"DNA Adenine Methyltransferase" created by Alexandra Wall
DNA adenine methyltransferase (pdb=2G1P) from Escherichia coli, also known as EcoDam, is an enzyme involved in the methylation of the exocyclic amino nitrogen (N6) of the adenine base in GATC (4). DNA-(adenine-N6)-MTases transfer a methyl group to the N6 positions of adenine residues embedded in specific recognition sequences (2). At specific GATC sites, this process plays a crucial role in the regulation of bacterial gene expression and DNA regulation and is essential for bacterial virulence of many gram-negative bacteria. DNA adenine methylation is critical to the post-replicative mismatch repair system. The methylation mark present on one DNA strand after replication is used to differentiate parental and daughter DNA strands. This allows for a directed repair of base mismatches (4). The enzyme that methylates DNA at GATC sites is involved in the coordination of DNA replication and of the cell cycle (2). The adenine base pair in GATC is the methylation target in this mechanism. It flips out from the DNA helix (4). S-adenosyl methionine is thought to be the methyl donor and to increase enzyme-binding specificity (6). In the absence of S-adenosyl methionine, adenine does not enter the active site pocket but binds to an alternative site on the surface of the enzyme. This resembes an intermediate in the base flipping pathway (8). It lies against the protein surface (side chains of Tyr-184 and His-228) outside the active site pocket. The imidazole ring of His-228 stacks edge-to-face on the adenine ring. N1 of adenine forms a hydrogen bond with the main-chain amide nitrogen atom, and N6 forms a hydrogen bond with the carbonyl oxygen atom of Val-261 (4).
The structure of EcoDam is important to the function of the enzyme. It is comprised of two monomers (molecule A and molecule B) and one DNA duplex. Molecule A binds primarily to a single DNA duplex, while molecule B binds to the joint between the two DNA duplexes. The secondary structure of EcoDam contains mainly alpha helices and beta sheets, but also contains some 3/10 helices and random coils. EcoDam has two domains. There is a seven-stranded catalytic domain, which harbors residues 1-56 and 145-270 and contains the binding site for S-adenosyl-L-homocysteine. A DNA binding domain contains a five-helix bundle (residues 57-144) and a beta hairpin loop (residues 118-139) (4). The β-hairpin is part of the DNA binding domain and an N-terminal extension of the catalytic domain. Key recognition contacts in EcoDam include the hydrogen bond between Lys-9 and guanine, contacts of Leu-122 and Pro-134 to base pair three, and an interaction of Arg-124 to guanine in base pair four. Exchange of any of these residues may lead to major changes in DNA recognition specificity of EcoDam (2). The molecular weight of EcoDam is 32,099.61 Da, and its isoelectric point (pI) is 8.78 (3).
The nucleotide analog 2-aminopurine (2AP) has been incorporated into synthetic oligodeoxynucleotide duplexes to probe DNA conformational changes because 2-aminopurine fluorescence noticeably increases when it is removed from the double helical DNA. Base flipping by EcoDam causes fluorescence changes of a hemi-methylated G-2AP-TC substrate occurring in two steps: 1) flipping the target base out of the DNA helix, and 2) binding the flipped base into the active site pocket of the enzyme. During the second step, the flipped base stacks with aromatic residues, leading to a reduction of 2-aminopurine fluorescence (4). If S-adenosyl-L-homocysteine is present, or if S-adenosyl methionine is absent from the enzyme, this reaction will not occur. Target base flipping is very fast, yet binding of the flipped base into the active site pocket of the enzyme is very slow (8). Full rotation of one dihedral bond drives the flipped adenine to insert into the active site pocket. Residues from the β-hairpin and the N-terminal loop are tightly connected through hydrogen bonding of the main-chain amide nitrogen atom and the carbonyl oxygen of the Lys-9 and Asn-115 side chains, respectively (4). The rotation of the dihedral bond may only take place after binding of S-adenosyl methionine, when the unstructured loop following the active site residues becomes ordered. This enables the formation of a closed active site (4).
Opposite of adenine, there is an orphan thymine that represents a major difference between the EcoDam-DNA complexes formed by molecule A versus those formed by molecule B. Orphan thymine in the middle of molecule A is flipped out of the DNA helix. Here, it is stabilized by the pi-stacking interactions with the guanine group of Arg-137. However, in molecule B, thymine is hydrogen bonded with the amide side chain of Asn-120. Asn-120 inserts its side chain into the helical space previously occupied by the flipped adenine. The orphan thymine is moved to an extrahelical position, disrupting the Thy-Asn-120 interaction. This mechanism causes a double base flipping (4). The Tyr-119 aromatic ring intercalates into the DNA duplex, stacking between the third base pair of GATC and the Thy-Asn-120 pair in molecule B or the side chain of Asn-120 in molecule A. This intercalation results in local doubling in helical rise. Tyr-119 intercalation is necessary for base flipping because it expands the middle and end of the DNA duplex. The length of the DNA corresponds to fourteen base pairs and matches the crystal α-axis with a length of forty-six angstroms (4).
A DNA adenine methylase (Dam)- mutant was created to determine involvement in pathogenicity. The mutant was out-competed by wild-type in establishing fatal injections in mice. Mice that had been previously injected with the mutant were less susceptible to further infection by the wild-type (4). In Salmonella, Haemophilus, and certain strains of Yersinia pseudotuberculosis, lack of Dam methylation causes reduction of virulence in model animals (7). A role for Dam as a virulence factor has been observed for a growing list of bacterial pathogens (4). Deletion of Dam erases DNA methylation patterns, which could alter the binding of regulatory proteins to regions on the bacterial chromosome (7). A Dam deletion strand of Salmonella used as live vaccines was able to raise cross-protective immunity. Humans do not have detectable adenine methylation, so this discovery may provide basis for development of a new class of antibiotics with broad antimicrobial action (4).
The purpose of the Dali server is to find similarities in tertiary structure to any protein. By submitting the coordinates of a query protein structure, Dali compares the entry to proteins in the Protein Data Bank. Comparing the 3D structures may help to detect similarities between two proteins that are not noticeable by simply comparing the sequences (5). The Z-score reflects whether or not an entry is a strong match for the protein searched. A score above 20% represents a strong match. The PSI-BLAST server is used to find biologically similar sequences to any protein. It works the same as gapped BLAST but at a faster rate, and it detects weaker sequences (1). An E-value below 0.5 indicates a high similarity between two proteins. DNA adenine methylase from Enterobacteria phage t4 (pdb ID=1Q0S) has an E value of 0.41 and a Z-score of 26.1, meaning T4Dam has strong primary and tertiary similarities to EcoDam; however, EcoDam has two monomers and T4Dam has one monomer. DNA recognition mediated by a hairpin loop in EcoDam is similar to that observed in T4Dam (4). Guanine is based on a bidentate hydrogen bond between Arg-130, located at the basis of the β-hairpin and guanine (2). The contacts of EcoDam to the third and fourth base pair (by Leu-122 and Pro-134 and by Arg-124, respectively) are equivalent to those observed in T4Dam. Here, two residues are intercalated into the DNA: Tyr-119 and Asn-120. In EcoDam, Tyr-119 is contacted by Lys-9 as opposed to Arg-130 interacting with the guanine base in T4Dam. There is an alanine in T4Dam corresponding to the EcoDam Lys-9 whose β-carbon atom points towards the DNA but does not contact it. Scientists changed the Lys-9 in EcoDam to alanine to investigate its role in DNA regulation. The variant showed slightly reduced catalytic activity (~60% of the wild-type) and DNA binding (~70%). Wild-type EcoDam methylated variants containing a single base substitution at the first position relative to GATC at a 100 to 1000-fold reduced rate. The Lys-9/alanine variant showed a loss of specificity at this site (4).