Deamination of 6-aminodeoxyfutalosine in menaquinone biosynthesis by distantly related enzymes

Article abstract of DOI:10.1021/bi400750a

Proteins of unknown function belonging to cog1816 and cog0402 were characterized. Sav2595 from Steptomyces avermitilis MA-4680, Acel0264 from Acidothermus cellulolyticus 11B, Nis0429 from Nitratiruptor sp. SB155-2 and Dr0824 from Deinococcus radiodurans R1 were cloned, purified, and their substrate profiles determined. These enzymes were previously incorrectly annotated as adenosine deaminases or chlorohydrolases. It was shown here that these enzymes actually deaminate 6-aminodeoxyfutalosine. The deamination of 6-aminodeoxyfutalosine is part of an alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminodeoxyfutalosine is deaminated by these enzymes with catalytic efficiencies greater than 10 ⁵ M^-1 s^-1, K_m values of 0.9-6.0 μM, and k_cat values of 1.2-8.6 s^-1. Adenosine, 2′-deoxyadenosine, thiomethyladenosine, and S-adenosylhomocysteine are deaminated at least an order of magnitude slower than 6-aminodeoxyfutalosine. The crystal structure of Nis0429 was determined and the substrate, 6-aminodeoxyfutalosine, was positioned in the active site on the basis of the presence of adventitiously bound benzoic acid. In this model, Ser-145 interacts with the carboxylate moiety of the substrate. The structure of Dr0824 was also determined, but a collapsed active site pocket prevented docking of substrates. A computational model of Sav2595 was built on the basis of the crystal structure of adenosine deaminase and substrates were docked. The model predicted a conserved arginine after β-strand 1 to be partially responsible for the substrate specificity of Sav2595.

Full text of DOI:10.1021/bi400750a

Article
pubs.acs.org/biochemistry
Deamination of 6‑Aminodeoxyfutalosine in Menaquinone
Biosynthesis by Distantly Related Enzymes
Alissa M. Goble,† Rafael Toro,∥ Xu Li,# Argentina Ornelas,† Hao Fan,‡,§,⊥ Subramaniam Eswaramoorthy,¶
Yury Patskovsky,∥ Brandan Hillerich,∥ Ron Seidel,∥ Andrej Sali,‡,⊥ Brian K. Shoichet,§,⊥ Steven C. Almo,∥
Subramanyam Swaminathan,¶ Martin E. Tanner,# and Frank M. Raushel*,†
†Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77843-3012, United States
§
⊥
‡Department of Bioengineering and Theraputic 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
¶Biology Department, Brookhaven National Laboratory, P.O. Box 5000, Upton, New York 11973-5000, United States
#Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada
∥Department of Biochemistry, Einstein College of Medicine, Bronx, New York 10461, United States
ABSTRACT: Proteins of unknown function belonging to
cog1816 and cog0402 were characterized. Sav2595 from
Steptomyces avermitilis MA-4680, Acel0264 from Acidothermus
cellulolyticus 11B, Nis0429 from Nitratiruptor sp. SB155-2 and
Dr0824 from Deinococcus radiodurans R1 were cloned, purified,
and their substrate profiles determined. These enzymes were
previously incorrectly annotated as adenosine deaminases or chlorohydrolases. It was shown here that these enzymes actually
deaminate 6-aminodeoxyfutalosine. The deamination of 6-aminodeoxyfutalosine is part of an alternative menaquinone
biosynthetic pathway that involves the formation of futalosine. 6-Aminodeoxyfutalosine is deaminated by these enzymes with
catalytic efficiencies greater than 105 M−1 s−1, Km values of 0.9−6.0 μM, and kcat values of 1.2−8.6 s−1. Adenosine, 2′-
deoxyadenosine, thiomethyladenosine, and S-adenosylhomocysteine are deaminated at least an order of magnitude slower than
6-aminodeoxyfutalosine. The crystal structure of Nis0429 was determined and the substrate, 6-aminodeoxyfutalosine, was
positioned in the active site on the basis of the presence of adventitiously bound benzoic acid. In this model, Ser-145 interacts
with the carboxylate moiety of the substrate. The structure of Dr0824 was also determined, but a collapsed active site pocket
prevented docking of substrates. A computational model of Sav2595 was built on the basis of the crystal structure of adenosine
deaminase and substrates were docked. The model predicted a conserved arginine after β-strand 1 to be partially responsible for
the substrate specificity of Sav2595.
Holm and Sander on the basis of the similarities in the three-
enaquinone can be biosynthesized through either the
Mshikimate pathway or the futalosine pathway. In dimensional structures of phosphotriesterase, adenosine deam-
inase, and urease.5 The common fold within the AHS is a
Escherichia coli, the well-studied shikimate pathway utilizes
distorted (β/α)8-barrel with conserved metal binding residues
at the C-terminal ends of β-strands 1, 4−6, and 8. Enzymes in
the AHS have either a mononuclear or binuclear metal center
that functions to activate a water molecule for nucleophilic
attack on amino acids, sugars, nucleic acids, and organo-
phosphate esters.6 The AHS is composed of 24 clusters of
orthologous groups (cog). Enzymes that are known to
deaminate aromatic bases are found in cog1001, cog0402,
and cog1816. Members of cog1001 include the prototypical
adenine deaminase (ADE) and N-6-methyladenine deaminase.7
Enzymes that deaminate guanine, cytosine, S-adenosylhomo-
cysteine, thiomethyl adenosine, N-formimino-L-glutamate, and
8-oxoguanine are found in cog0402.8 According to annotations
reported by NCBI, bacterial enzymes in cog1816 function in
seven enzymes (menA to menG) to convert chorismate into
menaquinone.1 Recently, menaquinone-producing organisms
were identified that lack menF, menD, menC, menE, and menB
orthologs.2,3 These organisms, including Streptomyces coelicolor
A3,2 Helicobacter pylori, Campylobacter jejuni, and Acidothermus
cellulolyticus, lack men gene homologues and were suspected of
employing a different pathway for the biosynthesis of
menaquinone.1,4 Using a combination of bioinformatics and
gene disruption experiments, the mqn genes involved in the
futalosine pathway were identified (Figure 1).1 Mqn genes are
found scattered throughout the genomes, with the exception of
A. cellulolyticusi, which has two clusters of menaquinone related
genes.4 The smaller cluster contains MqnC and MqnB orthologs
and the other cluster is made up of several genes, including
MqnA, another MqnC, and an adenosine deaminase ortholog
(Acel0264) (Figure 2).
Received: June 12, 2013
Revised: August 22, 2013
Published: August 23, 2013
Adenosine deaminase is a member of the amidohydrolase
superfamily (AHS) of enzymes, a superfamily first identified by
© 2013 American Chemical Society
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Figure 1. Biosynthetic pathways for the assembly of menaquinone. The deamination of AFL is shown in red. The pathway for menaquinone
biosynthesis in E. coli is shown in blue.
Figure 2. Genomic context for Acel0264. UbiD (Acel0255) is believed
to catalyze a decarboxylation reaction in the late stages of
menaquinone biosynthesis. UbiA (Acel0256) functions as a 4-
hydroxybenzoate polyprenyltransferase. UbiX (Acel0257) is a 3-
octaprenyl-4-hydroxybenzoate carboxy-lyase. Acel0261 and Acel0263
are MqnA and MqnC homologues, respectively. Acel0264 catalyzes the
deamination of 6-aminodeoxyfutalosine to futalosine and has been
characterized in this work. UbiE (Acel0265) is a methyltransferase
Figure 3. Sequence similarity network created using Cytoscape (www.
involved in menaquinone biosynthesis. cog-FixC (Acel0266) serves as a
cytoscape.org) of cog1816 from the amidohydrolase superfamily. Each
geranylgeranyl reductase in the biosynthesis of menaquinone. Genes
node in the network represents a single sequence, and each edge
depicted in gray have unknown function or function in an unrelated
(depicted as lines) represents the pairwise connection between two
pathway.
sequences at a BLAST E-value of better than 1 × 10−70. Lengths of
edges are not significant, except for tightly clustered groups, which are
more closely related than sequences with only a few connections.
Group 5 contains the adenosine deaminase from E. coli. Additional
groups containing adenosine deaminase enzymes are shown in green.
Group 3 contains adenine deaminase enzymes, with the exception of
the blue outliers, which are cytokinin deaminases. In groups 11 and 13,
the sequences depicted in pink contain all of the essential residues for
the deamination of AFL identified in this investigation.
the deamination of adenosine. The functional diversity of
cog1816 has recently been expanded to include enzymes that
deaminate adenine and cytokinins.9,10 The mechanism,
structure, and biological function of adenosine deaminase
enzymes from several organisms have been investigated.11−23
A
sequence similarity network for cog1816 is illustrated in Figure
3 at a BLAST E-value of 1 × 10−70, which corresponds to a
sequence identity within each group of ∼30%.24 In general,
members of a group share a common function, and the
substrate profiles differ between groups. Members of group 5 of
cog1816 are prototypical adenosine deaminases, including the
adenosine deaminase from E. coli K-12. Group 3 of cog1816
includes adenine deaminases with a single divalent cation and
enzymes that catalyze the deamination of cytokinins.
The initial targets for this investigation were the functional
annotation of Acel0264 from A. cellulolyticusi and Sav2595 from
Streptomyces avermitilis MA-4680. These enzymes belong to
groups 11 and 13 of cog1816, respectively. The substrate profile
for these two enzymes has been established and a three-
dimensional model has been proposed for how the best
substrate, 6-aminodeoxyfutalosine (AFL), binds in the active
site. On the basis of the absence of men genes in the genomes
of Deinococcus radiodurans R1 and Nitratiruptor sp. SB155-2,
two distantly related enzymes from cog0402, Dr0824 from D.
radiodurans R1 and Nis0429 from Nitratiruptor sp. SB155-2,
were identified as 6-aminodeoxyfutalosine deaminases. The
three-dimensional structures of Dr0824 and Nis0429 were
determined by X-ray crystallography. A sequence similarity
network of cog0402, which includes guanine deaminase,
cytosine deaminase, and other deaminases, is shown in Figure
4.
MATERIALS AND METHODS
■
Materials. All chemicals were purchased from Sigma-
Aldrich, unless otherwise stated. 6-Aminodeoxyfutalosine
(AFL) was synthesized as previously described.25 1-(6-Amino-
9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranuronamide
(NECA) was purchased from Ascent Scientific.
Cloning and Purification of Sav2595 from S.
avermitilis MA-4680 and Dr0824 from D. radiodurans
R1. The genes for Sav2595 and Dr0824 were cloned from the
genomic DNA of S. avermitilis MA-4680 and D. radiodurans R1,
respectively. The PCR products were amplified using the
primer pairs 5′-AGAGCCATGGTGACCGAGCACTTC-
GACGC-3′ and 5′-AGAGAAGCTTCAGGGCGCGAGCC-
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Figure 4. Sequence similarity network of cog0402. Groups 7 and 41 (pink) contain genes with all the essential residues to be AFL deaminases. Other
groups with known function include group 1 (5′-modified adenosine deaminases), group 2 (guanine deaminases), group 4 (isoxanthopterin
deaminases or 8-oxoguanine deaminases), group 6 (cytosine deaminases), group 8 (N-formimino-L-glutamate deiminases), and group 10 (pterin
deaminase). Groups with no known function are shown in gray.
ATGCC-3′, and pair 5′- GGGAATTACCATATGCGCTTTT-
CTGCCGTCAGCCG-3′ and 5′-CCCAAGCTTTCATCAC-
AAGTCGCGGCTCAACTCCCAACG-3′, respectively. The
restriction sites for NcoI and HindIII for Sav2595 and NdeI
and HindIII for Dr0824 were inserted into the forward and
reverse primers, respectively. The PCR products were purified
with a PCR cleanup system (Qiagen), digested with their
respective restriction enzymes, and ligated into a pET-30a(+)
vector, which was previously digested with the same two
restriction enzymes. The cloned gene fragments were
sequenced to verify fidelity of the PCR amplification. Dr0824
contains a C-terminal polyhistidine tag, whereas Sav2595 has an
N-terminal polyhistidine tag.
Sav2595-pET-30a(+) and Dr0824-pET30a(+) constructs
were transformed into BL21(DE3) cells (Novagen). A single
colony was used to inoculate a 5 mL overnight culture of LB
medium containing 50 μg/mL kanamycin. Each overnight
culture was used to inoculate 1.0 L of LB medium containing
50 μg/mL kanamycin. One liter cultures were grown at 37 °C,
supplemented with 1.0 mM ZnCl2, and induced with 0.5 mM
isopropyl D-thiogalactopyranoside (IPTG) when an OD600 of
0.6 was reached. Cultures of Dr0824 were induced with 1.0
mM IPTG. At the time of induction, the temperature was
lowered to 20 °C and the samples were allowed to shake for 18
h before the cells were harvested by centrifugation at 8000 rpm
for 10 min. The cells were resuspended in 20 mM HEPES
buffer, pH 7.5, containing 20 mM imidazole, 120 mM
ammonium sulfate, 10% glycerol, 0.1 mg/mL phenylmethane-
sulfonyl fluoride (PMSF), and 0.5 mg/mL deoxyribonuclease I
from bovine pancreas. Cells were lysed by sonication, and the
soluble protein was separated from the cell debris by
centrifugation. Soluble protein from cultures of Sav2595 was
loaded onto a HisTrap column (GE Healthcare) and eluted
with a linear gradient of a solution containing 20 mM HEPES,
pH 7.5, 500 mM imidazole, and 500 mM ammonium sulfate.
Soluble protein from cultures of Dr0824 was partitioned by
ammonium sulfate precipitation at 30, 50, and 70% saturation.
The precipitated protein (30−50% ammonium sulfate satu-
ration) was resuspended in 50 mM HEPES buffer (pH 7.7) and
applied to a High Load 26/60 Superdex 200 preparative grade
gel filtration column (GE Healthcare). Fractions containing
protein were pooled and passed through an anion exchange
column.
Cloning and Purification of Acel0264 from A.
cellulolyticus. The gene for Acel0264 was amplified from A.
cellulolyticus Strain 11B genomic DNA using 5′-TTAAGA-
AGGAGATATACCATGGTGATGACACCCCACGATCC-
CGTC-3′ as the forward primer and 5′- GATTGGAAG-
TAGAGGTTCTCTGCCAGCGACTCTCCCGCCGCTG-3′
as the reverse primer. PCR was performed using KOD Hot
Start DNA Polymerse (Novagen). The amplified fragment was
cloned into the C-terminal TEV cleavable C-terminal StrepII-
6x-His-tag containing vector CHS23 by ligation-independent
cloning.26
The gene for Acel0264 inserted in a CHS23 vector was
transformed into Rosetta2 (DE3) cells (Novagen). A single
colony was used to inoculate a 5 mL overnight culture of LB
medium containing 50 μg/mL kanamycin and 25 μg/mL
chloramphenicol. Each overnight culture was used to inoculate
1.0 L of LB medium containing 50 μg/mL kanamycin and 25
μg/mL chloramphenicol. One liter cultures were grown at 37
°C. At an OD600 of 0.6, 1.0 mM ZnCl2 was added followed by
induction with 50 μM IPTG. At the time of induction, the
temperature was lowered to 20 °C and the samples were
allowed to shake for 18 h before the cells were harvested by
centrifugation at 8000 rpm for 10 min. The cells were
resuspended in 20 mM HEPES buffer, pH 6.5, with 120 mM
ammonium sulfate and 20 mM imidazole. Cells were lysed by
sonication in the presence of 0.1 mg/mL PMSF and 0.5 mg/
mL deoxyribonuclease I from bovine pancreas. Soluble protein
was separated from the cell debris by centrifugation. The
protein was then loaded onto a HisTrap HP column (GE
Healthcare) and eluted with a gradient of buffer containing 20
mM HEPES, 500 mM imidazole, and 500 mM ammonium
sulfate, pH 6.5.
Cloning and Purification of Nis0429 from Nitratir-
uptor sp. SB155-2. The gene encoding Nis0429 was
chemically synthesized and cloned into the plasmid pUC57
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Table 1. Data Collection and Refinement Statistics (Data for High Resolution Shells)
PDB identifier
2IMR
3V7P
4M51
space group
C 1 2 1
C 1 2 1
C 1 2 1
unit cell dimension (Å)
a
113.83
71.30
51.34
91.515
75.169
75.646
92.163
75.788
76.291
b
c
cell angles (deg)
α
90.00
115.20
90.00
1
90.00
90.00
β
120.72
120.99
90.00
γ
90.00
molecules per ASU
solvent content
Matthew’s coefficient
ligands
1
1
40.24
2.06
Zn2+
47.01
47.03
2.32
2.33
benzoate
tartrate
benzoate
sulfate
HEPES
Fe2+
unknown
Fe2+
X-ray source
NSLS X12C
0.9795
NSLS X29A
1.075
NSLS X29A
1.075
wavelength (Å)
method of structure solution
resolution
SAD
SAD
molecular replacement
33.91−1.08 (1.10−1.08)
33.91−1.08
29.30−1.78 (1.84−1.78)
29.30−1.78
95.3 (64.7)
19.20 (3.30)
0.048 (0.364)
20.1 (23.0)
1321 (5.0%)
24.11
70.00−1.35 (1.38−1.35)
50.00−1.35
99.6 (94.3)
9.00 (1.30)
0.051 (0.730)
12.8 (16.1)
2889 (3.0%)
24.77
resolution/refinement
completeness (%)
I/σI
95.1 (91.1)
10.20 (2.50)
Rsym
0.061 (0.640)
11.8 (13.5)
Rwork (Rfree
)
Rfree reflections (%)
Average B factor
RMSD
5505 (3.0%)
16.48
bonds lengths
0.005
1.310
170
0.012
1.389
226
0.012
1.614
609
bond angles
number of solvent molecules
Ramachandran plot statistics
most favored regions
alowed regions
97.1%
96.9%
96.4%
2.6%
2.9%
3.4%
outliers
0.27% (His301)
0.25% (His257)
0.25% (His257)
(GenScript). Expression constructs were generated by PCR
amplification using 5′-TTAAGAAGGAGATATACCATGC-
GTATCATTAAGCCGTTCGCC-3′ as the forward primer
and 5′-GATTGGAAGTAGAGGTTCTCTGCTTCGCGG-
ACGTGTTCTTCGCC-3′ as the reverse primer. PCR was
performed using KOD Hot Start DNA Polymerase (Novagen).
The conditions were: 2 min at 95 °C, followed by 40 cycles of
30 s at 95 °C, 30 s at 66 °C, and 30 s at 72 °C. The amplified
fragment was cloned into the C-terminal TEV cleavable C-
terminal StrepII-6x-His-tag containing vector CHS30 by
ligation-independent cloning.26
Expression vectors were transformed into E. coli BL21(DE3)
containing the pRIL plasmid (Stratagene) and used to inoculate
a 25 mL M9 minimal media culture containing 25 μg/mL
kanamycin and 34 μg/mL chloramphenicol. The culture was
allowed to grow overnight at 37 °C in a shaking incubator. A 10
mL aliquot of the overnight culture was used to inoculate 2 L of
M9 SeMET high-yield medium (Shanghai Medicilon)
supplemented with 150 mM 2,2′-bipyridyl, 1.0 mM ZnCl2,
and 1.0 mM MnCl2. The culture was placed in a LEX48 airlift
fermenter and incubated at 37 °C until the OD600 reached 1.2.
Cells were induced by addition of 0.5 mM IPTG and allowed to
grow for 16 h at 22 °C overnight. The culture was harvested
and pelleted by centrifugation.
Cells were resuspended in lysis buffer containing 20 mM
HEPES, pH 7.5, 500 mM NaCl, 20 mM imidazole, and 10%
glycerol and lysed by sonication. Lysates were clarified by
centrifugation at 35000g for 30 min. Proteins were purified on
an AKTAxpress FPLC (GE Healthcare). Clarified lysates were
loaded onto a 5 mL Strep-Tactin column (IBA), washed with 5
column volumes of lysis buffer, and eluted with 20 mM HEPES,
pH 7.5, 500 mM NaCl, 20 mM imidazole, 10% glycerol, and 2.5
mM desthiobiotin. The eluent was loaded onto a 1 mL Histrap
HP column (GE Healthcare), washed with 10 column volumes
of lysis buffer, and eluted with 20 mM HEPES, pH 7.5, 500
mM NaCl, 500 mM imidazole, and 10% glycerol. The purified
sample was applied to a HiLoad S200 16/60 HR gel filtration
column, which was equilibrated in 20 mM HEPES, pH 7.5, 150
mM NaCl, 10% glycerol, and 5 mM DTT. Peak fractions were
collected and protein was analyzed by SDS-PAGE. Samples
were concentrated to 18 mg/mL using Amicon Ultra
centrifugal filters (Millipore), snap frozen in liquid nitrogen,
and stored at −80 °C.
Initial Activity Screens. Sav2595, Acel0264, Dr0824, and
Nis0429 (5 μM) were incubated for 16 h with AFL, adenine,
adenosine, 2′-deoxyadenosine, 3′-deoxyadenosine, 5′-deoxya-
denosine, 2′,5′-dideoxyadenosine, AMP, ADP, ATP, S-
adenosylhomocysteine, S-adenosyl methionine, N-6-methyl-2′-
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deoxyadenosine, 5′-thiomethyladenosine, formaminopyrimi-
dine-adenine, and NECA. The substrate concentration was 80
μM. Enzymatic activity was monitored by changes in
absorbance between 240 and 300 nm with a SPECTRAmax
Plus spectrophotometer (Molecular Devices).
Measurement of Kinetic Constants. Assays were
conducted with substrate concentrations of 4−200 μM. The
deamination of AFL, NECA, adenosine, AMP, 2′-deoxyadeno-
sine, 3′-deoxyadenosine, 5′-deoxyadenosine, SAH, and 5′-
methylthioadenosine was monitored by following the decrease
in absorbance at 263 nm. Differential extinction coefficients
were calculated by subtracting the extinction coefficient of the
product from that of the substrate for AFL, adenosine, NECA,
5′-methylthioadenosine, 5′-deoxyadenosine, SAH, and AMP
(Δε263 = 6900 M−1 cm−1).
Metal Analysis. Metal content of the proteins was
determined by ICP-MS.27 Protein samples for ICP-MS were
digested with HNO3 by refluxing for ∼45 min to prevent
protein precipitation during the measurement. Protein
concentration was adjusted to ∼1.0 μM with 1% (v/v) HNO3.
Data Analysis. Sequence alignments were created using
Clustal.28 Steady state kinetic data were analyzed using Softmax
Pro, version 5.4 (Molecular Devices). Kinetic parameters were
determined by fitting the data to eq 1 using the nonlinear least-
squares fitting program in SigmaPlot 11.0 (Systat Software
Inc.). In this equation, A is the substrate concentration, Km is
the Michaelis constant, v is the velocity of the reaction, and kcat
is the turnover number.
Structure Determination of Dr0824. Crystals were
grown by mixing 1 μL of protein with 1 μL of reservoir
solution via the sitting drop vapor diffusion method. Protein
was in HEPES buffer at pH 7.5 at a concentration of
approximately 14 mg/mL. Reservoir solution contained 30%
PEG MME500, 0.1 M Bis−Tris, pH 6.5, and 50 mM calcium
chloride. Harvested crystals were mounted in cryoloops and
immediately flash-cooled in liquid nitrogen. Diffraction from
these crystals was consistent with the monoclinic space group
C2 and data extending to 1.78 Å resolution collected on the
X12C beamline of the National Synchrotron Light Source
(NSLS), Brookhaven National Laboratory. Data were pro-
cessed, integrated, and scaled using HKL2000;40 data collection
parameters are given in Table 1. The structure was determined
by single anomalous dispersion (SAD) using Se-Met
substituted crystals. Se-Met crystals were obtained under the
same conditions that yielded native crystals. Se atoms were
located using SOLVE and the phases were refined with
SHARP.41,42 The initial protein model was built by an
automated procedure using ARP/wARP software.43 The
model was completed manually using O and water molecules
added on the basis of the Fourier difference map.44 The
structure was refined with CNS 1.1, and the refinement
statistics are presented in Table 1.45 The final model contains a
zinc ion in addition to protein atoms and 170 water molecules
(PDB ID: 2IMR).
Structure Determination of Nis0429. Se-Met derivatized
protein was crystallized by the sitting-drop vapor diffusion
method, as described below. A 0.5 μL aliquot of the protein
solution (18 mg/mL) was mixed with an equal volume of a
precipitant solution and equilibrated at room temperature (294
K) against the same precipitant solution in a clear tape-sealed
96-well INTELLI-plate. Crystallization was performed using a
TECAN crystallization robot. A single crystal appeared
approximately 11 months after setting up crystallization drops
in 0.1 M MES/NaOH, pH 6.0, 1 M potassium sodium tartrate.
This crystal was flash-cooled in liquid nitrogen prior to data
collection.
Two complete X-ray diffraction data sets were collected from
the same crystal at 100 K on the beamline X29A at NSLS using
wavelengths of 1.075 Å (the resolution cutoff was 1.35 Å) and
0.979 Å (the resolution cutoff was 1.5 Å), respectively. All data
were processed and scaled with HKL2000.40 The crystal
structure was determined by selenium-SAD applying anom-
alous X-ray diffraction data (collected at a wavelength of 0.979
Å) for phasing and SHELXD software (the CCP4 program
package suite).46,47 The structure was further refined using
higher resolution “native” data (resolution 1.35 Å) and the
program REFMAC.48 The model was rebuilt and fixed
manually using COOT visualization and refinement software.49
The data collection and refinement statistics are listed in Table
1. The coordinates and structure factors were deposited to the
Protein Data Bank (PDB ID: 3V7P).
Structure Determination of the S145A Mutant of
Nis0429. The purified S145A protein was concentrated to
about 38 mg/mL and crystallized as described above for the
wild-type Nis0429 using a PHOENIX crystallization platform
and four different MCSG crystallization screens. Protein
crystals appeared in two to three weeks. The crystals were
directly flash frozen in liquid nitrogen. The 1.08 Å resolution X-
ray diffraction data were collected on the beamline X29A
(NSLS) at a wavelength of 1.075 Å and a temperature of 100 K.
The data were processed and scaled with HKL2000.40 The
v/Et = kcatA/(Km + A)
(1)
Homology Modeling and Ligand Docking. The X-ray
structure of adenosine deaminase from Bos taurus with a
transition-state analogue bound in the active site (PDB ID:
1KRM, 26 and 25% sequence identity to Acel0264 and
Sav2595, respectively) was used as the template to build
homology models for Acel0264 and Sav2595. The sequence
alignment was computed with multiple sequence comparison
by log-expectation (MUSCLE)29 followed by generation of 500
homology models using the standard “automodel” class in
MODELER 9v12.30 The model with the best DOPE31 score
was selected, and this initial model was refined with the
“loopmodel” class in MODELER and the side chain prediction
protocol in PLOP.32 After refinement, the two homology
models of Acel0264 and Sav2595 were targeted by docking
screens using DOCK 3.633,34 with a virtual library35−37 that
contains NECA and AFL as well as 57 680 different high-
energy intermediates (HEI) of 6440 KEGG (Kyoto Encyclo-
pedia of Genes and Genomes) molecules.38,39 In docking
screens, a computed pose was selected if the O− moiety of the
HEI portion of the molecule was found within 4 Å of the metal
ion in the active site. The selected molecules were ranked by an
energy function consisting of protein−ligand van der Waals
interactions, protein−ligand electrostatic interactions, and a
correction for ligand desolvation. The top 500 ranked
molecules were inspected visually to ensure the compatibility
of the pose with the amidohydrolase reaction mechanism.
Mutation of Sav2595 and Nis0429. Single-site mutations
of Sav2595 were constructed using the standard QuikChange
PCR protocol according to the manufacturer’s instructions. The
mutants, R87A and R87M, were expressed and purified using
the protocol established for wild-type Sav2595. A single-site
mutation was made to Nis0429 through standard QuikChange
PCR protocols to create the S145A mutant.
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a
Table 2. Kinetic Constants for Sav2595, Acel0264, Dr0824, and Nis0429
Sav2595
Acel0264
Dr0824
Nis0429
kcat
Km
kcat/Km
kcat
Km
kcat/Km
kcat
Km
kcat/Km
kcat
Km
kcat/Km
(s−1
3.8
)
(μM)
(M−1 s−1
)
(s−1
)
(μM)
(M−1 s−1
)
(s−1
8.6
)
(μM)
(M−1 s−1
)
(s−1
1.2
)
(μM)
(M−1 s−1
)
AFL
4.8
7.9 × 105
34
4.3 × 103
2.2 × 104
6.8
6.0
1.1 × 106
110
28
1.8
4.8 × 106
1.0 × 104
3.4 × 104
1.8 × 104
7.0 × 103
0.9
1.3 × 106
110
2.8 × 103
11
79
NECA
0.007
61
5′-methylthioadenosine
adenosine
0.40
94
0.31
110
42
20
24
S-
310
adenosylhomocysteine
5′-deoxyadenosine
3′-deoxyadenosine
2′-deoxyadenosine
AMP
2.9 × 103
240
1.1 × 103
32
47
44
51
45
7.4 × 104
240
1.9 × 104
21
0.017
410
6
5
3
0.72
37
a
Standard errors on the kinetic constants are less than 15%. Assays were conducted at pH 7.5 and at 30 °C. Sav2595 is from group 13 of cog1816,
Acel0264 is from group 11 of cog1816, Dr0824 is from group 41 of cog0402, and Nis0429 is from group 7 of cog0402.
crystal of the S145S mutant of Nis0429 was essentially
isomorphous to that of the wild-type protein (PDB ID:
3V7P); thus, the coordinates of the latter were used to build
and subsequently refine the structure of the mutant using the
program REFMAC48 and a COOT visualization and refine-
ment software.49 The final refinement statistics with the data
collection parameters are listed in Table 1. The coordinates and
structure factors were deposited in the Protein Data Bank
(PDB ID: 4M51).
Nis0429 S145A mutant has a kcat of 4.5 s−1, a KM of 12 μM, and
a kcat/KM of 3.8 × 105 M−1 s−1.
a
Table 3. Kinetic Constants for Sav2595 Mutants
AFL
adenosine
kcat
Km
(μM)
kcat/Km
kcat
Km
(μM)
kcat/Km
(s−1
)
(M−1 s−1
)
(s−1
)
(M−1 s−1
)
WT
3.8
4.8
3.4
8
8 × 105
2.7 × 104
5.3 × 104
2.2 × 104
1.3 × 104
4.1 × 104
R87A
R87M
0.094
0.42
0.106
0.41
8.3
10
RESULTS
a
■
Standard errors on the kinetic constants are less than 15%. Assays
Protein Isolation. The enzymes studied in this inves-
tigation belong to groups 11 and 13 of cog1816 and groups 7
and 41 of cog0402 (Figures 3 and 4). Sav2595, Acel0264,
Dr0824, and Nis0429 were expressed in E. coli and purified to
homogeneity. Despite supplementing the growth medium with
1.0 mM ZnCl2, the metal content of all proteins was low.
Sav2595 contained an average of 0.1 equiv of Zn2+ per subunit.
Acel0264 contained an average of 0.4 equiv of Zn2+ and 0.1
equiv of Ni2+ per monomer. Both proteins were incubated
overnight with ZnCl2, but no increase in catalytic activity was
observed after incubation. Dr0824 contained 0.8 equiv of Mn2+.
Nis0429 contained 0.5 equiv of Zn2+ and 0.1 equiv each of Fe2+
and Mn2+.
were conducted at pH 7.5 and at 30 °C.
Structure of Nis0429. The crystal structure of Nis0429
(PDB ID: 3V7P) was determined at 1.35 Å resolution,
revealing a slightly distorted TIM-barrel domain with a metal
binding site and a much smaller β-barrel domain positioned
between two short α-helices. The latter is comprised of both N-
terminal (residues 1−54) and C-terminal (starting around
residue 354) sequences. The likely biological assembly of
Nis0429 is a C2-symmetrical homodimer (Figure 5), in which
both domains are involved in formation of the dimer interface
Substrate Specificity. The substrate profiles of Sav2595,
Acel0264, Dr0824, and Nis0429 were initially determined by
monitoring changes in absorbance between 240 and 300 nm
using a small library of modified adenosine derivatives. The best
substrate for these four enzymes is AFL. Less efficient
substrates are NECA, adenosine, 5′-methylthioadenosine, S-
adenosylhomocysteine, 2′-deoxyadenosine, 3′-deoxyadenosine,
5′-deoxyadenosine, and AMP. The kinetic constants are
presented in Table 2. The values of kcat/Km with AFL for
Sav2595, Acel0264, Dr0824, and Nis0429 are greater than 105
M−1 s−1.
Mutational Analysis. Single-site mutants of Sav2595 and
Nis0429 were constructed to help identify residues that may
interact with the side chain carboxylate of AFL. The R87A and
R87M variants of Sav2595 were expressed in E. coli and
subsequently purified. The S145A mutant of Nis0429 was
constructed, expressed in E. coli, and purified. The mutant
proteins were assayed, and the kinetic constants for the
deamination of AFL and adenosine were determined. The
results for the Sav2595 mutants are presented in Table 3. The
Figure 5. Cartoon model of Nis0429 C2-symmetrical homodimer
(based on the structure 3V7P). Each monomer consists of two
domains, a TIM-barrel domain (helices are red, β-sheets are yellow,
and loops are green) and a smaller domain 2 (a β-barrel flanked with
two short α-helices, all are blue). Iron and benzoic acid ligands are
shown as spheres. Both domains contribute to interactions between
amino acid residues along the dimer interface.
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Biochemistry
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and maintenance of the active site architecture. Besides protein
atoms and water molecules, the refined structure contains a few
tightly bound ligands (listed in Table 1). The metal is
coordinated to His-61, His-62, His-206, and water 608, which
was modeled and refined as Fe2+ on the basis of coordination
geometry and crystallographic refinement (Figure 6). However,
Figure 7. Cartoon model of Nis0429 with a bound substrate (6-
aminodeoxyfutalosine, AFL, the stick model). The substrate was
modeled using the location of a few smaller ligands detected inside of
the substrate-binding site in the structure 3V7P (the surface of these
small ligands is marked by yellow dots). The iron atom is drawn as a
sphere.
Figure 6. 2FoFc electron density map (1σ cutoff) around amino acid
residues involved in metal coordination (red) inside of the active site
of Nis0429 (PDB ID: 3V7P) and around small unknown ligands
(green). The residues are shown as stick models, and iron is a yellow
sphere. The unknown ligands were modeled and refined as benzoic
acid, glycine, and formate. They occupy the suspected substrate-
binding site of Nis0429. The overall protein structure in the
background is represented by a cartoon model.
the possibility exists that there is a mixture of metals in the
active site, but refinement parameters and coordination
geometry are more consistent with the presence of iron; thus,
the metal was modeled and refined as such. The difference
between the metal analysis of the purified protein and the metal
in the active site may be related to the crystallization
conditions.
Figure 8. Amino acid residues in the active site of Nis0429 (PDB ID:
3V7P) are shown with a modeled AFL presented as sticks. The
residues that are conserved among “typical” adenosine deaminases
have carbon atoms colored in magenta. The residues of the proposed
hydrophobic binding site are unique to Nis0429 and its homologues
and have carbon atoms drawn in yellow. The carbon atoms of AFL are
colored in cyan. The iron atom is shown as a gray sphere. The protein
in the background is presented by a cartoon model.
In addition to two small unknown ligands (refined as formic
acid and glycine) inside the active site, strong electron density
has revealed the presence of a larger molecule that was modeled
and refined as benzoic acid (Figure 6). On the basis of the
location of this particular ligand and using the coordinates of
previously reported structures of adenosine deaminases, one
AFL molecule was manually modeled into the active site of
Nis0429. In the crystal structure, the carboxylic acid moiety of
benzoic acid is coordinated by the side chain hydroxyl of Ser-
145 from β-strand 3 and a water molecule. The same
interactions can be mimicked by the 5′ side chain of AFL
(Figure 7). The analogous residue position in all members of
group 7 from cog0402 is either serine or threonine.
Comparison of the structure of Nis0429 (PDB ID: 3V7P)
with structures of adenosine deaminases revealed striking
similarities between their highly conserved (>90%) metal and
nucleoside binding sites (magenta, Figure 8). By contrast, the
hydrophobic portion of the binding site is represented by
mainly nonpolar residues specific for Nis0429 and its closest
homologues (yellow, Figure 8).
with one important difference: the orientation of the benzoic
acid ligand bound inside the active site next to a residue 145
(Figure 9). In the wild-type protein, the carboxyl moiety of the
ligand is hydrogen bonded to the Ser-145 side-chain hydroxyl
group and a Gly-144 amide nitrogen, whereas in the mutant
structure, the same group is hydrogen bonded to the side chain
from Arg-89. Another interesting feature of the Ser-145 mutant
is the presence of the metal ion (refined as Fe2+) in the active
site. The high resolution structure revealed a slightly distorted
tetrahedral metal ion coordination with varied distances
between bonded atoms. The distances are as follows: 1.63 Å,
Fe−water A962; 1.96 Å, Fe−His-61; 2.12 Å, Fe−His-63; 2.33
Å, Fe−His-206. The side chain of Asp-306 is positioned in two
alternate conformations, one of which may support a weak
coordination bond to the iron at a distance of 2.52 Å.
Structure of S145 Mutant of Nis0429. The crystals of
wild-type Nis0429 and the S145A mutant were essentially
isomorphous (Table 1), and their structures are very similar
Structure of Dr0824. The crystal structure of Dr0824 was
determined by the SAD method and refined to 1.78 Å
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Figure 10. Superposition of two structures, 3V7P (yellow) and 2IMR
(green and blue). Two sequence segments (residues 102−128 and
253−283) in the structure 2IMR are blue. Except for the small domain
2, these are the only parts in the Dr0824 structure that are significantly
different form their counterparts in the Nis0429 structure. AFL
(spheres) cannot be fitted inside of the active site of the structure
2IMR due to multiple clashes with residues residing on segments
colored in blue.
Figure 9. Superimposition of wild-type Nis0429 (PDB ID: 3V7P) and
the S145A mutant (PDB ID: 4M51). For the wild-type enzyme, the
carbon atoms are cyan, oxygen atoms are red, and nitrogen atoms are
blue. For the S145A mutant, the carbon atoms are green, with selected
residues drawn as sticks. Iron is drawn as a brown sphere. The 2FoFc
electron density maps (σ cutoff 1.0) are shown around the benzoic
acid ligands and interacting residues.
resolution (Table 1). Dr0824 consists of two domains, a
distorted TIM-barrel domain, and a small domain with a β-
sheet flanked by α-helices on both sides. The small domain is
made up of both the N-terminal (34−91) and C-terminal
(399−413) residues. Residues 1−33, 47−49, and 395−396
have poor or undefined electron density. Missing residues 47−
49 and 395−396 are located at the interface of the
intramolecular domains.
A metal ion is located at the C-terminal side of the β-sheet of
the distorted TIM-barrel domain, which is the catalytic domain.
The metal ion is coordinated with His-97 and His-99 of β-
strand 1, His-238 of β-strand 6, and a water molecule (O554).
Water molecule O554 also forms a hydrogen bond with OD1
of Asp-352 and is expected to be the nucleophilic center. Both
N- and C-terminal sides of the distorted β-barrel of this
catalytic domain are covered by α-helices, and the active site is
closed from outside interactions.
residues (His-32, His-34, and His-215) and one aspartate
residue (Asp-296). This catalytic machinery is conserved in
Dr0824 and Nis0429 from cog0402. The modeled active site of
Sav2595 shows the two hydroxyl groups from the ribose moiety
of AFL forming hydrogen bonds with the side chain carboxylate
group from Asp-154. This interaction is also identified in the
docking of AFL to Nis0429 (Glu-141). The N3 nitrogen from
the adenine moiety of AFL forms a hydrogen bond with the
backbone amine group from Gly-188, but this interaction is
replaced by a histidine residue in Nis0429 (His-179). The AFL
specificity of Sav2595 can be explained by the presence of an
arginine residue (Arg-87) that interacts with the carboxylate
group in the 3-ketobenzoic acid moiety of AFL (Figure 11). In
Nis0429, Ser-145 is predicted to hydrogen bond to the 3-
ketobenzoic acid moiety (Figure 8).
The crystallographic data support the conclusion that the
biological assembly for this protein is a monomer rather than a
homodimer. Excluding the unique N-terminal sequence
fragment (∼50 residues), the Dr0824 structure shows a high
degree of similarity to Nis0429 (Figure 10), with an RMSD
between Cα atoms along the aligned length (residues 54−274)
of about 2.1 Å. The only large difference between these two
structures is the conformation of the two segments near the
active site pocket (residues 102−128 and 253−283, colored
blue in the 2IMR structure in Figure 10). The observed
differences may be the result of crystal packing, loop flexibility,
or bound ligands present in the substrate-binding site of
Nis0429.
Docking Poses of AFL in Sav2595 and Acel0264. In
docking screens, the chiral tetrahedral intermediate for the
deamination reaction that was generated by the attack of
hydroxide on the re face of the adenine moiety of AFL was
ranked 51 and 30 among the 57 680 high-energy intermediates
(HEI) in the virtual substrate library for Acel0264 and Sav2595,
respectively. In the modeled active site of Sav2595 from
cog1816, the metal ion is coordinated by three histidine
Figure 11. Model of Sav2595 with AFL docked in the active site. The
HEI of AFL is shown in blue. The hydrophobic pocket that
accommodates the AFL side chain and substrate recognizing arginine
are shown in yellow. Residues in purple are conserved among
adenosine deaminases. The zinc ion is shown as a gray sphere.
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DISCUSSION
Article
guanine deaminases from group 2. Dr0824 has a sequence
identity of 25% or less with members of group 7 of cog0402
and has very low sequence similarity to well-characterized
members of cog0402 and groups 11 and 13 of cog1816. Active
site residues involved in metal binding are conserved among
group 7 and 41 members, including an HxH motif after β-
strand 1, another histidine after β-strand 5, and an aspartate
after β-strand 8. A histidine after β-strand 6 is associated with
binding a hydrolytic water molecule. A catalytically essential
glutamate is conserved after β-strand 5. Additional shared
substrate recognition elements include a glutamate after β-
strand 1 involved in binding of the 3′-hydroxyl group of the
ribose moiety and a histidine after β-strand 4 responsible for
hydrogen bonding to N3 of the purine ring system of a
nucleoside substrate.
The active site structure of Nis0429 (PDB ID: 3V7P) is
shown in Figure 6, and the complete structure is shown in
Figure 7 with AFL manually docked in the active site. The
placement of AFL in the active site of Nis0429 was guided by
the position of benzoic acid in the structure of this protein and
the binding of adenosine in the active site of adenosine
deaminases. This binding mode predicts an interaction with
Ser-145 after β-strand 3 to bind the side chain of AFL (Figure
8). A serine or threonine residue is conserved in this position in
all members of group 7 of cog0402 but not in group 41 or
groups 11 and 13 of cog1816. It is noteworthy that the S145A
mutation of Nis0429 has resulted in more than a 10-fold higher
KM value than that of the wild-type enzyme. These data support
the idea that Ser-145 is directly involved in binding of AFL.
We have demonstrated that Acel0264, Sav2595, Nis0429,
and Dr0824 are not adenosine deaminases or chlorohydrolases,
as currently annotated. On the basis of sequence similarity and
conservation of putative essential residues, all known members
of groups 11 and 13 of cog1816 and groups 7 and 41 of
cog0402 are probable 6-aminodeoxyfutalosine deaminases. It is
quite likely that these enzymes evolved from distinct
deaminases, which may explain the variation in observed
enzyme architecture. On the basis of our data, we propose a list
of proteins believed to be 6-aminodeoxyfutalosine deaminase
enzymes (Table 4). The presence of an AFL deaminase in
certain organisms is surprising to some degree because
Campylobacter jejuni has been reported to have a methyl-
thioadenosine nucleosidase (MTAN) that will hydrolyze AFL
directly to the MqnB product (Figure 1) but will not hydrolyze
futalosine.25 C. jejuni MTAN is the only close relative for
futalosine hydrolase (MqnB) activity in the genome. It is
unclear why organisms would have an AFL deaminase if they
do not also have a futalosine hydrolase. Perhaps the AFL
deaminase gene is a remnant of the original futalosine pathway
in these organisms.
■
Utilization of Bioinformatics, Structural Biology, and
Molecular Enzymology To Determine Substrate Specif-
icity. A group of bacterial deaminases from cog1816 and
cog0402 within the amidohydrolase superfamily of enzymes has
been characterized. These enzymes are currently incorrectly
annotated as adenosine deaminases and chlorohydrolases. The
most likely physiological substrate is 6-aminodeoxyfutalosine
(AFL). AFL is an intermediate in the biosynthesis of
menaquinone in certain bacteria that utilize the futalosine
pathway.4 Further evidence supporting a role in the futalosine
pathway is the location of Acel0264 in a cluster of genes related
to the biosynthesis of menaquinone (Figure 2). The gene for
Acel0264 is flanked by the genes for Acel0261 and Acel0263.
These two genes are orthologs to MqnA and MqnC,
respectively. Other members of group 11 from cog1816 have
only UbiE, a methyltransferase in menaquinone biosynthesis,
and cog-FixC, a geranylgeranyl reductase, as recurring
neighbors. Members of group 13 of cog1816 and groups 7
and 41 of cog0402 show no genomic hints related to
menaquinone biosynthesis, except for the lack of men gene
homologues and the presence of mqn homologues.
Sequence Comparison and Structural Analysis. The
four enzymes in group 11 of cog1816 share sequence identity
to one another of greater than 50% but are less than 30%
identical to the well-characterized adenosine deaminase from E.
coli (locus tag: b1623) from group 5 of cog1816. The same
degree of similarity is observed between members of group 13.
All of the residues that coordinate the divalent metal ion in the
active site are conserved among the group 5, 11, and 13
enzymes, which include the HxH motif following β-strand 1,
the histidine at the C-terminus of β-strand 5, and the aspartate
at the C-terminus of β-strand 8. Another mutually conserved
aspartate following β-strand 8 is responsible for hydrogen
bonding to N7 of the adenine ring. Also conserved among
these deaminases from cog1816 is the glutamate from the HxxE
motif at the C-terminus of β-strand 5. A functionally important
and conserved histidine at the C-terminus of β-strand 6 forms a
hydrogen bond with the putative hydrolytic water and may
participate in proton transfer reactions.
Unlike other members of cog1816, members of group 11 and
13 have a conserved arginine following β-strand 1. Using
adenosine deaminase (PDB ID: 1KRM) as a structural
template, a model was constructed in an attempt to understand
the substrate specificity of Acel0264 and Sav2595. An arginine
residue was identified that could possibly recognize the
carboxylate group of the 5′ side chain of AFL. The predicted
location of Arg-87 in Sav2595 is on a turn between two α-
helices after β-strand 1. This arginine was mutated to alanine or
methionine in Sav2595, resulting in slower rates for the
deamination of AFL, with essentially no effect on KM (Table 3).
The R87A and R87M mutants could be saturated with
adenosine, whereas the wild-type enzyme was not. From
these results, we propose that Arg-87 is likely near the substrate
binding site (Figure 11) because changing this residue leads to
the loss of substrate specificity. The remainder of the large
substrate binding pocket is made up of hydrophobic residues
and an aspartate or glutamate to interact with the 2′-hydroxyl
group.
Comparison to Adenosine Deaminase from cog1816.
The prototypical adeonosine deaminase from E. coli (locus tag:
b1623) is also a member of the amidohydrolase superfamily
and cog1816. This enzyme has a mononuclear metal center and
will deaminate adenosine, N-6-methyladenosine, and 2′-
deoxyadenosine as substrates with comparable rate constants.
With the high conservation of metal-binding and catalytically
essential residues between Acel0264, Sav2595, and b1623, it is
very likely that these enzymes will employ the same mechanism
for deamination. Dr0824 and Nis0429 have a slightly modified
nucleoside binding site compared to that of b1623, but the
structures and the biochemical data clearly support the notion
Members of group 7 of cog0402 share a sequence identity of
40% or more within the group but much less than 30% with
atrazine chlorohydrolase and less than 25% compared to that of
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Biochemistry
Article
were identified in clusters in the A. celluloyticus 11B genome,
and we have confirmed that Acel0264 was capable of
deaminating 6-aminodeoxyfutalosine. The genomes of Nitratir-
uptor sp. SB155-2, S. avermitilis MA-4680, and D. radiodurans
R1 lacked the men genes associated with the shikimate pathway,
but they do possess the mqn genes of the futalosine pathway. A
sequence similarity network was constructed and used to
identify additional proteins with similar sequences to Acel0264,
Sav2595, Dr0824, and Nis0429. Substrate profiles for these
proteins were determined using a small focused library, and a
model for substrate binding was proposed. The high resolution
crystal structure of Nis0429 with smaller ligands in the active
site helped us to predict the binding mode of AFL as a
substrate.
Table 4. 6-Aminodeoxyfutalosine Deaminase Enzymes
organism
locus tag
Acel_0264
GI
cog
A. cellulolyticus 11B
117927473
157736750
157164865
154174508
118475030
1816
0402
0402
0402
0402
Arcobacter butzleri RM018
Campylobacter concisus 13826
Campylobacter curvus 525.92
Abu_0489
CCC13826_0371
CCV52592_0362
CFF8240_1504
Campylobacter fetus subsp.
fetus 82-40
Campylobacter hominis ATCC
BAA_381
CHAB381_1212
154147820
0402
C. jejuni NCTC 11168
C. jejuni RM1221
Cj0067
15791459
57237077
121612374
153952082
157414381
222823270
226355533
94984683
0402
0402
0402
0402
0402
0402
0402
0402
CJE0064
C. jejuni subsp. 81-176
CJJ81176_0105
JJD26997_0077
C8J_0060
C. jejuni subsp. doylei 269.97
C. jejuni subsp. jejuni 81116
Campylobacter lari RM1200
Deinococcus deserti VCD115
AUTHOR INFORMATION
Cla_0229
■
Deide_06659
Dgeo_0576
Corresponding Author
*F. M. Raushel. E-mail: raushel@tamu.edu. Phone: (979) 845-
3373.
Deinococcus geothermalis DSM
11300
D. radiodurans R1
Frankia alni ANC14a
Frankia sp. Ccl3
Dr0824
15805850
111220494
86739247
158317840
158314030
32267263
0402
1816
1816
1816
1816
0402
FUNDING
FRAAL1027
Francci3_0534
Franean1_6098
Franean1_2196
HH1764
This work was supported in part by the Robert A. Welch
Foundation (A-840) and the National Institutes of Health (GM
71790). This work was made possible by a Center for
Synchrotron Biosciences grant, P30-EB-009998, from the
National Institute of Biomedical Imaging and Bioengineering
(NIBIB). Use of the National Synchrotron Light Source,
Brookhaven National Laboratory, was supported by the U.S.
Department of Energy, Office of Science, Office of Basic Energy
Sciences, under Contract No. DE-AC02-98CH10886.
Frankia sp. EAN1pec
Frankia sp. EAN1pec
Helicobacter hepaticus ATCC
51449
Nautilia profundicola AmH
Nitratiruptor sp. SB155-2
Nocardioides sp. JS614
NAMH_1355
NIS_0429
224373376
152990178
119715591
159037391
145594323
29829137
21224013
182435634
0402
0402
1816
1816
1816
1816
1816
1816
Noca_1355
Salinospora arenicola CNS-205 Sare_1766
Salinospora tropica CNB-440
S. avermitilis MA-4680
S. coelicolor A3(2)
Strop_1779
Sav_2595
Sco5662
Notes
The authors declare no competing financial interest.
Streptomyces griseus subsp.
griseus NBRC 13359
SGR_1841
ABBREVIATIONS
■
Sulfurimonas denitrificans DSM Suden_0686
1251
78776885
0402
ADE, adenine deaminase; ADD, adenosine deaminase; cog,
cluster of orthologous groups; IPTG, isopropyl-β-galactoside;
DTT, dithiothreitol; AHS, amidohydrolase superfamily; ICP-
MS, inductively coupled plasma mass spectrometry; NECA, 1-
(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranurona-
mide; SAH, S-adenosylhomocysteine; AFL, 6-aminodeoxyfuta-
losine
Sulfurovum sp. NBC37-1
SUN_1037
WS1477
152992630
34557817
0402
0402
Wolinella succinogenes DSM
1740
that they function through an activated water molecule to
facilitate the deamination reaction.
Acidothermus celluloyticus 11B has a protein from cog0402
(Acel1042) of unknown function, and no other proteins in
cog1816 or cog1001; thus, adenosine or adenine deaminases
are not likely to be found in this organism. The genome of S.
avermitilis MA-4680 contains five proteins from cog1816 and
four proteins from cog0402. Of the five proteins from cog1816,
Sav5577 is a confirmed adenine deaminase belonging to group
3, Sav1165 and Sav3358 are believed to be adenosine
deaminases from group 1, and Sav4906 from group 17 has an
unknown function. The genes from cog0402 include an
isoxanthopterin deaminase (Sav2017), Sav4899, Sav6652, and
Sav3494 of unknown function. D. radiodurans R1 has an
adenine deaminase from cog1001 (DrA0270) and a guanine
deaminase from cog0402 (DrA0180) in addition to Dr0824.
Nis0429 is the sole protein in Nitratiruptor sp. SB155-2 from
cog0402, cog1816, or cog1001.
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