Supporting
Information
to:
In
vitro
reconstitution
of
mycobacterial
ergothioneine
biosynthesis
Abteilung
Physikalische
Biochemie,
Max
Planck
Institut
für
molekulare
Physiologie,
Otto‐Hahn
Strasse
11,
44227
Dortmund,
Germany
*To
whom
correspondence
should
be
addressed.phone
+49
231
1332312.
Fax
+49
2311332399.
E‐mail:
florian.seebeck@mpi‐dortmund.mpg.de
Material
and
Methods
Materials.
All
standard
reagents
were
purchased
from
Aldrich/Sigma
if
not
otherwise
stated.
γ‐glutamyl
cysteine
and
α‐N,N‐dimethyl
histidine
were
purchased
from
Bachem.
2
was
prepared
from
α‐N,N‐dimethyl
histidine
and
methyl
iodide
according
to
previously
described
methods1.
Recombinant
S‐adenosylhomocysteine
nucleosidase
was
produced
in
E.
coli
and
purified
by
Ni(II)‐NTA
affinity
chromatography.
Gene
identification:
A
list
of
all
annotated
methyltransferases
from
M.
avium
was
compiled
relying
on
automatic
annotation
(http://www.ncbi.nlm.nih.gov)
(Table
S1).
Homologous
methyltransferases
from
N.
crassa
for
each
of
the
mycobacterial
sequences
was
searched
using
blastp.
A
pair
of
fungal/mycobacterial
proteins
was
considered
homologous
if
they
shared
mutually
the
highest
similarity
among
all
mycobacterial
and
fungal
methyltransferases.
The
same
criterion
was
applied
to
homologous
methyltransferases
in
E.
coli
and
B.
subtilis.
Table
S1.
Locus_tags
of
all
annotated
putative
methyltransferases
in
M.
avium.
Green:
Methyltransferases
found
in
M.
avium
and
N.
crassa.
Red:
Methyltransferases
found
in
M.
avium
and
in
E.
coli
and/or
in
B.
subtilis.
Yellow:
EgtD
from
M.
avium.
S1
MAV_0854
MAV_1536
MAV_2732
MAV_3501
MAV_3903
MAV_4441
MAV_4647
MAV_0130
MAV_0982
MAV_1715
MAV_2756
MAV_3570
MAV_4065
MAV_4442
MAV_4679
MAV_0219
MAV_1058
MAV_2110
MAV_2762
MAV_3580
MAV_4152
MAV_4444
MAV_4680
MAV_0301
MAV_1181
MAV_2111
MAV_2803
MAV_3642
MAV_4226
MAV_4516
MAV_4764
MAV_0313
MAV_1133
MAV_2216
MAV_2957
MAV_3703
MAV_4236
MAV_4517
MAV_4788
MAV_0402
MAV_1255
MAV_2280
MAV_2988
MAV_3760
MAV_4317
MAV_4539
MAV_4859
MAV_0404
MAV_1262
MAV_2328
MAV_2991
MAV_3811
MAV_4332
MAV_4557
MAV_4873
MAV_0574
MAV_1279
MAV_2424
MAV_3069
MAV_3871
MAV_4333
MAV_4558
MAV_4945
MAV_0750
MAV_1360
MAV_2425
MAV_3261
MAV_3889
MAV_4426
MAV_4573
MAV_0778
MAV_1364
MAV_2576
MAV_3373
MAV_3902
MAV_4435
MAV_4584
Cloning.
Genomic
DNA
from
M.
smegmatis
(DSM
#
43465)
was
prepared
using
standard
methods.
The
genes
coding
for
EgtB
(MSMEG_6249),
EgtC
(MSMEG_6248)
and
EgtD
(MSMEG_6247)
were
amplified
by
PCR
using
synthetic
oligonucleotides
(Table
S2).
The
gel
purified
fragments
were
digested
with
the
appropriate
restriction
enzymes
(Table
S2)
and
ligated
into
Nco1‐Xho1
digested
pET28a
expression
vector.
Table
S2.
Cloning
primers
Name
Sequence
Restr.
Enz.
EgtBs
5’- ATATCATATGATCGCACGCGAGACACT -3’
Nde1
EgtBa
5’- ATATCTCGAGTTAGACGTCCCAGGCCAG -3’
Xho1
EgtCs
5’- ATATACATGTGCCGGCATGTGGCGT-3’
Pci1
EgtCa
5’- ATATCTCGAGTCAATGATGATGATGATGATGGCTGCTCAGGGGTGTCACGACGA-3’
Xho1
EgtDs
5’- ATATTCATGACGCTCTCACTGGCCAA-3’
BspH1
EgtDa
5’- ATATCTCGAGTCAATGATGATGATGATGATGGCTGCTCCGCACCGCCAGCGA-3’
Xho1
S2
Protein
production.
EgtB
,
EgtC
and
EgtD
were
produced
in
E.
coli
BL21
cells
grown
in
LB
medium
(50
mg/l
Kanamycin)
and
induced
with
0.2
mM
IPTG
at
25°C
for
at
least
12
h.
Cells
were
pelleted
and
resuspended
in
50
mM
phosphate
buffer
pH
8.0,
300
mM
NaCl,
0.1%
Tween
and
1
mg/ml
lysozyme.
After
30
min
incubation
at
4°C
the
cells
were
lysed
by
sonication
and
the
cleared
lysate
was
mixed
with
Ni(II)‐ NTA
agarose
(Quiagen)
at
4°C
for
30
min.
The
agarose
beads
were
washed
with
phosphate
buffer
containing
10
mM
imidazole.
The
proteins
were
eluted
in
a
250
mM
imidazole,
300
mM
NaCl,
0.1
%
Tween
solution.
The
purified
eluted
proteins
were
dialyzed
into
20
mM
Tris‐HCl
buffer,
100
mM
NaCl,
0.1%
Tween,
20
%
glycerol
and
stored
at
‐80°C.
To
approximate
the
concentrations
of
the
prepared
proteins
calculated
molar
absorption
coefficients
were
used:
ε280nm,
ErgB
=
1.1
x
105
M‐1cm‐1
ε280nm,
ErgC
=
4.1
x
104
M‐1cm‐1
ε280nm,
ErgD
=
3.6
x
104
M‐1cm‐1
Figure
S1.
18%
SDS‐PAGE
of
EgtB,C,D.
1)
EgtD
(MSMEG_6247,
35.9
kDa)
2)
EgtC
(MSMEG_6248,
25.0
kDa)
3)
EgtB
(MSMEG_6249,
50.0
kDa)
4)
SeeBlue®
Plus2
Pre‐Stained
Standard.
S3
Substrate
specificity
of
EgtD.
A
solution
of
2
mM
histidine
or
either
of
the
proteinogenic
amino
acids
and
4
mM
SAM
at
pH
7.6
was
supplemented
with
1
mM
Mg(OAc)2,
5
mM
NaCl,
20
µg
ErgD
and
5
μg
recombinant
S‐adenosyl‐L‐ homocysteine
nucleosidase.
This
reaction
and
a
control
mixture
lacking
SAM
were
incubated
at
room
temperature
(RT)
for
2
h
and
then
subjected
to
ESI‐MS
analysis.
The
SAM
containing
reaction
produces
a
signal
at
the
mass
consistent
with
2
(Fig.
S2,
left)
but
non
of
the
other
amino
acids
led
to
methylation
products.
The
substrate
specificity
of
ErgD
was
also
tested
using
an
enzyme‐coupled
continuous
spectrophotometric
assay2.
100
μl
reactions
containing
100
mM
Tris‐ HCl
pH
7.6,
100
mM
NaCl,
0.5
mM
MnSO4,
100
uM
SAM,
20
μg
ErgD,
5
μg
recombinant
S‐adenosyl‐L‐homocysteine
nucleosidase,
and
10
u
adenine
deaminase
were
initiated
by
addition
of
10
mM
amino
acid.
In
this
screen,
tyrosine
and
tryptophan
were
left
out
due
to
their
inherent
strong
absorption
at
265
nm.
The
reactions
were
incubated
at
RT
and
monitored
at
265
nm
for
1
min
(Fig
S2,
right).
Figure
S2.
Left:
EgtD
transfers
three
methyl
groups
to
histidine.
Signals
for
the
intermediates
mono
and
dimethyl
histidine
are
not
detectable.
Right:
Continuous
spectrophotometric
assay
for
ErgD
substrate
preference.
Red:
Histidine;
blue:
α‐N,N‐dimethyl
histidine;
black:
Ala,
Cys,
Asp,
Glu,
Phe,
Ile,
Lys,
Leu,
Met,
Asn,
Pro,
Gln,
Arg,
Ser,
Thr,
Val.
S4
Figure
S3.
Sequence
Logo
of
a
putative
EgtB
2‐His‐1‐carboxylate
facial
triad
necessary
for
coordination
of
catalytic
iron
in
non‐heme
iron
(II)
enzymes7.
The
logo
was
generated
from
240
putative
EgtB
sequences
using
a
web‐based
logo
generator8.
Determination
of
the
extinction
coefficient
of
3.
Reactions
containing
100
mM
Tris‐HCl
pH
7.6,
100
mM
NaCl,
10
mM
2,
0.5
mM
tris(2‐carboxyethyl)
phosphine
(TCEP),
0,2
mM
FeSO4,
20
μg
ErgB
and
γ‐glutamyl
cysteine
in
concentrations
ranging
from
0.5
to
0.01
mM.
The
reaction
were
incubated
until
their
absorption
at
250
nm
reached
a
maximum.
Three
independent
sets
of
final
absorption
values
were
used
to
calculate
a
molar
extinction
coefficient
(ε250nm,
pH
7.6)
of
10600
±
230
M‐1
cm‐1
(Fig.
S4).
Figure
S4.
Determination
of
the
molar
extinction
coefficient
of
3.
S5
NMR
characterization
of
3.
A
2
ml
reaction
was
assembled
containing
2
mg
EgtB,
20
mM
Tris‐HCl
pH
7.6,
0.2
mM
FeSO4,
1
mM
TCEP,
2
mM
2
and
2
mM
γ‐glutamyl
cysteine.
After
12
h
incubation
at
RT
intermediate
3
was
isolated
from
the
reaction
mixture
by
HPLC
(C18
reversed
phase
semi‐prep
column,
Bischoff,
250
x
8.0
mm,
mobile
phase:
2
ml/min
flow
of
H20
containing
2%
acetonitrile,
0.03
%
trifluoroacetic
acid
(TFA)).
The
fractions
containing
3
were
concentrated
and
submitted
to
cation‐exchange
chromatography
(Varian,
PL‐SCX,
150
x
4.6
mm,
mobile
phase:
a
gradient
from
50
mM
to
1
M
TFA/NH4,
pH
2).
Purified
3
(250
ug)
was
dissolved
in
500
ul
D2O
and
characterized
by
1H,
COSY
and
HSQC
NMR
experiments.
NMR
spectroscopic
data
for
3
(600
MHz,
D2O,
20°C,
pD
=
5.0)
(Fig.
S5):
1H
NMR
δ
2.16
(m,
2
H,
3”),
δ
2.48
(t,
J
=
7.6
Hz,
2
H,
4”),
δ
3.15
(s,
9
H,
N‐methyl),
δ
3.28
–
3.38
(m,
2
H,
3pro‐R
and
3pro‐S),
δ
3.65
(m,
2
H,
3’pro‐R
and
3’pro‐S),
δ
3.92
(t,
J
=
6.33
Hz,
2
H,
2”),
δ
3.97
(m,
2
H,
2
and
2’), δ
7.29
(s,
1
H,
4*);
13C
NMR
chemical
shifts
are
assigned
from
1H‐13C
HSQC
correlations
δ
27
(3),
28
(3”),
33
(4”),
54
(N‐methyl),
55
(2”),
56
(2’
and
2),
79
(2),
124
(4*);
Figure
S5.
Intermediate
3.
S6
Iron
dependence
of
EgtB.
To
assess
the
iron
dependency
of
EgtB,
the
protein
was
pretreated
with
chelex
100
for
2
h
at
4°C.
Reactions
containing
0.2
mM
of
either
FeSO4
x
7
H2O,
ZnCl2,
CuNO3,
MnSO4
or
2
mM
EDTA
and
40
μg
chelex
treated
EgtB,
20
mM
Tris‐HCl
pH
7.6,
0.5
mM
TCEP,
5
mM
2
and
5
mM
γ‐glutamyl
cysteine
were
incubated
at
RT
for
2
h
and
also
subjected
to
HPLC
analysis
(C18
reversed
phase
column,
Bischoff,
250
x
4.6
mm,
and
1
ml/min
flow
of
H20
containing
2%
acetonitrile,
0.1
%
TFA
as
mobile
phase)
(Fig.
S6).
Figure
S6
Metal
depleted
EgtB
was
supplemented
with
a)
0.2
mM
FeSO4;
b)
ZnCl2;
c)
MnSO4;
d)
CuNO3;
e)
no
metal
and
f)
2
mM
EDTA.
The
residual
activity
observed
in
the
reactions
be
is
attributable
to
incomplete
removal
of
iron
(II)
in
the
ErgB
preparation.
Addition
of
EDTA
to
the
reaction
eliminates
any
trace
activity,
and
metal
ions
other
than
iron
(II)
reduce
EgtB
activity
as
compared
to
the
“metal
free”
reaction
e.
S7
Sulfur
acceptor
specificity
of
EgtB.
200
μl
reactions
were
assembled
containing
40
μg
EgtB,
20
mM
Tris‐HCl
pH
7.6,
0.2
mM
FeSO4,
0.5
mM
TCEP,
and
5
mM
of
either
2
(a),
α‐N,N‐dimethyl
histidine
(b)
or
histidine
(c).
After
2
h
incubation
at
RT
the
reactions
a‐d
were
analyzed
by
ESI‐MS
and
by
HPLC
(C18
reversed
phase
column,
Bischoff,
250
x
4.6
mm,
and
1
ml/min
flow
of
H20
containing
2%
acetonitrile,
0.1
%
TFA
as
mobile
phase)
(Fig.
S7).
Figure
S7.
In
comparison
with
2
and
α‐N,N‐dimethyl
histidine,
histidine
is
poor
EgtB
substrate.
Left:
HPLC
analysis
of
reactions
containing
a)
2,
b)
α‐N,N‐dimethyl
histidine,
c)
histidine.
Right:
ESI‐MS
identification
of
the
corresponding
EgtB
products.
S8
Sulfur
donor
specificity
of
EgtB.
200
μl
reactions
were
assembled
containing
40
μg
EgtB,
20
mM
Tris‐HCl
pH
7.6,
0.2
mM
FeSO4,
0.5
mM
TCEP,
5
mM
2
and
5
mM
of
either
(a)
γ‐glutamyl
cysteine,
(b)
N‐acetyl
cysteine,
(c)
cysteine
or
(d)
glutathione.
After
2
h
incubation
at
RT
the
reactions
a‐d
were
analyzed
by
HPLC
(C18
reversed
phase
column,
Bischoff,
250
x
4.6
mm,
and
1
ml/min
flow
of
H20
containing
2%
acetonitrile,
0.1
%
TFA
as
mobile
phase)(Fig.
S8).
Figure
S8:
Sulfur
donor
specificity
of
EgtB.
New
product
is
only
observed
in
the
presence
of
γ‐ glutamyl
cysteine.
a)
γ‐glutamyl
cysteine,
b)
N‐acetyl
cysteine,
c)
cysteine,
d)
glutathione.
Inset:
ESI‐ MS
spectrum
of
the
isolated
HPLC‐peak
at
4
min.
The
detected
mass
(462.1
Da)
corresponds
to
the
calculated
mass
(462.2
Da)
of
S9
Production
of
recombinant
βlyase
from
E.
tasmaniensis.
To
circumvent
the
solubility
problem
of
mycobacterial
EgtE,
a
number
of
unrelated
PLP‐binding
proteins
were
tested
for
β‐elimination
activity
with
4.
A
putative
β‐lyase
from
E.
tasmaniensis
appeared
to
catalyze
this
step
(see
below).
This
protein
shares
31
%
identity
with
cystalysin
from
Treponema
denticola
and
34
%
identity
with
MalY
from
E.
coli
both
of
which
are
well
characterized
β‐lyases4,5.
The
gene
for
this
enzyme
(ETA_14770)
was
amplified
from
E.
tasmaniensis
genomic
DNA
(DSM
#
17949)
using
the
following
primers
Erwin2s,ATATCATATGCTTCAATTAACGGAGAGC
Erwin2a,ATATCTCGAGTCAGTAACCGCTCGTCAG
The
gene
was
cloned
into
a
pET28a
vector.
Expression
and
purification
was
performed
as
described
above
(ε280nm,
βlyase
=
5.4
x
104
M‐1cm‐1).
In
vitro
reconstituted
biosynthesis
of
1.
Three
200
μl
reactions
(a
–
c)
containing
20
mM
Tris‐HCl
pH
7.6,
20
mM
NaCl,
0.2
mM
FeSO4,
0.5
mM
TCEP,
5
mM
2
and
5
mM
γ‐glutamyl
cysteine
were
initiated
by
the
addition
of
(a)
20
μg
EgtB,
(b)
20
μg
EgtB
and
EgtC
or
(c)
20
μg
EgtB
and
EgtC
and
β‐lyase
from
E.
tasmaniensis.
The
three
reactions
were
incubated
for
4
h
at
RT
and
analyzed
by
HPLC
(C18
reversed
phase
column,
Bischoff,
250
x
4.6
mm,
and
1
ml/min
flow
of
H20
containing
2%
acetonitrile,
0.03
%
TFA
as
mobile
phase).
The
peaks
with
absorption
at
265
nm
were
collected,
concentrated
and
submitted
to
standard
ESI‐MS
as
well
as
high
resolution
ESI‐FTMS.
Despite
the
apparent
elimination
activity
towards
4
by
this
β‐lyase,
E.
tasmaniensis
is
an
unlikely
producer
of
1
since
no
homolog
of
EgtD
could
be
found
in
its
genome.
Possibly
this
enzyme
has
evolved
to
turn
over
substrates
similar
to
4.
Furthermore,
the
conversion
from
4
to
1
is
a
rather
facile
reaction
for
a
PLP
binding
protein6.
A
solution
containing
4
supplemented
with
100
uM
PLP
in
place
of
the
β‐lyase
produces
significant
amounts
of
1
after
prolonged
(10
h)
incubation
(Fig.
S9).
S10
Figure
S9.
PLP
catalyzed
elimination
of
4
to
produce
1.
Identification
of
EgtB
and
EgtD
homologs.
The
M.
smegmatis
EgtD
sequence
was
blasted
against
the
genome
database
at
http://www.ncbi.nlm.nih.gov.
Genomes
with
a
significant
EgtD
homolog
(0.01
≥
e
value)
were
searched
for
EgtB
homologs.
Conservation
of
the
putative
iron
binding
motif
(His‐X3‐His‐X‐Glu)
was
used
as
a
criterion
to
distinguish
EgtB
homologs
from
unrelated
FGE‐like
proteins
.
Table
S3
presents
a
list
of
predicted
ergothioneine
producers
along
with
the
locus
tags
of
the
putative
biosynthetic
proteins.
The
majority
of
the
EgtB/EgtD
pairs
are
either
encoded
as
fusion
proteins
(predominantly
in
eukaryota)
or
in
close
genomic
neighborhood.
Eukaryota Fungi/Ascomycota:
Pezizomycotina Fungi/Ascomycota:
Schizosaccharomycetes Schizosaccharomyces japonicus yFS275 Schizosaccharomyces pombe Fungi/Basidiomycota Bacteria Actinobacteria Aeromicrobium marinum Catenulispora acidiphila Actinosynnema mirum Frankia sp. CcI3 Geodermatophilus obscurus Frankia sp. EuI1c Frankia alni ACN14a Frankia sp. EAN1pec Conexibacter woesei Gordonia bronchialis Micromonospora aurantiaca Micromonospora sp. Kribbella flavida Mycobacteria Nakamurella multipartita Rubrobacter xylanophilus Salinispora tropica CNB-440 Salinispora arenicola CNS-205 Saccharomonospora viridis Nocardioides sp. JS614
S11
Most species
Most species
SJAG_00832 SPBC1604.01 Most species
SJAG_00832 SPBC1604.01 Most species
HMPREF0063_1966 Caci_8031 Amir_2125 Francci3_4514 Gobs_0202 FraEuI1cDRAFT_3722 FRAAL6843 Franean1_7303 Cwoe_4694 Gbro_0612 MicauDRAFT_0340 MCAG_00315 Kfla_5160 All, except M. leprea Namu_1079 Rxyl_0685 Strop_2116 Sare_2259 Svir_22440 Noca_1639
HMPREF0063_1965 Caci_8029 Amir_2123 Francci3_4512 Gobs_0204 FraEuI1cDRAFT_6959 FRAAL6841 Franean1_7301 Cwoe_4693 Gbro_0611 MicauDRAFT_0342 MCAG_00317 Kfla_5162 All, except M. Namu_1081 leprea Rxyl_0687 Strop_2114 Sare_2257 Svir_22460 Noca_1640
Rhodococcus jostii RHA1 Rhodococcus erythropolis PR4 Rhodococcus opacus B4 Stackebrandtia nassauensis Nocardiopsis dassonvillei subsp Saccharopolyspora erythraea NRRL 2338 Nocardia farcinica IFM 10152
Streptomyces Streptosporangium roseum Thermomonospora curvata Cyanobacteria Bacteriodetes Polaribacter sp. MED152 Pedobacter sp. BAL39 Leeuwenhoekiella blandensis Spirosoma linguale Flavobacterium johnsoniae UW101 Chitinophaga pinensis unidentified eubacterium SCB49 Psychroflexus torquis Croceibacter atlanticus HTCC2559 Chryseobacterium gleum Robiginitalea biformata HTCC2501 Gramella forsetii KT0803 Dokdonia donghaensis MED134 Dyadobacter fermentans Microscilla marina Flavobacteria bacterium BBFL7 Firmicutes Exiguobacterium sibiricum 255-15 Acidobacteria Acidobacterium capsulatum Solibacter usitatus Ellin6076 acidobacteria bacterium Ellin345 Planctomycetes Gemmata obscuriglobus UQM 2246 Blastopirellula marina Rhodopirellula baltica SH 1 Chloroflexi Herpetosiphon aurantiacus Verrucomicrobia bacterium Ellin514 α-Proteobacteria: others Xanthobacter autotrophicus Py2 Methylobacterium nodulans ORS 2060 Phenylobacterium zucineum HLK1 Nitrobacter winogradskyi Nb-255 Rhodobacter sphaeroides Methylobacterium sp. 4-46 Caulobacter sp. K31 Bradyrhizobium sp. ORS278 Bradyrhizobium sp. BTAi1 Rhodobacter sphaeroides Rhodobacter sphaeroides 2.4.1 Rhodopseudomonas palustris TIE-1 β-Proteobacteria: Bortella Bordetella petrii Bordetella pertussis Tohama I Bordetella parapertussis 12822 β-Proteobacteria:
Burkholderiaceae Burkholderia ambifaria Burkholderia cenocepacia Burkholderia glumae BGR1 Burkholderia graminis C4D1M Burkholderia multivorans Burkholderia oklahomensis Burkholderia phymatum STM815 Burkholderia phytofirmans PsJN Burkholderia pseudomallei Burkholderia sp. 383 Burkholderia thailandensis Burkholderia ubonensis Bu Burkholderia vietnamiensis G4 Burkholderia xenovorans LB400 Cupriavidus taiwanensis Polynucleobacter necessarius
S12
RHA1_ro05703 RER_43170 ROP_57720 Snas_5171 NdasDRAFT_3411 SACE_3823 nfa48840 Most species Sros_0329 Tcur_3638 Most species
RHA1_ro05701 RER_43190 ROP_57700 Snas_5169 NdasDRAFT_2865 SACE_3825 nfa48860 Most species Sros_0331 Tcur_3640 Most species
MED152_03665 PBAL39_23182 MED217_01885 Slin_6101 Fjoh_3834 Cpin_4447 SCB49_08198 P700755_15396 CA2559_13323 HMPREF0204_2929 RB2501_03225 GFO_0284 MED134_00085 Dfer_1252 M23134_00338 BBFL7_01215
MED152_03660 PBAL39_05986 MED217_01890 Slin_6100 Fjoh_3833 Cpin_4446 SCB49_08203 P700755_15401 CA2559_13318 HMPREF0204_2928 RB2501_03220 GFO_0285 MED134_00090 Dfer_1251 M23134_00340 BBFL7_01214
Exig_1679
Exig_1680
ACP_1953 Acid_3752 Acid345_2835
ACP_1952 Acid_3753 Acid345_2836
GobsU_25869 DSM3645_29641 RB4230
GobsU_25874 DSM3645_29646 RB4229
Haur_0996
Haur_0997
Cflav_PD2520
Cflav_PD2519
Xaut_4562 Mnod_0355 PHZ_c1906 Nwi_1310 Rsph17025_3927 M446_2254 Caul_1581 BRADO2791 BBta_5395 Rsph17029_3803 RSP_3076 Rpal_1794
Xaut_4563 Mnod_0356 PHZ_c1906 Nwi_1311 Rsph17025_3926 M446_2253 Caul_1581 BRADO2792 BBta_5394 Rsph17029_3802 RSP_3075 Rpal_1795
Bpet2715 BP3515 BPP2522
Bpet2716 BP3514 BPP2520
Bamb_0015 BCAL3488 bglu_2g17730 BgramDRAFT_0684 BURMUCGD1_3502 BoklE_22859 Bphy_4432 Bphyt_4271 Bpse7_27702 Bcep18194_A3207 BTH_II1713 BuboB_06861 Bcep1808_0032 Bxe_B0605 RALTA_A1502 Pnuc_1661
Bamb_0016 BCAL3489 bglu_2g17720 BgramDRAFT_0873 BURMUCGD1_3503 BoklE_22864 Bphy_4124 Bphyt_7042 Bpse7_27707 Bcep18194_A3208 BTH_II1712 BuboB_06856 Bcep1808_0033 Bxe_B0024 RALTA_A2191 Pnuc_1660
Ralstonia eutropha JMP134 Ralstonia metallidurans CH34 Ralstonia pickettii β-Proteobacteria:
others Nitrosospira multiformis Variovorax paradoxus S110 Janthinobacterium sp. Marseille Rhodoferax ferrireducens T118] Gallionella ferruginea ES-2 Methylibium petroleiphilum PM1 Delftia acidovorans SPH-1 Aromatoleum aromaticum EbN1 Acidovorax sp. JS42 Diaphorobacter sp. TPSY Acidovorax avenae subsp. citrulli Sideroxydans lithotrophicus ES-1 Acidovorax avenae subsp. avenae Nitrosomonas sp. AL212 Candidatus Accumulibacter phos. clade Leptothrix cholodnii SP-6 Thiobacillus denitrificans Polaromonas sp. JS666 Azoarcus sp. BH72 Herminiimonas arsenicoxydans Comamonas testosteroni CNB-2 δ-proteobacteria Myxococcus xanthus DK 1622 Stigmatella aurantiaca DW4/3-1 Sorangium cellulosum 'So ce 56' Plesiocystis pacifica SIR-1 Haliangium ochraceum Geobacter sp. M21 Anaeromyxobacter sp. Fw109-5 Geobacter bemidjiensis Bem Anaeromyxobacter sp. K Anaeromyxobacter dehalogenans 2CP-C Anaeromyxobacter dehalogenans 2CP-1 γ-Proteobacteria: others Thioalkalivibrio sp. HL-EbGR7 Nitrococcus mobilis Nb-231 Nitrosococcus oceani Alcanivorax sp. DG881 Congregibacter litoralis KT71 gamma proteobacterium NOR51-B Alcanivorax borkumensis SK2 Thioalkalivibrio sp. K90mix Nitrosococcus halophilus Nc4 marine gamma proteob. HTCC2080 Allochromatium vinosum Saccharophagus degradans 2-40 Kangiella koreensis Pseudoalteromonas atlantica T6c Pseudoalteromonas haloplanktis Shewanella woodyi Hahella chejuensis KCTC 2396 Teredinibacter turnerae T7901 Beggiatoa sp. PS γ-Proteobacteria: Pseudomonadaceae Pseudomonas mendocina ymp Azotobacter vinelandii DJ Pseudomonas stutzeri A1501 γ -Proteobacteria: Xanthomonadaceae Xanthomonas axonopodis pv. citri Xanthomonas campestris pv. vesicatoria Xanthomonas oryzae pv. oryzae PXO99A Xanthomonas oryzae pv. oryzae KACC10331 Xanthomonas oryzae pv. oryzae Xanthomonas campestris pv. campestris Stenotrophomonas maltophilia R551-3
S13
Reut_A0918 Rmet_2565 Rpic_0821
Reut_A0917 Rmet_2566 Rpic12D_1461
Nmul_A1925 Vapar_3659 mma_1195 Rfer_1610 GalfDRAFT_2812 Mpe_A1380 Daci_4835 ebA7008 Ajs_2288 Dtpsy_1586 Aave_3091 SlitDRAFT_0423 AcavDRAFT_0897 NAL212DRAFT_1384 CAP2UW1_0454 Lcho_1724 Tbd_1020 Bpro_2909 azo1723 HEAR1060 CtCNB1_1703
Nmul_A1924 Vapar_3660 mma_1196 Rfer_1619 GalfDRAFT_2813 Mpe_A1380 Daci_4834 ebA1815 Ajs_2287 Dtpsy_1587 Aave_3090 SlitDRAFT_0422 AcavDRAFT_0898 NAL212DRAFT_1385 CAP2UW1_0455 Lcho_1725 Tbd_1019 Bpro_1756 azo0700 HEAR1061 CtCNB1_1704
MXAN_7473 STIAU_3072 sce7074 PPSIR1_35602 Hoch_6117 GM21_2631 Anae109_2422 Gbem_1582 AnaeK_2449 Adeh_1408 A2cp1_2545
MXAN_7471 STIAU_3073 sce7073 PPSIR1_19324 Hoch_6118 GM21_0053 Anae109_2423 Gbem_0055 AnaeK_2450 Adeh_1407 A2cp1_2546
Tgr7_2473 Noc_2028 Noc_2028 ADG881_1123 KT71_03107 NOR51B_1807 ABO_0654 TK90_0941 Nhal_2808 MGP2080_09773 Alvin_2660 Sde_3536 Kkor_1234 Patl_2433 PSHAa1438 Swoo_2444 HCH_06303 TERTU_3974 BGP_2342
Tgr7_2475 NB231_07522 Noc_2029 ADG881_2597 KT71_03112 NOR51B_1127 ABO_0655 TK90_0939 Nhal_2807 MGP2080_09778 Alvin_2661 Sde_3535 Kkor_1233 Patl_2434 PSHAa1437 Swoo_2445 HCH_06304 TERTU_3973 BGP_5711
Pmen_4038 Avin_19130 PST_1443
Pmen_4037 Avin_19140 PST_1444
XAC0186 XCV0170 PXO_03587 XOO4508 XOO_4247 XCC0168 Smal_3998
XAC0185 XCV0169 PXO_03588 XOO4509 XOO_4248 XCC0167 Smal_3999
References
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
Reinhold,
V.
N.;
Ishikawa,
Y.;
Melville,
D.
B.
J.
Bacteriol.
1970,
101,
881‐884.
Dorgan,
K.
M.;
Wooderchak,
W.
L.;
Wynn,
D.
P.;
Karschner,
E.
L.;
Alfaro,
J.
F.;
Cui,
Y.
Q.;
Zhou,
Z.
S.;
Hevel,
J.
M.
Anal.
Biochem.
2006,
350,
249‐255.
Ishikawa,
Y.;
Israel,
S.
E.;
Melville,
D.
B.
J.
Biol.
Chem.
1974,
249,
4420‐4427.
Clausen,
T.;
Schlegel,
A.;
Peist,
R.;
Schneider,
E.;
Steegborn,
C.;
Chang,
Y.
S.;
Haase,
A.;
Bourenkov,
G.
P.;
Bartunik,
H.
D.;
Boos,
W.
Embo
J.
2000,
19,
831‐ 842.
Krupka,
H.
I.;
Huber,
R.;
Holt,
S.
C.;
Clausen,
T.
Embo
J.
2000,
19,
3168‐3178.
Eliot,
A.
C.;
Kirsch,
J.
F.
Annu.
Rev.
Biochem.
2004,
73,
383‐415.
Hegg,
E.
L.;
Que,
L.
Eur.
J.
Biochem.
1997,
250,
625‐629.
Crooks,
G.
E.;
Hon,
G.;
Chandonia,
J.
M.;
Brenner,
S.
E.
Genome
Res.
2004,
14,
1188‐1190.
S14
10
H 2N 2”
O
3”
9
H N
OH
4”
O
Solvent: D2O Temp. 20.0 C / 293.1 K
Pulse Sequence: 1H NMR
O
8
3’
2’
O
4*
NH
7
N-methyl
N
4*
S
OH
N
3
6
2
O
OH
5
DOH
2 + 2’
4
2”
3’
3
3
N-methyl
4” 3”
2
1
0
ppm
10
9
8
7
6
5
4
3
2
1
F1 (ppm)
10
9
8
7
Solvent: D2O Temp. 20.0 C / 293.1 K
Pulse Sequence: gCOSY
6
O
H N
4
OH
4”
F2 (ppm)
5
H 2N 2”
3”
O
3
O
3’
2’
O
4*
NH
1
N-methyl
N
2
S
OH
N
3 2
0
O
OH
-1
140
130
120
110
100
90
80
70
60
10
O
H 2N 2”
40
50
3”
30
20
F1 (ppm)
9
H N
OH
4”
O
8
O
Solvent: D2O Temp. 20.0 C / 293.1 K
Pulse Sequence: HSQC
3’
2’
4*
S
O
4*
NH
7
N-methyl
N
OH
6
N
3 2
2’
2”
2
5 4 F2 (ppm)
O
OH
3
3’
N-methyl
3 4”
2
3”
1
0
-1
