued 9 December 1992, assigned to Benzon Pharma ASand Takeda Pharma AS
  11. ↑ "Benzonase® Nuclease HC, Purity > 99% - 71206". MilliporeSigma. Retrieved 2023-04-29.
  12. ↑ "UniProt". UniProt. Retrieved 2023-04-29.
  13. ↑ "UniProt". UniProt. Retrieved 2023-04-29.
  14. ↑ "Basemuncher Benzonase". Westburg. Retrieved 2023-04-29.
  15. ↑ "Benzo Nuclease". Tinzyme Ltd – Enzymes, dNTP and rNTP. 2021-12-24. Retrieved 2023-04-29.
  16. ↑ "Benz-Neburase™, His". GenScript. 2021-08-12. Retrieved 2023-04-29.
  17. ↑ "B-1400-5KU - Decontaminase™, 5 KU". AG Scientific. 2022-12-13. Retrieved 2023-04-29.
  18. ↑ "Denarase". c-LEcta. 2022-12-13. Retrieved 2023-04-29.
  19. ↑ "Benzonase Nuclease Alternative, DENARASE Nuclease Alternative". Syd Labs. 2020-05-01. Retrieved 2023-04-29.
  20. ↑ "GENIUS™Nuclease DMF Filed". ACROBiosystems. Retrieved 2023-04-29.
  21. ↑ "Pierce™ Universal Nuclease for Cell Lysis". Thermo Fisher Scientific. 2023-04-29. Retrieved 2023-04-29.
  22. ↑ "TurboNuclease". Accelagen. 2023-04-29. Retrieved 2023-04-29.
  23. ↑ Biolabs, New England. "DNA Modifying Enzymes & Cloning Technologies - Exonucleases and Non-specific Endonucleases". New England Biolabs. Retrieved 30 April 2023.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.