Discovery of a bacterial 5-methylcytosine deaminase

Article abstract of DOI:10.1021/bi5012767

5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a "discriminating" residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of k_cat/K_m of 1.4 × 10⁵, 2.9 × 10⁴, and 1.1 × 10³ M^-1 s^-1, respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of k_cat/K_m of 1.2 × 10⁵, 6.8 × 10⁴, and 2.0 × 10² M^-1 s^-1, respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85), 5-fluorocytosine (PDB id: 4R88), and phosphonocytosine (PDB id: 4R7W) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil.

Full text of DOI:10.1021/bi5012767

Subscriber access provided by DICLE UNIV
Article
Discovery of a Bacterial 5-Methylcytosine Deaminase
Daniel S. Hitchcock, Alexander A. Fedorov, Elena V. Fedorov, Steven C. Almo, and Frank M. Raushel
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/bi5012767 • Publication Date (Web): 10 Nov 2014
Downloaded from http://pubs.acs.org on November 14, 2014
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street
N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
Discovery of a Bacterial 5-Methylcytosine
Deaminase
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Daniel S. Hitchcockϕ, Alexander A. Fedorovψ, Elena V. Fedorovψ,
Steven C. Almoψ*, and Frank M. Raushelϕξ*
ϕDepartment of Biochemistry & Biophysics, ξDepartment of Chemistry, Texas A&M
University, College Station, Texas 77843; Department of Biochemistry, ψAlbert Einstein
College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461
This work was supported in part by the Robert A. Welch Foundation (A-840) and the
National Institutes of Health (GM 71790).
*To whom correspondence may be sent:
F. M. Raushel. Telephone: 979-845-3373; e-mail: raushel@tamu.edu
S. C. Almo. Telephone: 718-430-2746; e-mail: steve.almo@einstein.yu.edu
1
ACS Paragon Plus Environment

Biochemistry
Page 2 of 41
1
2
3
4
5
ABBREVIATIONS
6
7
8
9
5-FC – 5-fluorocytosine
5-FU – 5-fluorouracil
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
COG – cluster of orthologous groups
PDB – Protein Data Bank
CGSC – Coli Genetic Stock Center at Yale University
PMSF – phenylmethylsulfonyl fluoride
BLAST – Basic Local Alignment Search Tool
SSN – sequence similarity network
2
ACS Paragon Plus Environment

Page 3 of 41
Biochemistry
1
2
3
4
5
ABSTRACT
5-Methylcytosine is found in all domains of life but the bacterial cytosine deaminase
6
7
8
9
from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since
significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes,
this compound must eventually be catabolized and the fragments recycled by enzymes that
have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for
enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a
systematic analysis of sequence homologues of CodA from thousands of bacterial species,
we identified putative cytosine deaminases where a “discriminating” residue in the active
site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter
sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075)
were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of
kcat/Km of 1.4 x 105, 2.9 x 104,and 1.1 x 103 M-1s-1, respectively. These three enzymes also
catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 x
105, 6.8 x 104, and 2.0 x 102 M-1 s-1, respectively. The three dimensional structure of
Kpn00632 was determined by x-ray diffraction methods with 5-methylcytosine (PDB id:
4R85), 5-fluorocytosine (PDB id: 4R88) and phosphonocytosine (PDB id: 4R7W) bound in
the active site. When thymine auxotrophs of E. coli express these enzymes, they are
capable of growth in media lacking thymine when supplemented with 5-methylcytosine.
Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to
the efficient transformation to 5-fluorouracil.
3
ACS Paragon Plus Environment

Biochemistry
Page 4 of 41
1
2
3
4
5
6
7
8
9
5-Methylcytosine is a modified nucleobase formed by the methylation of cytosine in
DNA. The synthesis of 5-methylcytosine is catalyzed by DNA methyltransferases, and in
animals, plants, and fungi, this modification functions as an epigenetic marker.1-3 In
mammals, methylation occurs predominantly at CpG sites in ~1% of the human genome.4
In Escherichia coli and related bacteria, methylation occurs at CC(A/T)GG sites by the dcm
methylase.5 Methylation of cytosine in the DNA of bacteria is part of the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
restriction/modification system and has also been implicated in controlling gene
expression during stationary phase.6 There is no known direct demethylation reaction to
form cytosine from 5-methylcytosine in DNA. Instead, the methyl group is first
hydroxylated and then oxidized to form 5-carboxycytosine, which is excised from DNA by
base excision repair (Scheme 1).7, 8 This process is initiated by methylcytosine
dioxygenase 1 (TET1) to produce 5-hydroxymethylcytosine.9 Further oxidation of
hydroxymethyl cytosine by TET1 and methylcytosine dioxygenase 2 (TET2) yields 5-
formylcytosine and 5-carboxycytosine, respectively.10
The deamination of the cytosine moiety in nucleotides and nucleic acids is a
conserved metabolic step for the recycling of pyrimidines across all domains of life. This
reaction may occur through the deamination of cytosine,11, 12 cytidine,13 cytidine
monophosphate,14, 15 or cytidine triphosphate.16, 17 Cytidine deaminases from cog0295 are
found in both prokaryotes and eukaryotes.18, 19 The cytidine deaminases from E. coli and
yeast have been studied in some detail, and the E. coli enzyme has been shown to be
catalytically active with both cytidine and 5-methylcytidine. At least two variants of
cytosine deaminase exist. The yeast cytosine deaminase can deaminate 5-methylcytosine
in addition to cytosine and the active site of this enzyme is similar to that of cytidine
4
ACS Paragon Plus Environment

Page 5 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
deaminase.12, 20 These enzymes are members of the cytidine deaminase-like superfamily
and cog0590. In contrast, the unrelated bacterial cytosine deaminase (CodA) from E. coli
(locus tag: b0337) will not deaminate 5-methylcytosine at appreciable rates.21
CodA from E. coli is a member of cog0402 and the amidohydrolase superfamily
(AHS).22, 23 Other deaminases from this Cluster of Orthologous Groups (COG) include
guanine deaminase,24 S-adenosylhomocysteine deaminase,25 and 8-oxoguanine
deaminase26 among others. The structure of CodA from E. coli has been determined in the
absence of bound ligands (PDB id: 1K6W), and also in the presence of isoguanine (PDB id:
3RN6) and a phosphonate mimic of the transition-state (PDB id: 3O7U; Figure 1).
Substrate binding relies on Gln-156, which forms a pair of hydrogen bonds with the
carboxamide moiety of the pyrimidine or purine base. Glu-217 participates in substrate
recognition and catalysis by a direct interaction with the amidine moiety of the substrate
(Figures 1B and 1C). In this active site, Asp-314 provides an apparent steric boundary for
the binding of cytosine as a substrate, and participates in a hydrogen bond to N7 of the
purine ring for recognition of isoguanine (Figure 1B). CodA can accept pyrimidine
(cytosine) and purine (isoguanine) substrates but the active site is apparently not
configured to deaminate structurally related compounds such as 5-methylcytosine and 5-
fluorocytosine.21, 27
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Since significant amounts of 5-methylcytosine are produced in both prokaryotes
and eukaryotes, this compound must eventually be catabolized and the fragments recycled.
We therefore initiated a search for enzymes of unknown function related to the bacterial
cytosine deaminase with the catalytic ability to deaminate 5-methylcytosine. By analyzing
sequence homologues of CodA from thousands of bacterial species, we identified groups of
5
ACS Paragon Plus Environment

Biochemistry
Page 6 of 41
1
2
3
4
5
6
7
8
9
putative cytosine deaminases where the “discriminating” residue corresponding to Asp-
314 in CodA from E. coli was no longer conserved. Representative examples of these
enzymes were purified and found to efficiently deaminate cytosine, 5-methylcytosine, and
5-fluorocytosine. Expression of this enzyme in thymine auxotrophs of E. coli rescued
growth in the presence of 5-methylcytosine. Expression of this enzyme was toxic in the
presence of 5-fluorocytosine in strains of E. coli that also expressed uracil
phosphoribosyltransferase.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
M
ATERIALS and
METHODS
E. coli Cell Lines. Two gene knockout strains of E. coli were obtained from the Coli
Genetic Stock Center (CGSC) at Yale University. Both cell lines lack the genes for the
metabolism of arabinose, allowing the use of arabinose-inducible plasmids. The
pyrimidine auxotroph (CGSC-9145) lacks the gene for orotidine-5-phosphate
decarboxylase (F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, ΔpyrF789::kan, rph-1, Δ(rhaD-
rhaB)568, hsdR514)28. This cell line expresses pyrimidine phosphoribosyltransferase and
will convert added pyrimidine nucleotides to their monophosphorylated counterparts,
including 5-fluorocytosine and 5-fluorouracil. The thymine auxotroph (CGSC-4091) lacks
the gene for thymidylate synthetase (F-, araBAD-1?, tsx-77, thyA40, deoB15).
Cloning and Purification of Kpn00632, Rsp0371, NCgl0075 and CodA. The
genes for Kpn00632 from Klebsiella pneumoniae subsp. pneumoniae (gi|152969203),
Rsp0371 from Rhodobacter sphaeroides 2.4.1 (gi|77463913), and NCgl0075 from
Corynebacterium glutamicum ATCC 13032 (gi|19551325) were cloned into pET28 with a C-
terminal His6-tag using standard cloning practices. The plasmids were transformed into E.
6
ACS Paragon Plus Environment

Page 7 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
coli BL-21 DE3 cells and plated onto LB-agarose containing 50 μg/mL kanamycin. Cultures
of 1 L (LB broth, kanamycin 50 μg/mL) were inoculated with the resulting colonies and
grown at 37 °C until the optical density at 600 nm reached 0.6. Protein expression was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
induced with 1.0 mM isopropyl β-D-1-thiogalactopyranoside, and the cultures were shaken
for 20 hours at 25 °C. The cultures were centrifuged at 7,000 rpm for 15 minutes and the
isolated cell pellets disrupted by sonication in 35 mL of buffer A (20 mM HEPES, 250 mM
NaCl, 250 mM NH4SO4, 20 mM imidazole, pH 7.5) containing 2.5 mg phenylmethylsulfonyl
fluoride (PMSF). DNA was removed by dropwise addition of 100 mg protamine sulfate
dissolved in 15 mL of buffer A. Proteins with a His6-tag were loaded onto a 5-mL HisTrap
column (GE Heathcare) with running buffer A and eluted with a linear gradient of elution
buffer B (20 mM HEPES, 250 mM NaCl, 250 mM NH4SO4, 500 mM imidazole, pH 7.5).
The gene for cytosine deaminase (CodA , b0337) from E. coli K12 (gi|16128322),
was cloned into pBAD322C without purification tags.29 E. coli cells lacking arabinose
metabolizing genes (CGSC 9145) were transformed with the plasmid and plated onto LB-
agarose containing 25 μg/mL chloramphenicol. A 1 L culture (LB broth, chloramphenicol
25 μg/mL) was inoculated from the resulting colonies and grown at 37 °C until the optical
density at 600 nm reached 0.6. Protein expression was induced with 1.0 mM L-arabinose,
and the culture was shaken for 20 hours at 25 °C. The sample was centrifuged at 7,000
RPM for 15 minutes and the cell pellet was disrupted by sonication in 35 mL of running
buffer (50 mM HEPES, pH 7.5) and 2.5 mg of PMSF. DNA was removed by the dropwise
addition of 100 mg protamine sulfate dissolved in 15 mL of running buffer. The protein
was precipitated with 70% saturated NH4SO4 and the pellet resuspended in 4.0 mL of
7
ACS Paragon Plus Environment

Biochemistry
Page 8 of 41
1
2
3
4
5
6
7
8
9
running buffer. The protein solution was loaded onto a HiLoad 26/600 Superdex 200 gel
filtration column. Fractions containing active enzyme were pooled and concentrated.
Cloning of Enzymes for in vivo Assays. The genes for Kpn00632 and Rsp0341
were cloned into pBAD322C using standard cloning practices without purification tags.
The D314A mutant of cytosine deaminase from E. coli was constructed by standard site
directed mutagenesis methods using primer overlap extension from the pBAD322c-b0337
plasmid with the primers 5’-CGTCTGCTTTGGTCACGATGCTGTCTTCGATCCGTGGTATCC-3’
and 5’- GGATACCACGGATCGAAGACAGCATCGTGACCAAAGCAGACG-3’.30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Activity Screening and Determination of Kinetic Constants. Purified enzyme
was incubated in 50 mM HEPES, pH 7.5, at 25 oC for 1 hour in a 96-well UV-Vis quartz plate
with a small library of potential substrates (Scheme 2). The spectra were monitored as a
function of time from 240-350 nm. The substrate library included cytosine (1), 5-
methylcytosine (2), 5-hydroxymethylcytosine (3), 5-fluorocytosine (4), 5-aminocytosine
(5), creatinine (6), isoguanine (7), cytosine-5-carboxylate (8), N-methylcytosine (9), and 5-
formylcytosine (10). Initial reaction velocities were measured by a direct UV-Vis assay in a
96-well quartz plate using various enzyme/substrate combinations in 20 mM HEPES, pH
7.5 at 30 oC. Product formation was monitored at the following wavelengths using
experimentally-derived differential molar extinction coefficients for the following
substrates: cytosine (255 nm, Δε = 2,600 M-1 cm-1), 5-methylcytosine (262 nm, Δε = 3,700
M-1 cm-1), creatinine (240 nm, Δε = 6,100 M-1 cm-1), isoguanine (294 nm, Δε = -6,600 M-1 cm-
1), 5-fluorocytosine (262 nm, Δε = 3,200 M-1 cm-1), 5-aminocytosine (235 nm, Δε = -2,000
M-1 cm-1), 5-hydroxymethylcytosine (258 nm, Δε = 3,700 M-1 cm-1). Values of kcat and
8
ACS Paragon Plus Environment

Page 9 of 41
Biochemistry
1
2
3
4
kcat/Km were determined by fitting the data to equation 1 using SigmaPlot 11, where v is the
5
6
7
initial velocity, Et is enzyme concentration, and A is the substrate concentration.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
v/Et = kcat A/ (A + Ka)
(1)
Selection Medium for in vivo Experiments. The selection medium contained M9
minimal salts, 1.96 g/L yeast synthetic dropout medium without uracil, 0.1% glycerol, 100
µM CaCl2, 1.0 mM MgSO4, and trace elements. The 5000x mixture of trace elements was
prepared with 95 mL H2O, 5.0 g citric acid, 5.0 g ZnSO4ꢀ(7 H2O), 4.75 g FeSO4ꢀ(7 H2O), 1.0 g
Fe(NH4)2(SO4)2ꢀ(6 H2O), 250 mg CuSO4ꢀ(H2O), and 50 mg each of MnSO4ꢀ(H2O), and
Na2MoO4ꢀ(2H2O). Yeast synthetic dropout medium with glycerol (and cytosine when
supplemented) was autoclaved separately from the M9 medium. CaCl2 and MgSO4 were
sterile filtered and the mixture of trace elements was autoclaved before all components
were combined.
Determination of in vivo 5-Methylcytosine Deaminase Activity. The thymine
auxotroph of E. coli was transformed with pBAD322C and the plasmids containing cytosine
deaminase from E. coli, Kpn00326, and Rsp0341. These cells were plated onto LB-Agar
with chloramphenicol (25 µg/mL) supplemented with 200 µM thymine. Single colonies
were picked and 5 mL cultures of LB containing 100 µM thymine and 25 µg/mL
chloramphenicol were grown for 14 hours overnight. Cultures of selection medium (25
mL) containing 100 µM MnSO4, 100 µM zinc acetate, and 25 µg/mL chloramphenicol were
prepared in 100 mL flasks with the following experimental conditions: (a) 400 µM 5-
fluorocystosine (b) 100 µM arabinose, (c) 100 µM thymine and 100 µM arabinose, (d) 100
9
ACS Paragon Plus Environment

Biochemistry
Page 10 of 41
1
2
3
4
5
6
7
8
9
µM 5-methylcytosine and 100 µM arabinose, and (e) 500 µM 5-methylcytosine and 100 µM
arabinose. The cultures were inoculated with 25 µL from the overnight cultures and
shaken at 200 rpm for 12 hours at 37 oC. The absorbance at 600 nm was measured every 2
hours.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Determination of in vivo 5-Fluorocytosine Deaminase Activity. The E. coli
pyrimidine auxotroph was transformed with pBAD322C and the plasmids containing the
genes for cytosine deaminase (CodA, b0337), Kpn00326, and the D314A mutant of cytosine
deaminase from E. coli. Cells were grown on LB-agar plates in the presence of 25 µg/mL
chloramphenicol. Single colonies were picked and overnight cultures (5 mL) were grown
in selection medium containing 150 µM cytosine, 25 µg/mL chloramphenicol, 100 µM
MnSO4, and 100 µM zinc acetate in 100 mL flasks prepared with the following experimental
conditions: (a) 100 µM arabinose, (b) 50 µM 5-fluorocytosine and 100 µM arabinose (c)
500 µM 5-fluorocytosine and 100 µM arabinose, (d) 5.0 µM 5-fluorouracil and 100 µM
arabinose. The cultures were inoculated with 25 µL of the overnight cultures and shaken at
200 rpm for 12 hours at 37 oC. The absorbance at 600 nm was measured every 2 hours.
Crystallization and Data Collection. Crystals of Kpn00632 from Klebsiella
pneumoniae liganded with Fe2+ and 5-methylcytosine were grown by the sitting drop
method at room temperature. The protein solution contained Kpn00632 (18.6 mg/mL) in
20 mM HEPES (pH 7.5), 200 mM imidazole, 250 mM NaCl, 250 mM ammonium sulfate, 1.0
mM FeCl2, and 40 mM 5-methylcytosine. The precipitant contained 20% PEG 3350, 0.15 M
malic acid (pH 7.0) and 1.0 mM FeCl2. Crystals appeared in two weeks and exhibited
diffraction consistent with the space group P21, with six polypeptides per asymmetric unit.
Crystals of Kpn00632 liganded with Fe2+ and 5-fluorocytosine were also grown by the
10
ACS Paragon Plus Environment

Page 11 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
sitting drop method at room temperature. The protein solution contained enzyme (18.6
mg/mL) in 20 mM HEPES (pH 7.5), 200 mM imidazole, 250 mM NaCl, 250 mM ammonium
sulfate, 1.0 mM FeCl2, and 60 mM 5-fluorocytosine; the precipitant contained 30% PEG
3350, 0.1 M sodium citrate (pH 5.6), 0.2 M ammonium acetate, and 1.0 mM FeCl2. Crystals
appeared in five days and exhibited diffraction consistent with the space group P212121,
with six polypeptides per asymmetric unit. Crystals of Kpn00632 liganded with Fe2+ and
phosphonocytosine21 were grown by the sitting drop method at room temperature. The
protein solution contained enzyme (15 mg/mL) in 20 mM HEPES (pH 7.5), 1.0 mM FeCl2,
and 10 mM phosphonocytosine. The precipitant contained 20% PEG 3350, 0.15 M malic
acid (pH 7.0), and 1.0 mM FeCl2. Crystals appeared in two days and exhibited diffraction
consistent with the space group P212121, with six polypeptides per asymmetric unit.
Prior to data collection, crystals of the three forms of Kpn00632 were transferred to
cryoprotectant solutions composed of their mother liquids supplemented with 20%
glycerol, and flash-cooled in a nitrogen stream. Three X-ray diffraction data sets were
collected at the NSLS X29A beamline (Brookhaven National Laboratory) on the 315Q CCD
detector. Diffraction intensities were integrated and scaled with programs DENZO and
SCALEPACK.31 The data collection statistics are given in Table 1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Structure Determination and Model Refinement. The three Kpn00632
structures were determined by molecular replacement with BALBES,32 using only input
diffraction and sequence data. BALBES used the structure of cytosine deaminase from E.
coli complexed with phosphonocytosine (PDB id: 3O7U) as the search model. Partially
refined structures of the three Kpn00632 crystal forms were generated by BALBES.
Subsequent iterative cycles of refinement were performed for each crystal form including
11
ACS Paragon Plus Environment

Biochemistry
Page 12 of 41
1
2
3
4
5
6
7
8
9
model rebuilding with COOT,33 refinement with PHENIX 34, and automatic model rebuilding
with ARP.35 The quality of the final structures was verified with omit maps. The
stereochemistry was checked with WHATCHECK36 and MOLPOBITY.37 Program LSQKAB38
was used for structural superposition. Figures with electron density maps were prepared
using PYMOL.39 The final model of the monoclinic crystal form of Kpn00632 with 5-
methylcytosine contains residues 1-411 of the enzyme in all six polypeptides located in the
asymmetric unit. The C-terminal His-tag residues are not included in the final model. The
ferrous ion and ligand are well defined in the active site of every monomer.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The final models of the two orthorhombic crystal forms of Kpn00632 with 5-
fluorocytosine and phosphonocytosine in the active site contain residues 1-412 of the
enzyme in all six polypeptides of these crystal forms. The C-terminal His-tag residues have
weak electron density and are not included in the final models. The ferrous ions and the
corresponding ligand molecules are well defined and bound in the active sites of the
corresponding structures. The final crystallographic refinement statistics for all three
structures of Kpn00632 are provided in Table 1.
RESULTS
Phylogeny of the Cytosine Deaminase Group from cog0402. A BLAST search
using the sequence of cytosine deaminase from E. coli (b0337, CodA) was submitted, and
1377 sequence homologues were identified. An all-by-all BLAST was subsequently
performed with these protein sequences to create a sequence similarity network (SSN) at
an E-value stringency of 10-140 40, 41
.
At this level of protein sequence identity, three major
12
ACS Paragon Plus Environment

Page 13 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
subgroups and a number of minor subgroups could be identified as illustrated in Figure 2
and four representative proteins, one from each major cluster and one from a single minor
cluster, were selected for functional characterization. These proteins included cystosine
deaminase (CodA, b0337) from E. coli (subgroup-a); Kpn00632 from Klebsiella pneumonia
(subgroup-b); Rsp0341 from Rhodobacter sphaeroides 2.4.1 (subgroup-c); and NCgl0075
from Corynebacterium glutamicum ATCC 13032 (subgroup-d). The sequence identity
between any two proteins ranged from 35% (Rsp0341 and NCgl0075) to 58% (CodA and
Kpn00632).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
An amino acid sequence alignment of the proteins contained within each of the four
groups of proteins selected for this investigation identified a striking difference in the
amino acid residue that immediately follows the critical aspartate residue at the C-terminal
end of β-strand 8. In the amidohydrolase superfamily (AHS) of enzymes, this invariant
aspartate residue (Asp-313 in E. coli CodA) coordinates one of the divalent cations in the
active site and functions in proton transfer reactions.21 In subgroup-a, the residue that
follows the invariant aspartate is also an aspartate, whereas in subgroups-b, -c, and -d this
residue is a serine, cysteine, and serine, respectively (Figure 3).
Enzymatic Characterization. The genes for the four enzyme targets were cloned,
expressed in E. coli, and the proteins purified to homogeneity. The kinetic constants for the
purified proteins with a series of ten potential substrates were determined at pH 7.5 and
the results are present in Tables 2 and 3. The prototypical cytosine deaminase from E. coli
(CodA, b0337) was able to deaminate cytosine (1) and isoguanine (7) at appreciable rates
(kcat/Km = 8.4 x 104 and 1.1 x 105 M-1 s-1, respectively), whereas the ability to deaminate 5-
methylcytosine (2) was barely detectable (kcat/Km = 2.2 x 101 M-1 s-1). Kpn00632
13
ACS Paragon Plus Environment

Biochemistry
Page 14 of 41
1
2
3
4
5
6
7
8
9
deaminated 5-methylcytosine (2) approximately four orders-of-magnitude more efficiently
than CodA from E. coli (kcat/Km = 3.3 x 105 M-1 s-1). This enzyme also utilized 5-
fluorocytosine (4) and 5-aminocystosine (5) as substrates with kcat/Km values greater than
105 M-1 s-1. Rsp0341 was determined to have a similar catalytic profile as Kpn00632,
except that this enzyme preferentially deaminates cytosine (1) relative to 5-methylcytosine
(2). The best substrate for NCgl0075 was creatinine (kcat/Km = 6.3 x 104 M-1 s-1). The
D314A mutant of CodA of E. coli shared a similar substrate profile with Kpn00632,
including the dramatic increase in the catalytic activity using 5-methylcytosine (kcat/Km =
9.7 x 104 10 M-1 s-1). As previously reported, this enzyme also exhibited a significant
increase in the rate of deamination of 5-fluorocytosine, relative to the wild-type enzyme
(9.9 x 103 M-1s-1).27
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Three-Dimensional Structure of Kpn00632. The three-dimensional structure of
Kpn00632 was determined in the presence of 5-methylcytosine (2) (PDB id: 4R85), 5-
fluorocytosine (4) (PDB id: 4R88), and phosphonocytosine (PDB id: 4R7W). In each case,
the enzyme adopts a distorted (β/ɑ)8-barrel with a mononuclear metal center at the C-
terminal end of the β-barrel. In the complexes formed with 5-methylcytosine (Figure 4A),
5-fluorocytosine (Figure 4B) and phosphonocystosine21 (Figure 4C) the metal ion is
coordinated to His-58 and His-60 from the C-terminal end of β-strand 1, His-209 from the
C-terminal end of β-strand 5, and Asp-308 from the C-terminal end of β-strand 8. In all
three structures, the substrates and inhibitor form a pair of hydrogen bonds between the
carboxamide functional group and the side chain of Gln-151 (Figures 5 A-C). Additionally,
Glu-212 interacts with the –NH=C-NH2 moiety contained within each of these three ligands.
14
ACS Paragon Plus Environment

Page 15 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
Rescue of Thymine Auxotrophs with 5-Methylcytosine. Strains of E. coli that
lack thymidylate synthase cannot grow without the addition of exogenous thymine. These
cells cannot grow in the presence of added 5-methylcytosine since the wild-type cytosine
deaminase of E. coli lacks the ability to deaminate 5-methylcytosine to thymine.21 The E.
coli thymine auxotroph was transformed with pBAD322c vectors containing the genes for
b0337, Rsp0341, and Kpn00632 to determine whether the expression of enzymes capable
of deaminating 5-methylcytosine to thymine was sufficient to enable E. coli to grow in the
absence of added thymine. All four examples were capable of growth when supplemented
with 100 µM thymine (Figure 6). However, neither the empty vector nor additional CodA
from E. coli were capable of rescuing growth when supplemented with 400 µM 5-
methylcytosine (Figures 6A and 6B). Expression of either Kpn00632 or Rsp0341 allowed
growth in the presence of 400 µM 5-methylcytosine, and to a lesser extent in the presence
of 100 µM added 5-methylcytosine for Kpn00632(Figures 6C and 6D).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Enhanced Toxicity of 5-Fluorocytosine. The ability of Kpn00632 and the D314A
mutant of CodA of E. coli to deaminate 5-fluorocytosine to 5-fluorouracil in vivo was tested
in pyrimidine auxotrophs, lacking pyrF. This cell line will phosphoribosylate exogenous
pyrimidines and in the presence of 5-fluorouracil will form 5-fluorouridine
monophosphate, a potent inhibitor of thymidylate synthase. This strain of E. coli cannot
grow in the presence of 5 μM 5-fluorouracil (Figure 7). The growth rate is not affected by
the addition of 50 μM 5-fluorocytosine, but is measurably reduced in the presence of 500
μM 5-fluorocytosine (Figure 7A). Overexpression of the wild-type cytosine deaminase
from E. coli in these cells reduces the growth rate in the presence of 50 and 500 μM 5-
fluorocytosine (Figure 7B) and is reduced even further when the D314A mutant of CodA is
15
ACS Paragon Plus Environment

Biochemistry
Page 16 of 41
1
2
3
4
5
6
7
8
9
expressed (Figure 7C). Expression of Kpn00632 in these cells completely abolished
growth in the presence of 500 μM added 5-fluorocytosine and severely inhibited growth in
the presence of 50 μM added 5-fluorocytosine (Figure 7D).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
D
ISCUSSION
The methylation of the nucleobase cytosine is a common epigenetic modification of
DNA in both eukaryotes and prokaryotes.1, 6 However, certain bacterial enzymes
apparently actively exclude this metabolite from the active site. For example, wild-type
cytosine deaminase from E. coli (CodA) catalyzes the deamination of 5-methylcytosine with
a value of kcat/Km of ~22 M-1 s-1. This rate constant is more than three orders of magnitude
smaller than the value of kcat/Km for the deamination of cytosine (~105 M-1 s-1) by the same
enzyme. Since significant quantities of 5-methylcytosine are produced in bacterial cells
during the modification of DNA, a metabolic pathway must exist for the catabolism of this
compound. In our search to identify candidate enzymes that would be capable of
deaminating 5-methylcytosine to thymine, we assumed that these enzymes would be quite
similar in structure and sequence to the cytosine deaminase from E. coli. A similar
approach has previously lead to the successful identification of the first enzyme capable of
deaminating 8-oxoguanine to uric acid.26
A search for sequence homologues to CodA from E. coli identified approximately
1400 candidate sequences. These sequences cluster into three major groups and
numerous minor groups at an E-value threshold of 10-140 (Figure 2). The genes for three
proteins, in addition to CodA from E. coli, were cloned and expressed, and the proteins
16
ACS Paragon Plus Environment

Page 17 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
purified to homogeneity. Kpn00632 from Klebsiella pneumoniae efficiently catalyzes the
deamination of 5-methylcystosine to thymine with a value of kcat/Km that exceeds 105 M-1 s-
1. This rate constant is four orders of magnitude greater than the value of kcat/Km for the
deamination of 5-methylcytosine by CodA from E. coli. Similar results were obtained with
Rsp0341 from Rhodobacter sphaeroides.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The three-dimensional structure of Kpn00632 was determined in the presence of 5-
methylcytosine, 5-fluorocytosine, and the phosphonate mimic of the putative tetrahedral
intermediate bound in the active site of the enzyme. In the vicinity of the methyl- and
fluoro-substituents at C5 of the bound ligands, the closest two residues are Glu-273 and
Ser-309. In CodA from E. coli these residues correspond to Val-278 and Asp-314,
respectively. These two residue positions are highly conserved within each of the three
major subgroups identified in Figure 2. Sequence alignments indicate that these two
residue positions correspond to isoleucine and cysteine in Rsp0371, and with glutamate
and serine in NCgl0075.
Wild-type CodA from E. coli is essentially unable to catalyze the deamination of 5-
methylcytosine. In Kpn00632, the residue that follows Asp-308 is a serine. When Asp-314
in E. coli is mutated to alanine, this enzyme is now capable of deaminating 5-
methylcytosine, thus demonstrating that this residue is largely responsible for the
discrimination between 5-methylcytosine and cytosine in the active site. These changes in
the active site also enable these enzymes to deaminate 5-fluorocytosine to 5-fluorouracil, a
highly toxic metabolite. Similar results have previous been observed by Mahan et al. when
they demonstrated that the D314S mutation in CodA of E. coli enhanced the turnover ratio
of 5-fluorocytosine/cytosine by 4-fold.27
17
ACS Paragon Plus Environment

Biochemistry
Page 18 of 41
1
2
3
4
5
6
7
8
9
The addition of Kpn00632 enables the E. coli thymine auxotroph to grow in the
presence of 5-methylcytosine. The thymine auxotroph cannot produce thymidine since it
lacks thymidylate synthase and thus has no way to make thymine since wild-type CodA
cannot effectively catalyze the deamination of 5-methylcytosine to thymine. In the
presence of Kpn00632 these cells can catalyze the formation of thymine from 5-
methylcytosine and thymidylate can be made from the phosphoribosyltransferase reaction
(Figure 6). 5-Fluorocytosine is not normally toxic to E. coli since wild type CodA cannot
catalyze the formation of 5-flourouracil.27 5-Fluorouracil is a toxic metabolite that
irreversibly inactivates thymidyldate synthase. When Kpn00632 is expressed in E. coli, 5-
flourocytosine becomes toxic as exhibited by the substantial retardation of growth (Figure
7).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The discovery of enzymes capable of deaminating 5-methylcytosine and 5-
fluorocytosine reveals the difficulty of defining the substrate/sequence boundaries of
enzymes based on simple sequence similarity to proteins of known function. Essentially all
of the sequences depicted in the SSN of Figure 2 have been annotated as cytosine
deaminase. Given the inability of CodA from E. coli to deaminate 5-methylcytosine, these
enzymes would have been predicted to not catalyze the deamination of either 5-
methylcytosine or 5-fluorocytosine. However a careful examination of the residues that
reside in the active site reveals a significant perturbation of a conserved aspartate residue
to either a serine or cysteine residue.
Full-length sequence alignments indicate that the subgroups depicted in Figure 2
are highly specific for the amino acids that populate the active site. E. coli CodA is found in
subroup-a and most sequences in this subgroup possess an aspartate residue
18
ACS Paragon Plus Environment

Page 19 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
corresponding to Asp-314. Subgroup-b (including Kpn00632) conserves a serine residue
at this position and is expected to occupy the same role as Ser-309 in Kpn00632.
Subgroup-c and Rsp0341 strongly favor a cysteine residue at this position. Finally,
subgroup-d, previously characterized as creatinine deaminase, possesses a serine residue
at this position. While 5-methylcytosine is not known to be a metabolite produced in great
quantities for cell proliferation, it seems reasonable to assume there is an advantage for
having an enzyme that can catalyze the deamination of the free nucleobase. As
demonstrated by the in vivo experiment in Figure 6, it is possible to produce thymine by
this route. It remains mysterious why the cytosine deaminase from E. coli has evolved to
exclude 5-methylcytosine from the active site.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The original evidence that homologues of CodA from E. coli may have promiscuous
activity for the deamination of 5-methylcytosine is based on mutagenesis studies that
produced a more efficient 5-fluorocytosine deaminase, with the ultimate goal of
transfecting cancer cells with this enzyme.27 In suicide gene therapy, the gene for an
enzyme capable of deaminating the nontoxic prodrug 5-fluorocytosine is delivered to
cancer cells.42 The expressed deaminase subsequently converts 5-fluorocytosine to 5-
fluorouracil, which is ultimately transformed to 5-fluorouridine monophosphate, an
irreversible inhibitor of thymidylate synthase. DNA replication is ultimately blocked due
to the inhibition of deoxythymidine triphosphate synthesis.
The D314A/S/G mutants created by Mahan et al. each showed cytotoxicity in the
presence of 5-fluorocytosine in an experiment similar to that presented in Figure 7.27
However, Kpn0062 deaminates 5-fluorocytosine with a value of kcat/Km that is greater than
any of the D314 mutants of CodA reported previously.27 Kpn00632 also deaminates 5-
19
ACS Paragon Plus Environment

Biochemistry
Page 20 of 41
1
2
3
4
5
6
7
8
9
fluorocytosine more efficiently than it does cytosine. This enzyme may therefore provide a
novel starting point for the creation of even better enzymes for the deamination of 5-
fluorocytosine to 5-fluorouracil.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
20
ACS Paragon Plus Environment

Page 21 of 41
Biochemistry
1
2
3
4
5
REFERENCES
6
7
8
9
(1) Suzuki, M. M., and Bird, A. (2008) DNA methylation landscapes: provocative insights
from epigenomics. Nat. Rev. Genet. 9, 465-476.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(2) Bestor, T., Laudano, A., Mattaliano, R., and Ingram, V. (1988) Cloning and sequencing of
a cDNA encoding DNA methyltransferase of mouse cells: the carboxyl-terminal domain
of the mammalian enzymes is related to bacterial restriction methyltransferases. J. Mol.
Biol. 203, 971-983.
(3) Pavlopoulou, A., and Kossida, S. (2007) Plant cytosine-5 DNA methyltransferases:
structure, function, and molecular evolution, Genomics 90, 530-541.
(4) Jabbari, K., and Bernardi, G. (2004) Cytosine methylation and CpG, TpG (CpA) and TpA
frequencies. Gene 333, 143-149.
(5) Gomez-Eichelmann, M. C., Levy-Mustri, A., and Ramirez-Santos, J. (1991) Presence of 5-
methylcytosine in CC(A/T)GG sequences (Dcm methylation) in DNAs from different
bacteria. J. Bacteriol. 173, 7692-7694.
(6) Kahramanoglou, C., Prieto, A. I., Khedkar, S., Haase, B., Gupta, A., Benes, V., Fraser, G. M.,
Luscombe, N. M., and Seshasayee, A. S. N. (2012) Genomics of DNA cytosine
methylation in Escherichia coli reveals its role in stationary phase transcription. Nat
Commun 3, 886.
(7) He, Y.-F., Li, B.-Z., Li, Z., Liu, P., Wang, Y., Tang, Q., Ding, J., Jia, Y., Chen, Z., Li, L., Sun, Y., Li,
X., Dai, Q., Song, C.-X., Zhang, K., He, C., and Xu, G.-L. (2011) Tet-mediated formation of
5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303-
1307.
21
ACS Paragon Plus Environment

Biochemistry
Page 22 of 41
1
2
3
4
5
6
(8) Wu, S. C., and Zhang, Y. (2010) Active DNA demethylation: many roads lead to Rome.
Nat. Rev. Mol. Cell Biol. 11, 607-620.
7
8
9
(9) Tahiliani, M., Koh, K. P., Shen, Y., Pastor, W. A., Bandukwala, H., Brudno, Y., Agarwal, S.,
Iyer, L. M., Liu, D. R., Aravind, L., and Rao, A. (2009) Conversion of 5-methylcytosine to
5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930-
935.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(10) Ito, S., Shen, L., Dai, Q., Wu, S. C., Collins, L. B., Swenberg, J. A., He, C., and Zhang, Y.
(2011) TET proteins can convert 5-methylcytosine to 5-formylcytosine and 5-
carboxylcytosine. Science 333, 1300-1303.
(11) Katsuragi, T., Sakai, T., Matsumoto, K. y., and Tonomura, K. (1986) Cytosine deaminase
from Escherichia coli-production, purification, and some characteristics. Agric. Biol.
Chem. 50, 1721-1730.
(12) Kream, J., and Chargaff, E. (1952) On the cytosine deaminase of yeast1, J. Am. Chem.
Soc. 74, 5157-5160.
(13) Cohen, R. M., and Wolfenden, R. (1971) Cytidine Deaminase from Escherichia coli :
purification, properties, and inhibition by the potential transition state analog 3,4,5,6-
tetrahydrouridine. J. Biol. Chem. 246, 7561-7565.
(14) Weiner, K. X., Weiner, R. S., Maley, F., and Maley, G. F. (1993) Primary structure of
human deoxycytidylate deaminase and overexpression of its functional protein in
Escherichia coli. J. Biol. Chem. 268, 12983-12989.
(15) Hou, H.-F., Liang, Y.-H., Li, L.-F., Su, X.-D., and Dong, Y.-H. (2008) Crystal structures of
streptococcus mutans 2′-deoxycytidylate deaminase and its complex with substrate
analog and allosteric regulator dCTP·Mg2+. J. Mol. Biol. 377, 220-231.
22
ACS Paragon Plus Environment

Page 23 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
(16) Johansson, E., Fanø, M., Bynck, J. H., Neuhard, J., Larsen, S., Sigurskjold, B. W.,
Christensen, U., and Willemoës, M. (2005) Structures of dCTP deaminase from
Escherichia coli with bound substrate and product: Reaction mechanism and
determinants of mono-and bifunctionality for a family of enzymes. J. Biol. Chem. 280,
3051-3059.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(17) Wang, L., and Weiss, B. (1992) dcd (dCTP deaminase) gene of Escherichia coli:
mapping, cloning, sequencing, and identification as a locus of suppressors of lethal dut
(dUTPase) mutations. J. Bacteriol. 174, 5647-5653.
(18) Vita, A., Amici, A., Cacciamani, T., Lanciotti, M., and Magni, G. (1985) Cytidine
deaminase from Escherichia coli B. Purification and enzymic and molecular properties.
Biochemistry 24, 6020-6024.
(19) Xie, K., Sowden, M. P., Dance, G. S. C., Torelli, A. T., Smith, H. C., and Wedekind, J. E.
(2004) The structure of a yeast RNA-editing deaminase provides insight into the fold
and function of activation-induced deaminase and APOBEC-1. Proc. Natl. Acad. Sci. U. S.
A. 101, 8114-8119.
(20) Ireton, G. C., Black, M. E., and Stoddard, B. L. (2003) The 1.14 Å crystal structure of
yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications
for genetic chemotherapy. Structure 11, 961-972.
(21) Hall, R. S., Fedorov, A. A., Xu, C., Fedorov, E. V., Almo, S. C., and Raushel, F. M. (2011)
Three-dimensional structure and catalytic mechanism of cytosine deaminase.
Biochemistry 50, 5077-5085.
(22) Holm, L., and Sander, C. (1997) An evolutionary treasure: unification of a broad set of
amidohydrolases related to urease. Proteins Struct. Funct. Bioinformat. 28, 72-82.
23
ACS Paragon Plus Environment

Biochemistry
Page 24 of 41
1
2
3
4
5
6
(23) Seibert, C. M., and Raushel, F. M. (2005) Structural and catalytic diversity within the
amidohydrolase superfamily. Biochemistry 44, 6383-6391.
7
8
9
(24) Gupta, N. K., and Glantz, M. D. (1985) Isolation and characterization of human liver
guanine deaminase. Arch. Biochem. Biophys. 236, 266-276.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(25) Hermann, J. C., Marti-Arbona, R., Fedorov, A. A., Fedorov, E., Almo, S. C., Shoichet, B. K.,
and Raushel, F. M. (2007) Structure-based activity prediction for an enzyme of
unknown function. Nature 448, 775-779.
(26) Hall, R. S., Fedorov, A. A., Marti-Arbona, R., Fedorov, E. V., Kolb, P., Sauder, J. M., Burley,
S. K., Shoichet, B. K., Almo, S. C., and Raushel, F. M. (2010) The hunt for 8-oxoguanine
deaminase. J. Am. Chem. Soc. 132, 1762-1763.
(27) Mahan, S. D., Ireton, G. C., Knoeber, C., Stoddard, B. L., and Black, M. E. (2004) Random
mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene
therapy. Protein Eng. Des. Sel. 17, 625-633.
(28) Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K. A., Tomita,
M., Wanner, B. L., and Mori, H. (2006) Construction of Escherichia coli K-12 in-frame,
single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 0008.
(29) Cronan, J. E. (2006) A family of arabinose-inducible Escherichia coli expression vectors
having pBR322 copy control. Plasmid 55, 152-157.
(30) Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Site-directed
mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59.
(31) Otwinowski, Z., and Minor, W. (1997) Processing of X-ray diffraction data collected in
oscillation mode. pp 307-326, Elsevier.
24
ACS Paragon Plus Environment

Page 25 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
(32) Long, F., Vagin, A. A., Young, P., and Murshudov, G. N. (2008) BALBES: a molecular-
replacement pipeline. Acta Crystallogr. Sect. D-Biol. Crystallogr. 64, 125-132.
(33) Emsley, P., and Cowtan, K. (2004) COOT: model-building tools for molecular graphics.
Acta Crystallogr. Sect. D-Biol. Crystallogr. D60, 2126-2132.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(34) Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J.,
Hung, L.-W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner,
R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H.
(2010) PHENIX: a comprehensive Python-based system for macromolecular structure
solution. Acta Crystallogr. Sect. D-Biol. Crystallogr. 66, 213-221.
(35) Lamzin, V. S., and Wilson, K. S. (1993) Automated refinement of protein models. Acta
Crystallogr. Sect. D-Biol. Crystallogr. 49, 129-147.
(36) Hooft, R. W., Vriend, G., Sander, C., and Abola, E. E. (1996) Errors in protein structures.
Nature 381, 272.
(37) Chen, V. B., Arendall, W. B., 3rd, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J.,
Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all-atom
structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D-Biol.
Crystallogr.66, 12-21.
(38) Collaborative Computation Project, Number 4 (1994) The CCP4 suite: programs for
protein crystallography, Acta Crystallogr. Sect. D-Biol. Crystallogr. 50, 760-763.
(39) PyMOL (http://www.pymol.org)
(40) Atkinson, H. J., Morris, J. H., Ferrin, T. E., and Babbitt, P. C. (2009) Using sequence
similarity networks for visualization of relationships across diverse protein
superfamilies. PLoS ONE 4, e4345.
25
ACS Paragon Plus Environment

Biochemistry
Page 26 of 41
1
2
3
4
5
6
7
8
9
(41) Cline, M. S., Smoot, M., Cerami, E., Kuchinsky, A., Landys, N., Workman, C., Christmas, R.,
Avila-Campilo, I., Creech, M., Gross, B., Hanspers, K., Isserlin, R., Kelley, R., Killcoyne, S.,
Lotia, S., Maere, S., Morris, J., Ono, K., Pavlovic, V., Pico, A. R., Vailaya, A., Wang, P.-L.,
Adler, A., Conklin, B. R., Hood, L., Kuiper, M., Sander, C., Schmulevich, I., Schwikowski, B.,
Warner, G. J., Ideker, T., and Bader, G. D. (2007) Integration of biological networks and
gene expression data using Cytoscape. Nat. Protoc. 2, 2366-2382.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(42) Freytag, S. O., Khil, M., Stricker, H., Peabody, J., Menon, M., DePeralta-Venturina, M.,
Nafziger, D., Pegg, J., Paielli, D., Brown, S., Barton, K., Lu, M., Aguilar-Cordova, A., and
Kim J. H. (2002) Phase I study of replication-competent adenovirus-mediated double
suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer
Research 62, 4968-4976.
26
ACS Paragon Plus Environment

Page 27 of 41
Biochemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1.
27
ACS Paragon Plus Environment

Biochemistry
Page 28 of 41
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2.
28
ACS Paragon Plus Environment

Page 29 of 41
Biochemistry
1
2
3
4
5
Table 1. Data collection and refinement statistics for Kpn00632
5-methylcytosine
5-fluorocytosine
phosphonocytosine
Data collection
Space Group
Molecules in Asym. 6
6
7
8
9
P21
P21 2121
6
P212121
6
unit
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cell dimensions
a (Å)
b (Å)
c (Å)
117.477
101.957
162.430
184.051
102.169
147.807
185.327
137.665
112.096
118.03
1.80
βº
Resolution (Å)
Unique Reflections 265794
Rmerge 0.089
2.0
1.90
198447
0.091
96.07
214497
0.114
97.60
Completeness (%) 96.8
Refinement
Resolution (Å)
Rcryst
Rfree
No. atoms
Protein
Waters
Ligand RMS
deviations
25.0-1.80
0.159
0.188
25.0-2.0
0.163
0.202
25.0-1.90
0.198
0.243
19377
1743
234
19416
1160
254
19334
1523
195
Bond lengths (Å) 0.007
Bond angles (°)
PDB entry
0.007
1.05
4R88
0.007
1.05
4R7W
1.06
4R85
29
ACS Paragon Plus Environment