Re: What is site-directed mutagenesis?

What is site specific mutagenesis?

Site specific mutagenesis is the term used to describe when changes in DNA are made at a desired position. This means that someone has changed one (or more) of the bases in the DNA sequence of the gene which is being studied. This results in an amino acid change in the protein that the gene codes for. Apparently you're at least in college, so I'll just give a brief explanation of methods of site specific mutagenesis.

The sequence of the bases in DNA literally spell out the information which is used to make a protein. By changing one of these bases, a change in the DNA sequence is produced. This changes the protein that the DNA sequence "spells out".

DNA is a double stranded molecule. One strand will have the code for the protein, and the other strand will be complementary, that is, it will be the "antisense" strand. If these strands are separated, each strand can act as a template and direct the synthesis of its complementary strand.

In site specific mutagenesis, a single strand of DNA, usually 20 to 40 bases long, is synthesized in the lab. This strand of DNA, known as an oligonucleotide or "oligo", will be identical to the gene to be changed, except that the oligo will contain the desired base change.

There are two ways to proceed with this oligo.

One way to do site specific mutagenesis is to start with the gene you want to change in double strand form. The strands of the DNA are separated by heating. The oligo is present, and it will hybridize (pair) with its complementary strand. Then DNA polymerase converts the single strand DNA to double strand DNA. This newly made double strand DNA is like the original DNA double helix, except that one of the bases has been changed, and the base change has been directed by the sequence of the oligo that was used. The process is then repeated, and more mutated DNA is produced. This method is known as "PCR" mutagenesis.

Another way is to clone the gene into the bacteriophage M13 which produces its DNA in single strand form. This way, when the DNA is purified, it is already single stranded. The oligo can be hybridized, and the polymerase step can begin.

Site specific mutagenesis was invented in 1978 by Clyde Hutchison and Michael Smith. Michael Smith was given the Nobel Prize for discovering it in 1993. Many people (and me too) think that Clyde Hutchison deserved to share the prize with Smith, but the Nobel committee just honored Smith. Clyde Hutchison is my boss here at UNC, and he's a groovy dude!

There are many good sites that discuss site specific mutagenesis
http://biology.ecsu.ctstateu.edu/courses/molgen/ directed%20mutagen/SiteDiremut.html
http:// www.moorhead.msus.edu/chastain/html/mut.html
http:// www.nobel.se/chemistry/laureates/1993/illpres/site.html

You ask what is meant by the specific activity of an enzyme and how you could use site specific mutagenesis to improve the specific activity of the enzyme cellulase.

What is specific activity? Specific activity is the activity of a known amount of enzyme. Enzymes are catalysts. That means they cause a particular chemical reaction to go at a faster rate. Let's say you measure the rate of an enzyme reaction, like the breakdown of cellulose. The amount of cellulose being broken down per second by a sample of cellulase enzyme is the activity. But you would not know if your enzyme sample had a little bit of very active cellulase, or a lot of not very active cellulase. However, if you knew how much cellulase was in the sample, then you would know the amount of cellulose broken down per microgram of cellulase. This is known as specific activity. That way you can compare the activity of the cellulase in one enzyme sample to another.

Improving cellulase with site specific mutagenesis.

That's rather big order. It's being worked on by government and industry, including the CEA, funded by the DOE http://www.ceassist.com/assessment.htm. The CEA hopes for a 2x to 4x increase cellulase k_cat. The things they would do can be found at http:// www.ceassist.com/improvme.htm. Note that cellulase is a multienzyme system that contains three components: endo- and exo- glucanases and beta-glucosidase. These three enzymes work synergistically to degrade cellulose. The cellulases have been cloned and their structures are known. So definite changes to their amino acid sequence can be made.

The Biofuels Program is another DOE project. They increased the activity of the endoglucanase (part of the three component cellulase mixture) by changing an amino acid at the active site with site specific mutagenesis.
http://www.ott.doe.gov/ biofuels/cellulase.html

The CEA believes the most useful increases in cellulase activity can be made through increasing its thermal stability. At higher temperatures, reactions go faster. This has been done for other enzymes. For example, substituting a surface arginine with lysine using site specific mutagenesis resulted in a more thermostable glucose isomerase.
http://www.ejb.org/ content/vol2/issue1/full/2/

But before you make lots of changes, you need a high throughput (quick and easy) assay for the enzyme. Observing cleared areas on cellulose petri plates from colonies producing cellulase have been used to see cellulase efficiencies (http:// www.fao.org/docrep/w7241e/w7241e08.htm). This group also produced mutant strains with elevated activity by mutating with UV, nitrosoguanidine (NTG), etc., and growing cells with only a cellulose analog as a food source. This way, clones with a more active cellulase will grow faster and will be selected. Mutations produced this way are random and could be anywhere in the cellulase gene.

Back to site specific mutagenesis, I doubt that either of the two examples of site specific mutagenesis mentioned earlier were just lucky guesses. So how could it be done?

First, you would get some random mutagenesis data throughout the protein by mutating with UV or NTG. Then you would use site specific mutagenesis to make some mutations in an area such as the active site. These would give you an idea of what residues are important and you might (or might not) find mutations with increased activity. You can also make random changes over an area with an oligonucleotide, and then select for mutants. But while you're doing this, you can do some molecular modeling.

How do you increase the activity of an enzyme? An enzyme stabilizes the binding of the transition state ligand over that of the substrate. Crystal structures of cellulases complexed with substrates are available. For example, see 1CEN in the protein data bank at http://www.rcsb.org/pdb/ (and search cellulase for other examples). See J Mol Biol (1996) 257:1042-1051 for details on 1CEN.

You can model amino acid substitutions with expasy http://www.expasy.ch/swissmod/SWISS- MODEL.html
You can construct ligands with InsightII or Sybyl or CAChe (they have a free demo) http://www.cachesoftware.com/
You can dock ligands with mutated enzyme structures using these programs or use DOCK and score your matches. Information on DOCK can be found at http:// www.cmpharm.ucsf.edu/kuntz/dock.html
You may be able to make predictions of how to increase transition state binding. As you get your initial mutagenesis data, you can see how your predictions are working.

Increasing the thermal stability of an enzyme also gives more activity since a higher reaction temperature can be used. One way you could get a more stable protein is to increase its SNAPP score. The SNAPP score is a measure of the likelihood of tetrads of amino acids being found together. SNAPP scores are being examined by some of my fellow Tar Heels here at UNC. You can see how they do it at: J Mol Biol (2001) 311:625-638.

Increasing the activity of an enzyme can be big project. But if you get something useful, that would great. Mike Conrad