HhaI (cytosine-5-)-methyltransferase (M.HhaI) is a bacterial enzyme taken from Haemophilus haemolyticus. Its function is the same as methyltransferases (MTase) in a wide variety of other organisms such as the DNA (cytosine-5-)-methyltransferase found in eukaryotes such as humans. The enzyme requires the methyl donor S-adenosyl-L-methionine (AdoMet) in order to transfer a methyl onto the C5 position of cytosine in the target DNA. The methylation of DNA is thought to be involved in many cellular process including transcription of DNA, genomic imprinting, developmental regulation, mutagenesis, transposition, DNA repair, and chromatin organization. For these reasons, there is a great need for functional and structural understanding of the enzyme.
All DNA MTases have two domains with a cleft between them where the DNA and cofactor bind. The large domain is responsible for the catalytic activity of the enzyme. It contains six highly conserved residues and serves as the binding site for the cofactor. When this domain binds to th eminor groove of DNA, it undergoes a conformational change that brings the catalytic nucleophile Cys-81 into close proximity with the C5 cystine.
The small domain is composed of the variable residues and is responsible for recognition of the target DNA. It forms two glycine rich loops that bind only to the major groove. The first loop makes six specific contacts with the recognition bases and the second loop forms 3 hydrogen bonds with them. In addition to these specific contacts, there are also several non-specific interactions with the sugar-phosphate backbone.
Depicted below is the enzyme bound to DNA with the active site highlighted.
When the MTase is bound to the DNA, the target cytosine is flipped out of the helix and fits into the active site of the enzyme. The cofactor S-adenosyl-L-methionine (AdoMet) then binds to the proein in the active site, which causes the enzymes affinity for DNA to increase 900 fold. The Cys-81 then initiates catalysis by attacking the C6 of cytosine 18 which activates the C5 position for a nucleophillic attack on the AdoMet methyl group. The newly formed S-adenosyl-L-homocysteine (AdoHcy) then dissociates followed by the methylated DNA.
The picture below illustrates the active site with labeled residues.
M.HhaI was the first protein in which a base pair was observed to flip out of the DNA when it bound to it. This enzyme mediated nucleosome flipping occurs in other methyltransferases and also in DNA repair enzymes. The removal of the cytosine from within the helix causes major distortions of the phosphodiester backbone even though the two residues Gly-237 and Ser-87 fill in the gap by undergoing a conformational change in the active site loop. The target cytosine is stabilized by several specific ineractions with conserved residues in the MTase family. Salt bridges, ion pairing and several hydrogen bonds hold the cystine in place.
In short, the binding of M.HhaI to substrate DNA results in large conformational changes. The target cytosine is flipped into the active site which is followed by the insertion of protein residues into its vacant space and the bond formation between the Cys-81 and the C6 of the cytosine. This brings the three necessary components, target cytosine, catalytic nucleophile, and the methyl donor together in the active site of the of the enzyme so the methylation reaction can proceed.
Works Consulted
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