Structural Biochemistry/Enzyme Catalytic Mechanism/BamHI

BamHI

BamHI( from BAcillus amyloli) is a type II restriction enzyme that derived from B. amyloliquefaciens , having the capacity for recognizing short sequences of DNA and specifically cleaving them at a target site. In order to understand how these restriction endonuclease bind to their specific sequence, BamHI became a popular research target. By crystallizing BamHI endonuclease, scientists understood how DNA binding proteins select their specific target from a variety of nonspecific sequences in the cell.

Introduction

In order for restriction endonuclease to bind to their target sequence, they have to first bound to a nonspecific DNA. These nonspecific endonuclease-DNA complexes are believed to be more hydrated at their endonuclease-DNA interface, stabilized by electrostatic interactions and resulted a low heat capacity change during binding. Without strong interaction or binding to the nonspecific sequence, these weak interactions contributed to the sliding ability of these endonuclease. Instead of steady bound to a sequence, the endonuclease slide along the sequence until a specific match of sequence is reached. The following is a summary of a research conducted by two scientists, Hector Viadiu and Aneel K. Aggarwal in 2000.

Research

Background

The purpose of this research is to determine how BamHI binds to its nonspecific DNA sequence and interact with its target sequence. BamHI tends to bind to the following sequence at a particular cleavage site, as indicated below with the straight line.

5'-G|GATCC-3' 3'-CCTAG|G-5'

As a restriction enzyme, a change in the specific sequence, even a change as small as one base pair, is sensitive enough for the sequence to become nonspecific. The experiment starts by crystallizing nonspecific crystals with DNA sequence 5'-ATGAATCCATA-3' from solutions. In the sequence, GAATCC is similar to the target cleavage of BamHI. Study reveals the structure of BamHI in the presence of this similar sequence.

Catalytic Mechanism

BamHI is a catalysis mechanism that involves three different states. The first state, which is before the reaction occurs, is called the pre-reactive state. Next it's the intermediate state, known as the transition state. The last state is the post-reactive state. The following are the steps that of how the mechanism occurs: There is a water molecule and a glutamic acid present. This allows the glutamic acid to take a proton of water and extract the proton. This leaves a hydroxyl group which will have a nucleophilic attack on the phosphate atom. Since there are two metals, the negative charges get stabilized. Then, there will be a pentavalent phosphate, and the extra negative charge are stabilize by the metal ions. The phosphate will have an extra oxygen which causes the water in the solution to behave as a donor. This allows the tendency of phosphate to recover its coordination. Finally, the oxygen will attack the water molecule and the proton will go to the leaving group, resulting to the post-reactive state. Overall, when comparing the pre-reactive and post-reactive states, the slight movement phosphate is observed.

Catalytic Mechanism

Contact between BamHI and DNA backbone

BamHI at free, nonspecific and specific state

Although the DNA sequence is similar, BamHI behaves completely different from a specific complex. Not only that the bottom of the BamHI dimer is loose, but the enzyme is also tilted 20 degree from its axis. As a consequence, substitution in base pair does not only affect interaction at that specific base pair, but the entire sequence and the conformation of the BamHI endonuclease. The change of conformation is indicated in figure1. In the specific complex, the alpha helix at the C terminus of one monomer unfold to interact with the other monomer. However, when the targeted sequence is altered, the complex becomes nonspecific. In a nonspecific complex, these monomers do not unfold and interact. Since there is no conformational change, amino acids from the BamHI sequence are far from the DNA and do not involve in any binding. Thus, cleavage does not occur.

Catalytic Mechanism of Restriction Endonuclease BamHI

DNA Cleavage

BamHI cleaves DNA in two ways. First, BamHI cleaves DNA by co-crystallizing with divalent cations. For example, BamHI will bind to two divalent metals. Second, BamHI cleaves DNA by directly binding to the DNA itself. In the active site, the charge of the enzyme is negative. The metals are positioned between the active site and DNA. Inhibitors of this reaction include calcium. BamHI cleaves the phosphodiester bond by donating a proton the to the second water molecule. There is a pre-reactive site and post-reactive site that indicates which sequences have been cleaved. The phosphate is in the 5' direction while the oxygen is in the 3' direction.

Conclusion

Conformational change of BamHI is only triggered when it detects a specific DNA sequence. Otherwise, the BamHI, where active site residues are pointing outward, does not interact or bind with the nonspecific sequence. The specificity of this BamHI endonuclease is vital because it avoids lethal cleavages at similar DNA sequence.

Everytime DNA replicates in bacteria, it is methylated. Restriction endonucleases like BamHI cannot cleave DNA sequences that are methylated. However, DNA from viruses will not be methylated, so they will eventually be cleaved by BamHI. If bacteria want to survive, they have to be adept at recognizing which sequences are methylated. When the enzyme recognizes the right site, the DNA is in proximity. When the DNA changes conformation, it is far from the active site so catalysis is not carried out. Many residues carry the catalysis and many are close to the active site in order to recognize certain substrates and make sure they are the correct ones.

BamHI is able to recognize particular sequences of DNA in a pool of millions of possible sequences by simply binding the any DNA. Then, they slide along the DNA strand to find the right sequence. When it finds the particular sequence, it will change conformation and embrace the DNA.

Reference

1. Viadiu H, Kucera R, Schildkraut I, Aggarwal AK, "Crystallization of restriction endonuclease BamHI with nonspecific DNA." J Struct Biol 1(81-5):, 2000.

2. Viadiu, Hector. Examples of Catalytic Mechanism. Biochemistry Lecture. Dec. 3,2012