Structures of bacterial kynurenine formamidase reveal a crowded binuclear zinc catalytic site primed to generate a potent nucleophile

Article abstract of DOI:10.1042/BJ20140511

Tryptophan is an important precursor for chemical entities that ultimately support the biosynthesis of key metabolites. The second stage of tryptophan catabolism is catalysed by kynurenine formamidase, an enzyme that is different between eukaryotes and prokaryotes. In the present study, we characterize the catalytic properties and present the crystal structures of three bacterial kynurenine formamidases. The structures reveal a new amidase protein fold, a highly organized and distinctive binuclear Zn²⁺ catalytic centre in a confined, hydrophobic and relatively rigid active site. The structure of a complex with 2-aminoacetophenone delineates aspects of molecular recognition extending to the observation that the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing. The cations occupy a crowded environment, and, unlike most Zn²⁺ -dependent enzymes, there is little scope to increase co-ordination number during catalysis.We propose that the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism.

Full text of DOI:10.1042/BJ20140511

Biochem. J. (2014) 462, 581–589 (Printed in Great Britain) doi:10.1042/BJ20140511
581
Structures of bacterial kynurenine formamidase reveal a crowded binuclear
zinc catalytic site primed to generate a potent nucleophile
1
´
´
Laura DIAZ-SAEZ*, Velupillai SRIKANNATHASAN*, Martin ZOLTNER* and William N. HUNTER*
*Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
Tryptophan is an important precursor for chemical entities that
ultimately support the biosynthesis of key metabolites. The
second stage of tryptophan catabolism is catalysed by kynurenine
formamidase, an enzyme that is different between eukaryotes and
prokaryotes. In the present study, we characterize the catalytic
properties and present the crystal structures of three bacterial
kynurenine formamidases. The structures reveal a new amidase
restricted to assist binding in the confined space of the active site
and for subsequent processing. The cations occupy a crowded
environment, and, unlike most Zn -dependent enzymes, there is
little scope to increase co-ordination number during catalysis. We
propose that the presence of a bridging water/hydroxide ligand in
conjunction with the placement of an active site histidine supports
a distinctive amidation mechanism.
2
+
2
+
protein fold, a highly organized and distinctive binuclear Zn
catalytic centre in a confined, hydrophobic and relatively rigid
active site. The structure of a complex with 2-aminoacetophenone
delineates aspects of molecular recognition extending to the
observation that the substrate itself may be conformationally
Key words: amidase, binuclear metal site, kynurenine
formamidase, tryptophan catabolism, X-ray crystallography, zinc
enzyme.
INTRODUCTION
[5,6]. In KFase, an ordered mechanism results when the catalytic
triad positions histidine to activate serine into a nucleophile that
attacks the substrate carbonyl carbon. Subsequent engagement of
a water molecule then allows for completion of the reaction.
Our attention was drawn to the prokaryotic KFase KynB (EC
Tryptophan catabolism via the kynurenine pathway supports
the biosynthesis of NAD , important neuroactive intermediates
+
in eukaryotes, then anthranilate, quinolate and antibiotics in
prokaryotes [1–4]. The proteins involved in tryptophan catabolism
are in general highly conserved across species, but with the notable
exception of the second enzyme in the pathway: kynurenine
formamidase. This enzyme catalyses the conversion of N-formyl-
L-kynurenine (NFK) into formate and L-kynurenine [2].
3
.5.1.9). Analysis of KynB sequences indicated that they are
unrelated to KFase and indeed do not match any known amidase or
amidohydrolase. Clearly, KynB is different and so, intrigued, we
sought to investigate the structure–activity relationships in this
distinctive enzyme. We now report the preparation of efficient
recombinant expression systems for KynB from three bacterial
sources and their purification. The catalytic properties of the
enzymes are characterized and tryptophan fluorescence is used
to investigate ligand binding. High-resolution crystal structures
have been determined that allow us to describe the architecture
of the enzyme extending to the details of the active site, to
address metal ion identification and to investigate key features of
molecular recognition at the catalytic centre. Taken together, our
structural data then allow us to propose a plausible and distinctive
mechanism for KynB.
In eukaryotes, the enzyme is termed kynurenine formamidase
KFase) and displays relatively low sequence conservation,
(
although the structure itself is well maintained. This is evident
from studies with Sacharyomyces cerevisiae and Drosophila
melanogaster KFase, which, although they only share 10%
sequence identity, are structurally conserved α/α hydrolases [5,6].
The KFase fold is dominated by a core eight-stranded α-sheet
decorated on either side with a number of α-helices. This creates
a well-defined cavity at one end of the sheet, leading to a catalytic
triad consisting of a serine, histidine and aspartate combination.
Overall, there are close structural similarities between KFase
and carboxylesterases, but also intriguingly to another enzyme
ꢀ
involved in kynurenine biology, namely a 5 -pyridoxyl phosphate-
dependent kynurenine aminotransferase [5,6].
EXPERIMENTAL PROCEDURES
Expression plasmid preparation
The chemistry behind an amidation reaction involves either
acidic or basic hydrolysis. It is the latter that applies to these
metal-free amidases, which exploit the properties of reactive
cysteine or serine residues to create covalent acyl-intermediates
during catalysis. Cysteine-dependent amidases are exemplified by
trypanothione synthetase amidase [7], nicotinamidase [8] and the
nitrilases [9], whereas the eukaryotic KFases from S. cerevisiae
and D. melanogaster present examples of the serine-dependent
enzymes with a mechanism that correlates with known amidases
Genes encoding KynB in Pseudomonas aeruginosa (PaKynB,
UniProt: Q9I234), Burkholderia cenocepacia (BcKynB, UniProt:
B4E9I9) and Bacillus anthracis (BaKynB, UniProt: Q81PP9)
were purchased (Genscript) having been codon optimized for
expression in Escherichia coli K12. PaKynB and BcKynB
encoding genes were cloned into the NdeI/XhoI and BaKynB into
the NdeI/BamHI site of a modified pET15b vector (Novagen)
Abbreviations: ACMSD, α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase; BaKynB, Bacillus anthracis KynB; BcKynB Burkholderia
cenocepacia KynB; CV, column volume; PaKynB, Pseudomonas aeruginosa KynB; KFase, kynurenine formamidase; NCS, non-crystallographic symmetry;
NFK, N-formyl-L-kynurenine; SEC, size-exclusion chromatography; TEV, tobacco etch virus; XANES, X-ray absorption near-edge structure.
1
To whom correspondence should be addressed (email w.n.hunter@dundee.ac.uk).
Atomic co-ordinates and structure factors have been deposited in the PDB under codes 4COG, 4COB, 4CO9 and 4CZ1.
ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

582
L. D ´ı az-S a´ ez and others
to create plasmids that produce an N-terminal hexahistidine-
tagged (His-tagged) protein and a tobacco etch virus (TEV)
protease cleavage site. All constructs were sequenced to check
their integrity.
Protein concentration determination
Protein concentrations were measured by absorbance at 280 nm
using the predicted molar absorption coefficients by ExPASy
bioinformatics resource portal ProtParam [10]:
−
1
−1
−1
−1
ε(PaKynB) = 28 210 M · cm
−
1
ε(BcKynB) = 28 210 M · cm
Expression and protein purification
−
1
Gene expression and purification of the proteins started with
freshly transformed E. coli BL21(DE3)pLysS in the case of
PaKynB and BcKynB systems, and E. coli BL21(DE3) for
ε(BaKynB) = 20 970 M · cm
◦
BaKynB. Bacteria were cultured at 37 C in 10 ml of LB
Enzymatic assay and determination of catalytic parameters
−
1
broth containing 50 μg· ml carbenicillin and, for PaKynB and
A spectrophotometric assay was developed to obtain K , Vmax
m
−
1
BcKynB cultures, 20 μg· ml chloramphenicol was also present.
and kcat values for KynB. The assay is based on the increase
in absorbance due to L-kynurenine formation [11,12] which is
detected by measuring the absorbance at 365 nm with a UV-
These cultures were used as inoculum for 1 litre of LB/antibiotic
◦
mixtures. The bacteria were cultured at 37 C until an attenuance
of 0.6–0.8 at λ = 600 nm was achieved. The temperature was
2
450 Shimadzu spectrophotometer over a period of 160 s. The
◦
lowered to 16 C in the case of PaKynB and BcKynB, and to
reactions were performed in triplicate using 1 ml final volume,
at 25 C, using 0.025, 0.15, 0.25, 0.5, 0.7, 1, 1.5, 2 and 3 mM
◦
2
0 C for BaKynB. Gene expression was induced with 1 mM
◦
IPTG and cultures were incubated overnight. Cells were then
NFK substrate (Dalton Pharma Services), and 500 ng of enzyme
◦
harvested by centrifugation (4000 g at 4 C for 10 min). The
(
20 nM). The buffer used contained 0.1 M NaH
2
PO
4
/Na
2
HPO ,
4
PaKynB and BcKynB cultures were resuspended in buffer A1
25 mM Tris/HCl, pH 7.5, and 100 mM NaCl), and BaKynB in
buffer A2 (20 mM Tris/HCl, pH 7.4, and 200 mM NaCl) with
the addition of complete EDTA-free protease inhibitor cocktail
tablets from Roche. The cells were disrupted using a sonicator in
the case of PaKynB and BcKynB, and a French press for BaKynB,
pH 7.4, and 20 μM ZnCl . Calculations of the initial velocity
2
(
for each substrate concentration were made by fitting a linear
equation to the data of the increase in L-kynurenine concentration
during 1 min, and data were analysed using the Michaelis–
Menten equation (SigmaPlot, Systat Software). L-Kynurenine
concentration was calculated using the Beer–Lambert law and
◦
and homogenates were centrifuged at 40000 g for 30 min at 4 C.
− 1
− 1
ε
365 of 4220 M · cm [12].
The resulting supernatants were passed through a 0.45 μm filter,
and samples containing PaKynB and BcKynB were adjusted to
contain 5 mM imidazole. Proteins were loaded on a 5-ml HisTrap
HP column (GE Healthcare). After a 10-column volume (CV)
washing step using 90% buffers A1 or A2 and 10% of buffer B1
25 mM Tris/HCl, pH 7.5, 100 mM NaCl and 0.5 M imidazole)
for PaKynB and BcKynB, or buffer B2 (20 mM Tris–HCl pH 7.4,
00 mM NaCl, 0.8 M imidazole) for BaKynB, the recombinant
proteins were eluted applying a linear imidazole gradient (50–
50 mM for PaKynB and BcKynB, and 80–400 mM for BaKynB
Crystallization, diffraction and structure determinations
−
1
PaKynB and BcKynB were concentrated to 7.5 mg · ml in buffer
−
1
(
A1 and BaKynB to 4 mg· ml in buffer A2 to provide a stock
solution for crystallization experiments. The first crystallization
trials used commercial screening sets from Molecular Dimensions
and Qiagen and were set up in 96-well sitting drop plates with a
Phoenix liquid handling system (Rigaku-MSC) using a ratio of
1:1 for protein/reservoir and final volumes of 0.2 and 0.4 μl for
every condition. Plates were incubated at room temperature in
a Gallery DT plate hotel (Rigaku-MSC). PaKynB crystallization
occurred using the reservoir condition 0.1 M Hepes, pH 7.5, 20%
(w/v) PEG 4000 and 10% (v/v) propan-2-ol. Crystals grew to a
maximum dimension of approximately 0.25 mm, over 5 days. The
BcKynB crystal was obtained using a reservoir comprising 10–
2
2
over 20 CV). The eluted proteins were dialysed into buffer A1
or A2 and incubated overnight with 1 mg of His-tagged TEV
protease per 20 mg of protein at 4 C, then applied to a HisTrap
◦
HP column equilibrated with buffer A1 or A2 to remove the
TEV protease, cleaved peptide and non-cleaved material. Initial
experiments indicated that the His-tag of PaKynB was not cleaved
efficiently by TEV protease, and protein aggregation/precipitation
was pronounced. We left the His-tag in place for further
experiments with that protein. The enzyme assay (see later)
indicated that the His-tagged PaKynB possessed a level of activity
comparable with that of other samples. Fractions containing
the proteins were collected, concentrated and applied to a size-
exclusion chromatography (SEC) column (HR 16/60, Superdex75
prep grade, GE Healthcare, CV = 120 ml) equilibrated with
16% (w/v) PEG 3350, 5 mM CoCl
2
, 5 mM CdCl
2
, 5 mM MgCl
2
and 5 mM NiCl . Before data collection, the crystals were soaked
2
for a few seconds in the reservoir liquid with added 25% (v/v)
glycerol and then flash frozen in liquid nitrogen. Diffraction data
for PaKynB and BcKynB were collected using the Diamond Light
Source beamline IO3 using a Pilatus 6M-F detector, indexed and
integrated using XDS [13], scaled and analysed with SCALA and
POINTLESS [14] from the CCP4 suite [15].
buffer C (10 mM NaH
2
PO
4
/Na
2
HPO , pH 7.8, 20 mM NaCl and
4
0
.5 mM Tris/HCl) in the case of PaKynB and BcKynB, and buffer
The initial crystallization conditions for BaKynB gave only
small mechanically twinned samples and optimization was carried
out in 24-well hanging drop plates using a ratio of 1:1 in final
A1 for BaKynB. The SEC columns had been calibrated with
molecular mass standards (thyroglobulin, 670 kDa; γ -globulin,
1
1
weight were assessed by SDS/PAGE and MALDI–TOF-MS
analyses performed at the University of Dundee ‘Fingerprints’
Proteomics Facility using an Applied Biosystems Voyager DE-
STR spectrometer. Protein quaternary structure was investigated
by native PAGE and SEC.
◦
58 kDa; serum albumin, 67 kDa; ovalbumin, 44 kDa; myoglobin,
7 kDa; vitamin B12, 1 kDa). Protein purity and molecular
volumes of 2 and 4 μl at 20 C. BaKynB was incubated with
8 mM L-kynurenine for 10 min at room temperature, and a
structure was obtained from a crystal grown in a drop equilibrating
with a reservoir containing 100 mM Tris/HCl, pH 8.5, 150 mM
MgCl , 30% (w/v) PEG 4000 and 1.5% (v/v) dioxane. The
2
presence of dioxane was crucial to improve the morphology
such that single-crystal blocks could be obtained. Crystals of
ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Bacterial kynurenine formamidase
583
BaKynB were flash frozen directly from the drops in which
they grew. Diffraction data were obtained using the in-house
X-ray facility (Rigaku M007HF X-ray generator with a Saturn
crystallization, then it is most likely to be derived from the media
used to culture the E. coli.
2
+
2 +
The presence of Cd and Zn in the active site of BcKynB
was confirmed on the basis of the electron density and anomalous
scattering. Positive density in a difference Fourier map calculated
for the 12 sulfur atoms in the asymmetric unit gave 1.1 σ per
9
44HG + CCD detector). Data were indexed and integrated
using iMOSFLM [16] and scaled and analysed by AIMLESS
from the CCP4 suite [15]. To obtain crystals of the BaKynB–
2
2
-aminoacetophenone complex, protein was incubated with 5%
electron. The average B-factor for these 12 atoms is 16.4 Å .
(
v/v) 2-aminoacetophenone before the crystallization plate was set
This was used for the determination of the number of electrons
present at the BcKynB metal ion sites. The difference Fourier
map revealed peaks with heights of 46.5 and 68.4 σ, respectively.
These height values and the difference between them are constant
within the four active sites in the asymmetric unit, and indicate the
presence of two different metal atoms per active site. Comparing
atomic numbers between the different metals present in the
up. The ligand was previously dissolved to a final concentration
of 20% (v/v) into buffer A2 containing 20% (w/v) PEG 4000.
The reservoir contained 140 mM MgCl , 30% (w/v) PEG 4000
2
and 100 mM Tris/HCl, pH 8.5. Diffraction data for the BaKynB–
-aminoacetophene complex were obtained at the Diamond Light
2
Source using beamline IO3.
2
+
2 +
The structure of PaKynB was determined by molecular
replacement (MOLREP) [17] using a single polypeptide
crystallization condition, one Cd and one Zn were added
to these positions. The average B-factors refined to 11.4 and
2
(molecule A) for a putative metal-dependent hydrolase from
10.1 Å , respectively. A B-factor-adjusted calculation suggests
Geobacillus stearothermophilus (PDB code 1R61). All water
molecules and ions were removed from the model. The search
model, which shares 25% sequence identity with PaKynB,
that one site is occupied by an ion consisting of approximately
−
−
−
28 e and the other about 48 e . Values of 28 and 46 e would
2
+
2 +
be expected for Zn and Cd , respectively. An anomalous
dispersion difference Fourier calculation provided a further
check. At the wavelength used for diffraction measurements,
2
+
is at 2.5 Å resolution, and although Zn is assigned in the
possible active site, it is with low occupancy (0.5), with an
2
ꢀꢀ
inflated thermal parameter (B-factor approximately 90 Å ) and
λ = 0.9795 Å, the theoretical f values for zinc and cadmium
−
distances to potential co-ordinating groups that are more typical of
hydrogen-bonding interactions than metal ion ligand associations.
are 2.480 and 2.132 e [20]. In an anomalous difference
Fourier a slightly larger signal should therefore occur at the
2
+
2 +
There is no information concerning why Zn was assigned here,
and there are no publications on this structure. The BcKynB
structure was solved using a partially refined PaKynB as the
search model (64% sequence identity), and then the BaKynB
structure was solved using BcKynB (40% sequence identity)
as the search model. Similar protocols were applied in the
refinement of all structures. This involved first a rigid body
refinement as part of the molecular replacement calculations, then
iterative cycles of restrained refinement combining REFMAC5
Zn . Indeed this is observed in all four active sites of the
asymmetric unit. The average heights of the peaks are 24.2 σ
2
+
2 +
at Zn
and 22.5 σ at Cd
(Supplementary Figure S2 at
http://www.biochemj.org/bj/462/bj4620581add.htm).
MolProbity [21] was used to assess geometry of all models.
Secondary-structure determination involved use of DSSP [22] and
visual inspection, and subunit interface surface area calculations
were carried out with PISA [23]. Figures were prepared using
ALINE [24] and PyMOL (http://www.pymol.org). The DALI
server [26] was used to search the PDB for structural homologues,
whereas superpositions were calculated using DALILITE [27].
Relevant crystallographic statistics and geometric details of the
refined models are reported in Table 1.
[
18] with electron and difference density map inspections, and
model manipulations in COOT [19]. The starting B-factors
for each model were derived from the Wilson B-factor. There
are multiple polypeptide chains in the asymmetric units (two
subunits for PaKynB and four subunits for BcKynB, BaKynB
and the BaKynB–2-aminoacetophenone complex), and tight non-
crystallographic symmetry (NCS) restraints were imposed at the
onset of refinement, which were gradually released during the
process. Although L-kynurenine was present in the crystallization
mixture used for BaKynB, there was no evidence for the enzyme
product in electron density maps. Even with a much greater
amount of 2-aminoacetophenone present, we only observed
ordered binding of ligand in one active site. Once the protein
models were complete, alternative side-chain conformers, water
molecules, metal ions and ligands were incorporated into the
models. A correction for a twinning component of 0.15, calculated
in REFMAC, was applied in refinement of the BaKynB–2-
aminoacetophenone complex. Refinements were terminated when
there were no significant changes in Rwork and Rfree values and
inspection of the difference density maps suggested that no further
corrections or additions were justified.
Fluorescence spectroscopy with BaKynB
Fluorescence measurements for BaKynB were performed
using a LS-55 PerkinElmer spectrometer to investigate ligand
association/disassociation. To determine the peak of maximum
emission due to tryptophan, the excitation wavelength used was
2
80 nm and the emission wavelength detection ranged from 300
to 400 nm. The sensitivity of the detector was fixed to 800 V, and
the peak of maximum emission was experimentally determined
at 337 nm.
The sample (2 ml) contained 20 μg of BaKynB (400 nM),
.02 M Tris/HCl, pH 7.4, buffer and 0.2 M NaCl. The two
0
products of the KynB catalysed reaction, L-kynurenine and
formate, and 2-aminoacetophenone were investigated separately.
Concentrations ranged from 0 to 800 μM in the case of L-
kynurenine and 2-aminoacetophenone and from 0 to 500 μM
for formate. No fluorescence emission from either L-kynurenine
or 2-aminoacetophenone was observed under the experimental
conditions. In the presence of increasing concentrations of L-
kynurenine and 2-aminoacetophenone, a decrease and shift of
the maximum fluorescence intensity was observed. In the case of
formate, no variation in the fluorescence intensity was detected.
Data are represented as percentage of active site saturation
within the different compound concentrations (Supplementary
2
+
The presence of Zn
was first suggested by anomalous
difference Fourier maps (not shown) and subsequently
confirmed in the PaKynB and BaKynB crystals by X-ray
absorption near-edge structure (XANES) spectra measured at
Diamond Light Source on beamline IO3 using a Vortex
Silicon Drift detector and an excitation energy range
from 9626.7 to 9690.14 eV (Supplementary Figure S1 at
http://www.biochemj.org/bj/462/bj4620581add.htm). Since no
2
+
Zn
was added during protein production, purification or
ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

584
L. D ´ı az-S a´ ez and others
Table 1 Crystallographic statistics
Structure
PaKynB
4COB
BcKynB
4COG
BaKynB
4CO9
BaKynB–ligand
4CZ1
PDB code
Space group
P3
0.9795
112.7, 112.7, 90.76
28.90–2.37
133546
1
21
P2
0.9795
1
P2
1.5418
1
P2
1
0.9791
Wavelength ( A˚ )
Unit cell dimensions a, b, c ( A˚ )
Resolution range* ( A˚ )
Number of reflections
Unique reflections
Completeness (%)
76.86, 50.12, 35.2, β = 94.14
28.37–1.60
479924
◦
73.17, 66.02, 83.76, β = 90.32
◦
73.69, 66.56, 84.06, β = 90.24
◦
42.48–1.95
202165
42.64–2.25
104599
27365
134904
58336
38046
99.2 (94.5)
0.057 (0.571)
4.9
16.8 (2.4)
43.87
99.3 (99.8)
0.068 (0.477)
3.6
12.8 (3)
14.97
99.4 (94.1)
0.063 (0.166)
3.5
11.1 (4.3)
11.84
98.1 (97.7)
0.164 (0.551)
2.7
4.8 (2.2)
15.33
R
merge
†
Redundancy
I/σ(I)>
<
Wilson B ( A˚ )
2
R
work‡/Rfree
§
0.1519/0.1945
412/181/6
0.198
0.018/1.890
51.4
0.1489/0.1842
831/1068/38
0.068
0.025/2.429
16.4
0.1715/0.2066
829/906/18
0.137
0.008/1.322
15.3
0.1837/0.2241
825/793/1/11
0.086
0.017/1.684
20.9
Number of residues/waters/ligands and metals
Diffraction precision indicatorꢀ ( A˚ )
Bond lengths ( A˚ )/angles¶ ( )
Average B-factors ( A˚ )
◦
2
Protein atoms
Water molecules
Metal ions
3207
6533
181
4 Zn
1068
2
+
2 +
2 +
2 +
2 +
2 +
4 Zn , 4 Cd , 8 Mg
Ligands
12 Glycerol, 10 1,2-ethanediol,
2 Dioxane, 3 1,2-ethanediol
1
PEG
Ramachandran analyses
Favoured regions (%)
Allowed regions (%)
96.1
100
96.1
100
97.2
100
96.5
100
*
Values in parentheses refer to the highest resolution shell.
†
R
R
R
merge = ꢀhkl
ꢀ
i
|I
i
(hkl) − <I(hkl)>|/ꢀhkl
ꢀ
i
I
i
(hkl); where I
i
(hkl) is the intensity of the ith measurement of reflection hkl and <I(hkl)> is the mean value of I
i
(hkl) for all i measurements.
‡
work = ꢀhklꢀF
o
| − |F
c
ꢀ/ꢀ|F |, where F is the observed structure factor and F
o
o
c
is the calculated structure factor.
§
free is the same as Rwork except calculated with a subset, 5%, of data that are excluded from the refinement calculations.
ꢀ
Diffraction Precision Index [42].
[43].
¶
Table 2 Catalytic parameters
max, maximum catalytic velocity; K
V
m
, Michaelis-Menten constant; specific activity (SA), amount of active enzyme from the total protein quantity; kcat, number of substrate molecules that are
transformed per active site and per time unit. kcat/K defines the catalytic efficiency.
m
max (nmol · min− 1)
K
(mM)
k
cat (s
− 1
)
k
cat/K
− 1
(M · s− 1) × 10
4
SA (μM · min− 1 · mg− 1
)
Enzyme
V
m
m
BaKynB
BcKynB
PaKynB-His
65.41 + 2.64
0.40 + 0.05
50.56
43.94
114.21
12.64
7.71
11.65
130.82
116.30
295.98
−
58.15 + 0.98
−
−
0.57 + 0.02
−
147.99 + 8.42
−
0.98 + 0.13
−
Figures S3 and S4 at http://www.biochemj.org/bj/462/
bj4620581add.htm), and disassociation constants were determ-
ined.
Enzyme assays
The kinetic properties of the enzymes were determined using a
spectrophotometric assay and are reported in Table 2. The assays
confirmed that active bone fide KFases had been purified; for
example, in the case of BaKynB, approximate values for K
m
RESULTS AND DISCUSSION
−
1
− 1
0
1
.4 μM, Vmax 65 nmol· min , kcat 50 s and specific activity
Enzyme purification and quaternary structure
− 1 − 1
30 μM min · mg were derived. No substrate inhibition was
Highly efficient recombinant protein expression systems for the
enzymes from B. anthracis (BaKynB), B. cenocepacia (BcKynB)
and P. aeruginosa (PaKynB) were prepared. The total protein
yields, after purification, from the bacterial cultures were about
observed under the assay conditions and NFK concentrations
that were used. In the case of PaKynB, assays were carried
out using protein still carrying the His-tag due to aggregation
problems when removal of the tag was attempted. The kinetic
parameters for PaKynB, BaKynB and BcKynB are similar to
each other. Comparisons of kinetic properties of enzymes is
often complicated by the use of different assay conditions and
this applies to studies with KynB from Streptomyces parvulus
[11] and Ralstonia metallidurans [1] which were conducted at
−
1
− 1
1
0 mg· l for PaKynB and BcKynB, and 50 mg· l for BaKynB.
The theoretical polypeptide mass of KynB is about 23 kDa. During
the final chromatography step of purification, i.e. SEC, all three
proteins were eluted as a single species with approximate mass of
4
0 kDa and native gels also identified that a single dimeric species
◦
◦
was observed in each case.
30 C and 37 C, respectively. In these cases, the activities are
ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

Bacterial kynurenine formamidase
585
Figure 1 Sequence alignments and assignment of secondary structure for BaKynB
Protein sequence alignment from BaKynB, PaKynB and BcKynB. Blue arrows (β-sheets) and red cylinders (α-helixes) indicate the secondary structure of BaKynB. Triangles mark the metal-binding
6
0
amino acids. Circles mark amino acids in the active site. The star marks a key amino acid for the reaction, His . The diamond marks the tryptophan located at the active site pocket.
lower than we report with reduced specific activities of about
than to the Gram-positive BaKynB (rmsd 1.25 Å in each case).
This maps to sequence identities of about 64% for the enzymes
from the Gram-negative organisms to 40% when compared with
Gram-positive KynB (Figure 1). The structures are, however, so
similar, particularly the detail in the active site which is highly
conserved (see below), that we primarily detail BaKynB mainly
because for that example we also have data that inform on the
molecular recognition of ligands in the active site.
−
1
2
7 and 4 units· mg noted. A more similar room-temperature
assay was reported for KynB from Bacillus cereus [12] and this
−
1
− 1
gave a specific activity of 68.5 μM min · mg with kcat/K
m
4
− 1
− 1
of 18.3 × 10 M · s , values comparable with those from our
observations.
Comments on crystallographic analyses and metal ion
identification
The KynB subunit, approximate mass 23 kDa, comprises just
over 200 residues with about 50% in 12 β-strands and 15%
in four α-helices (Figures 1 and 2). Three short segments of
The crystal structures of all three enzymes were determined
(
Table 1). The first analysis, PaKynB at about 2.4 Å resolution
3
10-helix occur between β4 and β5. The molecule gives the
identified two cations in the active site, tentatively assigned as
appearance of a distorted eight-stranded β-barrel by treating β1
and β4 as a single strand. Three α-helices cluster on one side
of the β-barrel, and on the other side is the dimer interface.
The fold is not common and a search of the PDB reveals a
significant similarity to only three other proteins for which there
are little or no published biochemical data. These orthologues
are a putative metal-dependent hydrolase/cyclase (PDB codes
2
+
a binuclear Zn site. A more accurate model was sought, and
the structure of BcKynB was determined at 1.6 Å resolution.
However, these highly ordered crystals could only by obtained
in the presence of different transition metal cations including
Co , Ni and Cd . In addition, glycerol, the optimized cryo-
protectant, was bound to the metal ions, blocking the active site,
so preventing ligand studies. A structure was required without
2
+
2 +
2 +
3
KRV and 1R61, Z-scores 27, 26, sequence identity 25%), isatin
2
+
Cd being present, or indeed any transition metal ions other than
2
+
hydrolase carrying a single Mn (PDB code 4J0N, Z-score 23,
sequence identity 23%) and a hypothetical protein with no metal
ion present (PDB code 2B0A, Z-score 20, sequence identity
2
+
Zn and for which we could obtain data to inform on substrate
recognition. Highly ordered structures of BaKynB and a complex
with 2-aminoacetophenone at resolutions of 1.9 and 2.3 Å resulted
and confirmed the binuclear Zn environment. XANES scans
confirmed the presence of Zn in crystals of PaKynB and in
BaKynB (Supplementary Figure S1).
2
3%).
2
+
The KynB dimer (Figure 2) displays comparatively high values,
2
+
between 22% and 27%, for the accessible surface area of
subunits that interact with the partner. This is consistent with
the observation of only dimeric species in SEC and native gels.
The dimer is stabilized by hydrophobic interactions primarily
involving side chains from strands β1, β11 and β12, together
with a contribution from short helical segments between residues
The overall structure of KynB
The secondary and subunit structure of KynB, NCS of the dimer
and detailed architecture of the active site are all highly conserved
for the three enzymes. The average NCS values for overlay of
Cα atoms range from 0.22 Å, for BcKynB, to 0.32 Å for both
BaKynB and PaKynB. The Gram-negative KynB structures are
more similar to each other (rmsd overlay of Cα atoms is 0.70 Å)
6
8 and 74. The formation of an antiparallel four-stranded β-sheet
involving β2–β3 from each subunit is a pronounced feature of the
dimer. The N-terminal segment of β3 contributes to formation of
the active site, which is mainly created by the partner subunit. In
the dimer, the two catalytic sites are separated by about 30 Å.
ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

586
L. D ´ı az-S a´ ez and others
stabilize the active site configuration. In similar fashion, the three
co-ordinating histidine side chains are held in place by hydrogen
1
51
198
161
50
bonds that link Asp , Asp with His and His , respectively,
5
2
then His54 interacts with the carbonyl group of Gly . All of these
residues and their contributions to the organization of the active
site are highly conserved in the three types of KynB structures
characterized (Figures 1 and 4).
There are 380 unique sequences annotated as a bacterial KFase
in UniProt [29]. The list is reduced to 336 entries when filtered
for those with ꢀ30% sequence identity with BaKynB using the
NCBI BLAST server [30]. The sequences were aligned (Clustal
Omega) [31,32] and the conservation of key active site residues
5
4
56
60
investigated. His , Asp and His are strictly conserved in
50
161
173
all sequences, whereas His , His and Glu are maintained
20
in 99.7% of the entries. Trp , which contributes a significant
hydrophobic character to the substrate-binding site, is strictly
conserved in 81% of the entries. Conservative substitutions for
phenylalanine and tyrosine account for a further 17.8% of the
entries, confirming the importance of an aromatic group at this
position.
Tryptophan fluorescence
20
Having identified Trp in the active site, we used fluorescence
spectroscopy to investigate binding of L-kynurenine and
formate, the products of the formamidase reaction. We also
investigated 2-aminoacetophenone given chemical similarities
to part of L-kynurenine. This ligand provides the aromatic
component of substrate and product and primarily lacks
only the 2-amino-4-oxobutanoic acid moiety. Formate did
not elicit any spectroscopic change but L-kynurenine and 2-
Figure 2 Ribbon diagram of the BaKynB and location of the active site,
orthogonal views
aminoacetophenone gave comparable K
d
values of approximately
6
0 and 50 μM, respectively (Supplementary Figures S3
Zn2 ions are shown as grey spheres. For one subunit, the terminal positions of the polypeptide
+
and S4). A BaKynB–2-aminoacetophenone complex structure
then revealed aspects of molecular recognition in the
active site (Figure 3 and Supplementary Figure S5 at
http://www.biochemj.org/bj/462/bj4620581add.htm). As we will
show, this is consistent with the ligands binding in the vicinity of
the active site tryptophan with comparable affinity and suggests
that 2-aminoacetophenone represents a suitable molecule from
which we can derive information on aspects of substrate
recognition. Formic acid did not elicit any change in tryptophan
fluorescence, suggesting that this moiety of substrate and one
product of the KynB-catalysed reaction does not interact with the
active site tryptophan.
are labelled N and C, helices are labelled and β-strands numbered. (A) Top view, (B) side view.
The active site of KynB
The enzyme active site is a narrow cavity approximately
7
Å × 12 Å and 10 Å in depth, lined by hydrophobic walls
20
60
158
40
composed of Trp , His and Leu from one subunit, and Val
and Val from the partner (Figure 3). Four of these residues
are strictly conserved in BcKynB and PaKynB, whereas Leu1
corresponds to Met in BcKynB (Figure 1). At the base of the
cavity is a polar floor, where two Zn ions, separated by a
distance of 3.1 Å, are bound. This separation is unusually close
for a binuclear site when compared with other systems [28].
42
58
157
2
+
2
+
The co-ordination environment at the binuclear Zn site is
2
+
56
Recognition of a conformationally restricted substrate is implied
highly organized (Figure 3). One Zn is co-ordinated by Asp ,
161
173
His , Glu and a water molecule/hydroxide ion in a tetrahedral
fashion with distances less than 2.3 Å. The water/hydroxide
co-ordinates with the other cation and acts as a bridge for the
binuclear site. A water molecule and the other oxygen of the side
We were unable to obtain the structure of a complex of KynB
with the products of catalysis, formate or kynurenine. However,
the characterization of a BaKynB–2-aminoacetophenone complex
structure at 2.25 Å resolution provides a suitable template to
inform on aspects of molecular recognition in the active site
(Figure 3). Key to ligand, and by implication substrate, binding
173
chain of Glu are about 2.5 Å from the cation, and together
2
+
with the adjacent metal ion crowd around this Zn to provide a
distorted octahedral environment. In a similar fashion, the other
cation also has four ligands co-ordinating with distances less than
20
is the interaction with Trp . There are also van der Waals
40
associations with Val from the partner subunit. The 2-amino
50
54
56
2 +
2
.3 Å. These are His , His , Asp and the bridging water/
group forms a hydrogen bond to the Zn bridging water or
173
hydroxide. Then, another water and OE1 of Glu are 2.4 Å
hydroxide. In this respect, the complex derived may closely mimic
the configuration at the completion of the catalytic reaction and
immediately prior to the replacement of product by the incoming
substrate for another round of catalysis. An intramolecular
hydrogen bond between the amine and carbonyl groups of 2-
aminoacetophenone appeared to be significant. A 3D search of the
Cambridge Structural Database [33] using 2-aminoacetophenone
2
+
distant from this Zn . The water molecules that co-ordinate the
metal ions also interact by hydrogen-bond formation with the
enzyme. This involves interactions with the main chain carbonyl
of Pro and NE1 of Trp in one case and the side chain of
Ser in the other. The bridging water or hydroxide forms a
148
20
149
60
hydrogen bond with NE2 His . Such interactions may help to
ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Bacterial kynurenine formamidase
587
Figure 3 Active site of the BaKynB–2-aminoacetophenone complex
Zn2 is a grey sphere, grey broken lines mark co-ordination to amino acid side chains and waters/hydroxide (red spheres). Amino acid atomic positions are coloured with C in grey or yellow
depending on subunit, N in blue, O in red, and 2-aminoacetophenone is shown with C in black, N in cyan and O in red. Blue broken lines represent potential hydrogen bonds and a single red broken
line indicates the intramolecular interaction in the ligand.
+
Figure 4 The active site of KynB is highly conserved
(
A) Superimposition of BaKynB (violet), BcKynB (blue) and PaKynB (yellow) active site structures. (B) The BaKynB active site with difference density for the three water molecules (red spheres) in
the active site depicted as dark green chicken wire and contoured at 5σ. (C) The BcKynB active site with glycerol bound to the metal ions. (D) The PaKynB active site. Continuous lines represent
2
+
co-ordinating contacts to the Zn ions (grey spheres).
ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

588
L. D ´ı az-S a´ ez and others
Figure 5 A proposed mechanism for the amidase KynB
S)-2-Amino-4-(2-formamidophenyl)-4-oxobutanoic acid (N-formylkynurenine) is converted into (S)-2-amino-4-(2-aminophenyl)-4-oxobutanoic acid (kynurenine).
(
as the template identified 28 similar compounds with structures
determined at atomic resolution and all of which display the same
interaction. Therefore, such an intramolecular hydrogen bond
probably defines or constrains the substrate conformation, and
orients the scissile bond such that the formyl moiety would be
increase from four to five or six in the intermediate state of
catalysis [34,35].
KynB immediately struck us as distinct with a binuclear Zn
2
+
site, cations only 3.1 Å apart, which is closer than in other
cases, in a more crowded environment and with little scope to
increase the co-ordination number given that six co-ordinating
groups already surround each ion. This suggests then that
straightforward replacement of a co-ordinating water ligand might
be important. Furthermore, unlike other metallohydrolases where
conformational flexibility appears to be important [34,36,37], the
catalytic site of KynB is relatively rigid with an average thermal
60
149
directed over between His and Ser . These are two strictly
conserved residues in KynB sequences. The complex structure
suggests then that when substrate binds the oxygen of the formyl
2
+
substituent could displace a water that binds Zn weakly and
149
perhaps also interact directly with Ser . The orientation or
alignment is then fixed for nucleophilic attack to occur at carbon
as is described further below.
2
parameter (B-factor) of 9.6 Å compared with an overall average
2
of 15.1 Å in BaKynB.
Based on the structural data and consistency with the
fluorescence measurements, we can now suggest a plausible and
distinctive mechanism (Figure 5). The fixed conformation of a
planar substrate would assist binding in the small rigid active
Mechanistic considerations and comparisons with
metallohydrolases
2
+
60
Although the enzyme fold is different, the binuclear Zn catalytic
centre of KynB suggests some similarity to mononuclear and
binuclear metalloenzymes in the amidohydrolase superfamily.
Such enzymes exploit the Lewis acid properties of the cation,
site cavity. His , a strictly conserved active site residue, in
conjunction with the powerful Lewis acid activating binuclear
2
+
Zn
environment, is ideally positioned to acquire a proton
from the bridging water to generate a potent nucleophilic
hydroxide for attack at the carbonyl component of substrate.
decrease the pK of water and generate a hydroxide nucleophile
a
2
+
to initiate catalysis [34,35]. They bind and polarize substrate
during an intermediate state in the catalytic cycle and generally,
then use an acidic glutamate or aspartate to donate a solvent-
acquired proton as the intermediate collapses to release products.
Examples of such metallohydrolases include type B β-lactamases
Direct interaction between the substrate and Zn as a catalytic
intermediate is formed would probably involve replacement of
1
49
weakly co-ordinated water assisted by interaction with Ser
.
60
The intermediate would collapse as His , which in the complex is
3.6 Å from the 2-amino group of the ligand, donates the proton to
form the amine. The C–N bond breaks and products are released.
[
28,36], phosphotriesterases [37], leucine aminopeptidases [38]
and dihydro-orotases [39]. Constant features of this superfamily
34] include using an acidic component as proton donor and
the presence of flexible loops to engage with substrate are in
particular noted in the (β/α) triose phosphate isomerase-type
[
8
AUTHOR CONTRIBUTIONS
barrel structures. A relevant example actually occurs further
down in the kynurenine pathway, it is the enzyme α-amino-
β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD)
Laura D ´ı az-S a´ ez, Velupillai Srikannathasan and Martin Zoltner performed the research;
Laura D ´ı az-S a´ ez, Velupillai Srikannathasan and William N. Hunter designed the research,
analysed the data and wrote the paper. William N. Hunter supervised the research.
2
+
[
40,41]. ACMSD possess a single Zn ion co-ordinated by three
histidine residues and an aspartate. Other (β/α) barrel enzymes
8
such as specific phosphodiesterases and dihydro-orotases also
possess a carboxylated lysine, which binds two metal ions,
typically Zn , holding them about 3.5–4.0 Å apart and with
enough room around the metal ions to allow for a co-ordination
ACKNOWLEDGEMENTS
2
+
We thank Paul Fyfe, Alice Dawson and Thomas Eadsforth for advice, and the Diamond
Light Source Synchrotron Radiation Facility for beam time.
ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Bacterial kynurenine formamidase
589
FUNDING
20 Sasaki, S. (1989) Numerical Tables of Anomalous Scattering Factors Calculated by the
Cromer and Liberman’s Method (KEK Report 88–14). National Laboratory for High Energy
Physics, Tsukuba
This research was supported by the Wellcome Trust [grant numbers 082596, 094090 and
100476] and a studentship from the Defence Science and Technology Laboratory (U.K.).
2
1
Chen, V. B., Arendall, W. B., 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. D Biol.
Crystallogr. 66, 12–21 CrossRef PubMed
REFERENCES
2
2
Kabsch, W. and Sander, C. (1983) Dictionary of protein secondary structure: pattern
recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637
CrossRef PubMed
1
Kurnasov, O., Jablonski, L., Polanuyer, B., Dorrestein, P., Begley, T. and Osterman, A.
2003) Aerobic tryptophan degradation pathway in bacteria: novel kynurenine
(
formamidase. FEMS Microbiol. Lett. 227, 219–227 CrossRef PubMed
Zummo, F. P., Marineo, S., Pace, A., Civiletti, F., Giardina, A. and Puglia, A. M. (2012)
Tryptophan catabolism via kynurenine production in Streptomyces coelicolor:
identification of three genes coding for the enzymes of tryptophan to anthranilate pathway.
Appl. Microbiol. Biotechnol. 94, 719–728 CrossRef PubMed
23 Krissinel, E. and Henrick, K. (2007) Inference of macromolecular assemblies from
2
crystalline state. J. Mol. Biol. 372, 774–797 CrossRef PubMed
24 Bond, C. S. and Sch u¨ ttelkopf, A. W. (2009) ALINE: a WYSIWYG protein-sequence
alignment editor for publication-quality alignments. Acta Crystallogr. D Biol. Crystallogr.
65, 510–512 CrossRef PubMed
3
4
5
Stone, T. W., Stoy, N. and Darlington, L. G. (2013) An expanding range of targets for
kynurenine metabolites of tryptophan. Trends Pharmacol. Sci. 34, 136–143
CrossRef PubMed
Kurnasov, O., Goral, V., Colabroy, K., Gerdes, S., Anantha, S., Osterman, A. and Begley,
T. P. (2003) NAD Biosynthesis: identification of the tryptophan to quinolinate pathway in
bacteria. Chem. Biol. 10, 1195–1204 CrossRef PubMed
Wogulis, M., Chew, E. R., Donohoue, P. D. and Wilson, D. K. (2008) Identification of
formyl kynurenine formamidase and kynurenine aminotransferase from Saccharomyces
cerevisiae using crystallographic, bioinformatic and biochemical evidence. Biochemistry
25 Reference deleted
26 Holm, L. and Rosenstrom, P. (2010) Dali server: conservation mapping in 3D. Nucleic
Acids Res. 38, W545–W549 CrossRef PubMed
27 Hasegawa, H. and Holm, L. (2009) Advances and pitfalls of protein structural alignment.
Curr. Opin. Struct. Biol. 19, 341–348 CrossRef PubMed
28 Schenk, G., Miti c´ , N., Gahan, L. R., Ollis, D. L., McGeary, R. P. and Guddat, L. W. (2012)
Binuclear metallohydrolases: complex mechanistic strategies for a simple chemical
reaction. Acc. Chem. Res. 45, 1593–1904 CrossRef PubMed
29 The UniProt Consortium (2014) Activities at the universal protein resource (UniProt).
Nucleic Acids Res. 42, 191–198 CrossRef PubMed
47, 1608–1621 CrossRef PubMed
6
7
8
Han, Q., Robinson, H. and Li, J. (2012) Biochemical identification and crystal structure of
kynurenine formamidase from Drosophila melanogaster. Biochem. J. 446, 253–260
CrossRef PubMed
Fyfe, P. K., Oza, S. L., Fairlamb, A. H. and Hunter, W. N. (2008) Leishmania trypanothione
synthetase-amidase structure reveals a basis for regulation of conflicting synthetic and
hydrolytic activities. J. Biol. Chem. 283, 17672– 17680 CrossRef PubMed
Fyfe, P. K., Rao, V. A., Zemla, A., Cameron, S. and Hunter, W. N. (2009) Specificity and
mechanism of Acinetobacter baumanii nicotinamidase: implications for activation of the
front-line tuberculosis drug pyrazinamide. Angew. Chem. Int. Ed. 48, 9176–9179
CrossRef PubMed
30 Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., McGinnis, S. and Madden, T. L.
(2008) NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5–W9
CrossRef PubMed
31 Sievers, F., Wilm, A., Dineen, D. G., Gibson, T. J., Karplus, K., Li, W., Lopez, R.,
McWilliam, H., Remmert, M., S o¨ ding, J., Thompson, J. D. and Higgins, D. G. (2011) Fast,
scalable generation of high-quality protein multiple sequence alignments using Clustal
Omega. Mol. Syst. Biol. 7, 539 CrossRef PubMed
32 Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. and Barton, G. J. (2009)
Jalview version 2-a multiple sequence alignment editor and analysis workbench.
Bioinformatics 25, 1189–1191 CrossRef PubMed
9
0
Pace, H. C. and Brenner, C. (2001) The nitrilase superfamily: classification, structure and
function. Genome Biol. 2, 1–9 CrossRef PubMed
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D. and
Bairoch, A. (2005) Protein identification and analysis tools on the ExPASy serve. In The
Proteomics Protocols Handbook (Walker, J. M., ed.), pp. 571–607, Humana Press,
Totowa CrossRef
Katz, E., Brown, D. and Hitchcock, M. J. (1987) Arylformamidase from Streptomyces
parvulus. Methods Enzymol. 142, 225–234 CrossRef PubMed
Bougie, J. M. (2011) Expression, Purification, and Characterization of Kynurenine
Formamidase from Bacillus cereus. Masters Thesis, George Manson University, Fairfax,
VA, U.S.A.
Kabsch, W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132
CrossRef PubMed
Evans, P. R. (2006) Scaling and assessment of data quality. Acta Crystallogr. D Biol.
Crystallogr. 62, 72–82 CrossRef PubMed
Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan,
R. M., Krissinel, E. B., Leslie, A. G., McCoy, A. et al. (2011) Overview of the CCP4 suite
and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242
CrossRef PubMed
Leslie, A. G. W. and Powell, H. R. (2007) In Evolving Methods for Macromolecular
Crystallography (Read, R. J. and Sussman, J. L., eds), pp. 41–51, Springer, Dordrecht
CrossRef
33 Thomas, I. R., Bruno, I. J., Cole, J. C., Macrae, C. F., Pidcock, E. and Wood, P. A. (2010)
WebCSD: the online portal to the Cambridge Structural Database. J. Appl. Crystallogr. 43,
362–366 CrossRef PubMed
34 Holm, L. and Sander, C. (1997) An evolutionary treasure: unification of a broad set of
amidohydrolases related to urease. Proteins 28, 72–82 CrossRef PubMed
35 Seibert, C. M. and Raushel, F. M. (2005) Structural and catalytic diversity within the
amidohydrolase superfamily. Biochemistry 44, 6383–6392 CrossRef PubMed
36 Bebrone, C. (2007) Metallo-β-lactamases (classification, activity, genetic organization,
structure, zinc coordination) and their superfamily. Biochem. Pharmacol. 74, 1686–1701
CrossRef PubMed
37 Bigley, A. N. and Raushel, F. M. (2013) Catalytic mechanisms for phosphotriesterases.
Biochim. Biophys. Acta 1834, 443–453 CrossRef PubMed
38 Str a¨ ter, N., Sun, L., Kantrowitz, E. R. and Lipscomb, W. N. (1999) A bicarbonate ion as a
general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase.
Proc. Natl. Acad. Sci. U.S.A. 96, 11151–11156 CrossRef PubMed
39 Lee, M., Maher, M. L., Christopherson, R. I. and Guss, J. M. (2007) Kinetic and structural
analysis of mutant Escherichia coli dihydroorotases: a flexible loop stabilizes the
transition state. Biochemistry 46, 10538–10550 CrossRef PubMed
40 Huo, L., Fielding, A. J., Chen, Y., Li, T., Iwaki, H., Hosler, J. P., Chen, L., Hasegawa, Y.,
Que, L. Jr and Liu, A. (2012) Evidence for a dual role of an active site histidine in
alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase. Biochemistry
24, 5811–5821 CrossRef PubMed
1
1
1
1
2
1
1
1
3
4
5
1
6
1
1
7
8
Vagin, A. and Teplyakov, A. (1997) MOLREP: an automated program for molecular
replacement. J. Appl. Crystallogr. 30, 1022–1025 CrossRef
Murshudov, G. N., Skubak, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A.,
Winn, M. D., Long, F. and Vagin, A. A. (2011) REFMAC5 for the refinement of
macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367
CrossRef PubMed
41 Garavaglia, S., Perozzi, S., Galeazzi, L., Raffaelli, N. and Rizzi, M. (2009) The crystal
structure of human alpha-amino-beta-carboxymuconate-epsilon-semialdehyde
decarboxylase in complex with 1,3-dihydroxyacetonephosphate suggests a regulatory
link between NAD synthesis and glycolysis. FEBS J. 276, 6615–6623 CrossRef PubMed
42 Cruickshank, D. W. J. (1999) Remarks about protein structure precision. Acta Crystallogr.
D Biol. Crystallogr. 55, 583–601 CrossRef PubMed
1
9
Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K. (2010) Features and development of
Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 CrossRef PubMed
43 Engh, R. A. and Huber, R. (1991) Accurate bond and angle parameters for X-ray protein
structure refinement. Acta Crystallogr. A 47, 392–400 CrossRef
Received 17 April 2014/27 May 2014; accepted 19 June 2014
Published as BJ Immediate Publication 19 June 2014, doi:10.1042/BJ20140511
ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Biochem. J. (2014) 462, 581–589 (Printed in Great Britain) doi:10.1042/BJ20140511
SUPPLEMENTARY ONLINE DATA
Structures of bacterial kynurenine formamidase reveal a crowded binuclear
zinc catalytic site primed to generate a potent nucleophile
1
´
´
Laura DIAZ-SAEZ*, Velupillai SRIKANNATHASAN*, Martin ZOLTNER* and William N. HUNTER*
*Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
Figure S2 Anomalous difference Fourier map for BcKynB
The map, blue chicken wire, is contoured at 6σ. Zn2 and Cd are shown as grey and purple
+
2 +
spheres respectively. Water molecules and glycerol have been omitted for the purpose of clarity.
Figure S1 XANES spectra
K-edge XANES spectra from BaKynB (A) and PaKynB (B) showing the anomalous scattering
ꢀ
ꢀꢀ
factor f and f values within a range of X-ray energy (from 9626.7 to 9690.14 eV).
1
To whom correspondence should be addressed (email w.n.hunter@dundee.ac.uk).
Atomic co-ordinates and structure factors have been deposited in the PDB under codes 4COG, 4COB, 4CO9 and 4CZ1.
ꢁc The Authors Journal compilation ꢁc 2014 Biochemical Society

L. D ´ı az-S a´ ez and others
Figure S3 L-Kynurenine binding
Plot derived from fluorescence spectroscopy showing the percentage of active site saturation at different L-kynurenine concentrations.
Figure S4 2-Aminoacetophenone binding
Plot derived from fluorescence spectroscopy showing the percentage of active site saturation at different 2-aminoacetophenone concentrations.
ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Bacterial kynurenine formamidase
Figure S5 Omit difference density for 2-aminoacetophenone
The F omit map is shown as green chicken wire and contoured at 1.5σ.
− F
o
c
Received 17 April 2014/27 May 2014; accepted 19 June 2014
Published as BJ Immediate Publication 19 June 2014, doi:10.1042/BJ20140511
ꢀc The Authors Journal compilation ꢀc 2014 Biochemical Society

Copyright of Biochemical Journal is the property of Portland Press Ltd. and its content may
not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's
express written permission. However, users may print, download, or email articles for
individual use.