Featured Article - December 2013
Short description: The identification of the first N-6-methyladenine deaminase from the NCBI database of prokaryotic genomes highlights the value of the database to the experimental annotation of novel enzymes.
Bacterial DNA contains methylated adenine (N-6-methyladenine or 6mA) as well as methylated cytosine to protect the genome from endogenous restriction endonucleases that degrade invading DNA, e.g. from bacteriophages. While DNA methylases and methyltransferases that modify adenine and cytosine are well characterized, no bacterial enzymes that metabolize 6mA have been identified.
The NCBI database of fully sequenced bacterial genomes organizes its entries as Clusters of Orthologous Groups (COGs). The 250 proteins of cog1001 are members of the amidohydrolase superfamily and share a metal cofactor binding site within a (β/α)8-barrel fold that confers the ability to hydrolyze amides, amines and esters. Adenine deaminases (ADEs) are highly represented in cog1001, whose subgroup 2 includes 32 genes from Bacillus species encoding proteins of unknown function that share low sequence identity (∼35%) with ADEs. One of these proteins, Bh0637, is located next to a known ADE from subgroup 1 in the B. halodurans genome, suggesting that it may have a related function. Raushel and colleagues (PSI NYSGRC) have demonstrated that Bh0637 catalyzes deamination of 6mA, thus identifying the first N-6-methyladenine deaminase (6-MAD).
The authors purified recombinant Bh0637 in parallel with B. subtilis protein Bsu06560, with which it shares 71% sequence identity. The active sites were reconstituted by the addition of metal cofactors, and the enzymes were screened against a library of 20 adenine-related compounds for deamination activity. Both enzymes catalyze deamination of 6mA to produce hypoxanthine and methylamine, as confirmed by mass spectrometry.
The structural basis of the enzyme's methyl-substrate specificity was probed by constructing a three-dimensional model of Bh0637 using the crystal structure of ligand-free adenine deaminase Atu4426 (PDB 3NQB) as a template and computationally docking 6mA into the active site. While residues comprising the metal binding center show substantial overlap between the subject and template enzymes, the Phe residues that likely interact with substrate are not conserved in Atu4426. The more hydrophobic binding site of Bh0637 thus promotes favorable van der Waals interactions with the methyl group of 6mA. Indeed, comparative kinetic analysis of deamination rates reveals that N-6-methyladenine is a 100-fold better substrate than adenine or other related compounds that could be modeled in the active site, further suggesting that 6mA is the physiologically relevant substrate of Bh0637.
In conclusion, identification of an adenine deaminase that specifically targets 6mA helps determine adenine methylation's essential contributions to bacterial cell viability.
S.S. Kamat et al. Enzymatic Deamination of the Epigenetic Base N-6-Methyladenine.
J. Am. Chem. Soc. 133, 2080-2083 (2011). doi:10.1021/ja110157u