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Enzymatic Deamination of the Epigenetic Base N-6-Methyladenine
Siddhesh S. Kamat,† Hao Fan,|| J. Michael Sauder,‡ Stephen K. Burley,‡ Brian K. Shoichet,§ Andrej Sali,|| and
Frank M. Raushel*,†
†Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77842-3012, United States
‡Eli Lilly & Company, Lilly Biotechnology Center, 10300 Campus Point Drive, Suite 200, San Diego, California 92121, United States
Department of Bioengineering and Therapeutic Sciences, §Department of Pharmaceutical Chemistry, and California Institute for
Quantitative Biosciences, University of California, San Francisco, 1700 Fourth Street, San Francisco, California 94158, United States
S Supporting Information
b
a dozen of these microorganisms also have an authentic adenine
ABSTRACT: Two enzymes of unknown function from the
amidohydrolase superfamily were discovered to catalyze the
deamination of N-6-methyladenine to hypoxanthine and
methyl amine. The methylation of adenine in bacterial DNA
is a common modification for the protection of host DNA
against restriction endonucleases. The enzyme from Bacillus
halodurans, Bh0637, catalyzes the deamination of N-6-
methyladenine with a kcat of 185 s-1 and a kcat/Km of
2.5 ꢀ 106 M-1 s-1. Bh0637 catalyzes the deamination of
N-6-methyladenine 2 orders of magnitude faster than adenine.
A comparative model of Bh0637 was computed using the
three-dimensional structure of Atu4426 (PDB code:
3NQB) as a structural template and computational docking
was used to rationalize the preferential utilization of N-6-
methyladenine over adenine. This is the first identification
of an N-6-methyladenine deaminase (6-MAD).
deaminase from subgroup 1 in their genome. Most of the genes
in subgroup 2 from cog1001 lie adjacent to the pur operon for the
biosynthesis of purine nucleotides. Moreover, in Bacillus halo-
durans, the genes for ADE from subgroup 1 (Bh0640) and
Bh0637 from subgroup 2 are nearly adjacent to one another. The
co-localization of these two genes suggests that these enzymes
may use the same or similar substrates and that the products of
their reactions are similar. On the basis of these observations, we
have thus predicted that Bh0637 is likely to have an adenine-like
substrate and that the reaction product resembles hypoxanthine.
There are a limited number of purine moieties in Nature that
closely resemble adenine. A prime substrate candidate for enzymes
in subgroup 2 is N-6-methyladenine because this compound is
found in the genomes of all bacteria.7 This compound is often
considered as the sixth DNA base with 5-methylcytosine being
the fifth base. The occurrence of N-6-methyladenine in most
bacterial genomes varies from 1.4 - 2.0% of the total adenine
content.8 This nucleotide modification functions to protect host
DNA against restriction endonucleases and is critical for cell
viability.8 More recently, N-6-methyladenine has been implicated
in DNA repair and replication, regulation of gene expression,
control of transposition, and host-pathogen interactions.7,8 In
bacteria, DNA methylases and cell-cycle regulated methyltrans-
ferases are responsible for the methylation of adenine in DNA.7,8
Previous studies with cell free extracts from various sources have
implicated a cryptic enzyme activity for the conversion of N-6-
methyladenine to hypoxanthine.9,10 However, no enzyme is
currently known to metabolize N-6-methyladenine. Other mod-
ified adenine moieties that could serve as potential substrates
include 7-methyladenine and 2-hydroxyadenine (isoguanine).11,12
Bh0637 from B. halodurans C-125 was selected from enzymes
clustered in subgroup 2 for substrate interrogation. The gene for
Bh0637 was cloned into a pET30a(þ) plasmid (Novagen) with
NdeI and HindIII restriction sites, expressed in Escherichia coli
and purified to homogeneity. Bsu06560 from Bacillus subtilis
(gi|16077724) was also purified to homogeneity. These two
proteins share 71% sequence identity. The gene for Bsu06560
was codon-optimized for expression in E. coli, synthesized, and
then cloned into an expression vector that yields a protein with a
noncleavable C-terminal histidine tag. The sequence and expres-
sion/purification details for Bsu06560 are available from PepcDB
CBI has classified proteins from completely sequenced
Nbacterial genomes that perform the same or similar func-
tions into Clusters of Orthologous Groups (COG).1 One of
these clusters, cog1001, consists of ∼250 proteins, all of which
are members of the amidohydrolase superfamily (AHS). The
AHS is an ensemble of evolutionarily related enzymes capable of
hydrolyzing amide, amine, or ester functional groups at carbon
and phosphorus centers.2 Enzymes within this superfamily pos-
sess a mononuclear or binuclear metal center embedded within a
(β/R)8-barrel structural fold.2
A sequence similarity network for cog1001 is presented in
Figure 1 at an E-value cutoff of 10-80 3,4
Adenine deaminase
.
(ADE) is the predominant enzyme in cog1001 and is represented
in Figure 1 by subgroups 1 and 4. Subgroups 3 and 5 consist of
enzymes that are currently annotated as isoaspartyl dipeptidases
and enamidases, respectively.5,6 The functional annotations for
most members of subgroup 2 in Figure 1 are currently listed as
unknown in the NCBI database but some of them have been
labeled as adenine deaminases without apparent experimental
validation. Proteins in subgroup 2 have less than 35% sequence
identity to the prototypical adenine deaminases found in sub-
groups 1 and 4.
There are sequences available for ∼32 proteins from subgroup
Received: November 11, 2010
Published: January 28, 2011
2. These enzymes are found in various species of Bacillus. At least
r
2011 American Chemical Society
2080
dx.doi.org/10.1021/ja110157u J. Am. Chem. Soc. 2011, 133, 2080–2083
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