Micrococcal nuclease
Encyclopedia
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.
has a molecular weight of 16.9kDa.
The pH
optimum is reported as 9.2. The enzyme
activity is strictly dependent on Ca2+ and the pH
optimum varies according to Ca2+ concentration. The enzyme
is therefore easily inacitvated by EGTA
.
of Staphylococcus aureus
. Two strains, V8 and Foggi, yield almost identical enzymes. A common source is E.coli
cells carrying a cloned nuc gene
encoding Staphylococcus
aureus extracellular nuclease (micrococcal nuclease).
, in 1969, deposited as now-obsolete Protein Data Bank
file 1SNS. Higher-resolution, more recent crystal structures are available for the apo form as Protein Data Bank file 1SNO: http://www.rcsb.org/pdb/explore/explore.do?structureId=1SNO and for the thymidine-diphosphate-inhibited form as Protein Data Bank file 3H6M: http://www.rcsb.org/pdb/explore/explore.do?structureId=3H6M. As seen in the ribbon diagram
above, the nuclease molecule has 3 long alpha helices
and a 5-stranded, barrel-shaped beta sheet
, in an arrangement known as the OB-fold (for oligonucleotide-binding fold) as classified in the SCOP
database.
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
-exonuclease
Exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end 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 ...
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
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
and all sequences will be ultimately cleaved.
Characteristics
The enzymeEnzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
has a molecular weight of 16.9kDa.
The pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
optimum is reported as 9.2. The enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
activity is strictly dependent on Ca2+ and the pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
optimum varies according to Ca2+ concentration. The enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
is therefore easily inacitvated by EGTA
EGTA (chemical)
EGTA is a polyamino carboxylic acid, a chelating agent that is related to the better known EDTA, but with a much higher affinity for calcium than for magnesium ions...
.
Source
This enzyme is the extracellular nucleaseNuclease
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase"....
of Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus is a facultative anaerobic Gram-positive coccal bacterium. It is frequently found as part of the normal skin flora on the skin and nasal passages. It is estimated that 20% of the human population are long-term carriers of S. aureus. S. aureus is the most common species of...
. Two strains, V8 and Foggi, yield almost identical enzymes. A common source is E.coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
cells carrying a cloned nuc gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
encoding Staphylococcus
Staphylococcus
Staphylococcus is a genus of Gram-positive bacteria. Under the microscope they appear round , and form in grape-like clusters....
aureus extracellular nuclease (micrococcal nuclease).
Structure
The 3-dimensional structure of micrococcal nuclease (then called Staphyloccal nuclease) was solved very early in the history of protein crystallographyX-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
, in 1969, deposited as now-obsolete Protein Data Bank
Protein Data Bank
The Protein Data Bank is a repository for the 3-D structural data of large biological molecules, such as proteins and nucleic acids....
file 1SNS. Higher-resolution, more recent crystal structures are available for the apo form as Protein Data Bank file 1SNO: http://www.rcsb.org/pdb/explore/explore.do?structureId=1SNO and for the thymidine-diphosphate-inhibited form as Protein Data Bank file 3H6M: http://www.rcsb.org/pdb/explore/explore.do?structureId=3H6M. As seen in the ribbon diagram
Ribbon diagram
Proteins are biological macromolecules made up of a long polypeptide chain of amino acids linked by peptide bonds...
above, the nuclease molecule has 3 long alpha helices
Alpha helix
A common motif in the secondary structure of proteins, the alpha helix is a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier...
and a 5-stranded, barrel-shaped beta sheet
Beta sheet
The β sheet is the second form of regular secondary structure in proteins, only somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet...
, in an arrangement known as the OB-fold (for oligonucleotide-binding fold) as classified in the SCOP
Structural Classification of Proteins
The Structural Classification of Proteins database is a largely manual classification of protein structural domains based on similarities of their structures and amino acid sequences. A motivation for this classification is to determine the evolutionary relationship between proteins...
database.
Applications
- Hydrolysis of nucleic acids in crude cell-free extracts.
- Sequencing of RNARNARibonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
. - Preparation of rabbit reticulocytes lysates
- Studies of chromatinChromatinChromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
structure. - Remove nucleic acids from proteinProteinProteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
preparation allowing for folding and structure-function studies. - Research on the mechanism of protein foldingProtein foldingProtein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil....
.