Keywords aminotransferase; crystal structure; kynurenic acid; kynurenine aminotransferase; mosquito Correspondence J.. Received 2 February 2005, accepted 7 March 2005 doi:10.1111/j.1742-
Trang 1Qian Han1,*, Yi Gui Gao1,2,*, Howard Robinson3, Haizhen Ding1, Scott Wilson2and Jianyong Li1
1 Department of Pathobiology, University of Illinois, Urbana, IL, USA
2 School of Chemical Sciences, University of Illinois, Urbana, IL, USA
3 Biology Department, Brookhaven National Laboratory, Upton, NY, USA
Aedes aegypti kynurenine aminotransferase (AeKAT)
is a multi-function aminotransferase [1] Its
mamma-lian homolog, KAT-I can catalyze several amino
acids and many biologically relevant keto acids [2,3],
and is identical to glutamine transaminase K and
also a cysteine S-conjugate b-lyase [4–8] KAT-I is
present in the brain [9,10], and also in the kidney
and liver [11,12], which indicates the important role
of KAT-I in the bioactivation of environmental pol-lutants that contribute to liver- and kidney-associated carcinogenesis [2] Although the kidney and liver show much greater KAT activity than the brain, the emphasis of KAT research has been almost exclu-sively on enzymes in the CNS, paralleling the investi-gation into the pivitol role of kynurenic acid (KYNA) therein
Keywords
aminotransferase; crystal structure;
kynurenic acid; kynurenine
aminotransferase; mosquito
Correspondence
J Li, Department of Pathobiology,
University of Illinois, 2001 South Lincoln
Avenue, Urbana, IL 61802, USA
Fax: +217 2447421
Tel: +217 244–3913
E-mail: jli2@uiuc.edu
*Qian Han and Yi Gui Gao contributed
equally to this work.
Note
The atomic coordinates and structure
fac-tors (PDB codes 1YIZ and 1YIY) have been
deposited in the Protein Data Bank,
Research Collaboratory for Structural
Bioin-formatics, Rutgers University, New
Bruns-wick, NJ, USA (http://www.rcsb.org).
(Received 2 February 2005, accepted 7
March 2005)
doi:10.1111/j.1742-4658.2005.04643.x
Aedes aegyptikynurenine aminotransferase (AeKAT) catalyzes the irrevers-ible transamination of kynurenine to kynurenic acid, the natural antagonist
of NMDA and 7-nicotinic acetycholine receptors Here, we report the crys-tal structure of AeKAT in its PMP and PLP forms at 1.90 and 1.55 A˚, respectively The structure was solved by a combination of single-wave-length anomalous dispersion and molecular replacement approaches The initial search model in the molecular replacement method was built with the result of single-wavelength anomalous dispersion data from the Br-AeKAT crystal in combination with homology modeling The solved struc-ture shows that the enzyme is a homodimer, and that the two subunits are stabilized by a number of hydrogen bonds, salts bridges, and hydrophobic interactions Each subunit is divided into an N-terminal arm and small and large domains Based on its folding, the enzyme belongs to the prototypical fold type, aminotransferase subgroup I The three-dimensional structure shows a strictly conserved ‘phosphate binding cup’ featuring PLP-dependent enzymes The interaction between Cys284 (A) and Cys284 (B) is unique in AeKAT, which might explain the cysteine effect of AeKAT activity Further mutation experiments of this residue are needed to eventu-ally understand the mechanism of the enzyme modulation by cysteine
Abbreviations
AeKAT, Aedes aegypti kynurenine aminotransferase; KAT, kynurenine aminotransferase; KYNA, kynurenic acid; MAD, multiwavelength anomalous dispersion; PLP, pyridoxal 5-phosphate; PMP, pyridoxamine 5-phosphate; rmsd, root mean square deviation; SAD, single wavelength anomalous dispersion.
Trang 2KYNA is a metabolite in the tryptophan metabolic
pathway In mammals, it is synthesized by irreversible
transamination of kynurenine by KATs KYNA
repre-sents the only known endogenous antagonist of the
excitatory action of ionotropic excitatory amino acids,
showing the highest affinities for the glycine
modula-tory site of the NMDA subtype of glutamate receptor
[13–15] and the a7-nicotinic acetylcholine receptor [16–
18] In mammals, it protects the CNS from
oversti-mulation by excitatory cytotoxins [19,20] The
inhibi-tory actions of KYNA at excitainhibi-tory amino acid KAI
receptors underlie its neuroprotective [19–22] and
anticonvulsant effects [23,24] Fluctuations in
endo-genous brain KYNA levels significantly influence
neur-onal excitation and vulnerability to excitotoxic attack
[25–28] In addition, KYNA is also involved in
main-taining physiological arterial blood pressure [29–33]
The role of activity-dependent synaptic plasticity in
learning and memory is a central issue in neuroscience
Much of the relevant experimental work concerns the
possible role of long-term potentiation in learning
Most forms of long-term potentiation are
glutamater-gic and the most prominent form is induced following
activation of the NMDA receptor [34] KYNA, as the
only natural antagonist of NMDA, may, therefore, be
involved in the processes of memory and learning in
the CNS Savvateeva et al [35] demonstrated that the
mutant cardinal fly (3-hydroxykynurenine excess, local
KYNA level might be affected) shows a decline in
learning and memory, which implies a possible role for
KYNA in the formation of long-term potentiation
Direct evidence is, however, still missing The
physiolo-gical importance of KYNA has attracted a
consider-able amount of attention towards understanding the
molecular regulation of KYNA production in living
organisms KATs have become the target enzymes
when studying modulation of the KYNA level in a
number of pathological conditions in animals
A aegypti KAT (AeKAT) shares 45–50% sequence
identity with mammalian or human KAT-Is [36]
Functional characterization of its recombinant protein,
expressed in a baculovirus⁄ insect cell-expression
sys-tem, showed that the protein is active to kynurenine
[36] The protein showed high activity towards many
biologically relevant keto acids Interestingly, most
keto acids showed substrate inhibition at relatively
high concentrations Cysteine had an intriguing effect
on the enzyme activity towards kynurenine, inducing
enhancement at relative low concentrations and
inhibi-tion at higher concentrainhibi-tions [1] Moreover, AeKAT is
mainly expressed in adult heads, indicating its major
function in the CNS [36] A bacterial homolog and
human KAT-I have been systematically characterized
using their respective recombinant enzymes [3,37], and both three-dimensional structures have recently been solved [38,39] The biochemical comparison of the three enzymes has been discussed previously [3] To under-stand the catalytic mechanism and structural basis underlying these biochemical differences, it is essential that the three-dimensional structure of AeKAT is deter-mined and a comparative study with mammalian KATs
is carried out Here, we provide data that describe the crystal structure of AeKAT as obtained using macro-molecular crystallography
Results and Discussion
Crystallization, single wavelength anomalous dispersion modeling, and homology modeling Single wavelength anomalous dispersion (SAD) diffrac-tion data for a Br-AeKAT (PMP form) derivative were collected at cryogenic temperature at X12C at the National Synchrotron Radiation Source in Brookha-ven National Laboratory (BNL) (k ¼ 0.97 A˚) The Br-AeKAT (PMP form) crystal has an orthorhombic unit cell with parameters of a¼ 55.34 A˚, b ¼ 95.32 A˚,
c¼ 167.67 A˚, and diffracts to 1.90 A˚ resolution The space group has been determined as P212121 from an auto-index using gadds program Two molecules of AeKAT are in an asymmetric unit, based on calcula-tion of the Matthews coefficient [40] SAD diffraccalcula-tion data of the Br-AeKAT (native⁄ PLP form) derivative were collected in the same manner (k¼ 1.1 A˚) The PLP form crystal has an orthorhombic unit cell with parameters of a¼ 55.28 A˚, b ¼ 94.98 A˚, c ¼ 167.60 A˚ and diffracts to 1.55 A˚ resolution (Table 1)
An initial atomic model with 520 residues of two molecules was obtained based on the SAD data of Br-AeKAT crystals with a resolution at 1.90 A˚ (Fig 1A) Because the SAD model has only 60% residues assigned, we turned to a molecular replacement method using homology modeling The first model was built by homology modeling using the initial SAD model and two homology structures (PDB codes: 1gck and 1v2d)
as template structures Briefly, SAD coordinates were assigned to target residues of AeKAT, the structures of other residues were built based on two search models Loop areas were highly optimized to target protein Ae-KAT using the program insight ii (Accelrys) In total
100 models were built, 10 of which were used in the ini-tial refinement tests based on the procheck [41] results Only one model (Fig 1B) was used in further refine-ment using shelx-97, o and x-plor This strategy enabled us to solve the three-dimensional structure of AeKAT Figure 1 shows the initial SAD model (A), a
Trang 3homology model (B) and the final refined structure of
the PMP form (C) of AeKAT
Overall structure
The PMP form of AeKAT was solved at a resolution of
1.90 A˚ The three-dimensional structure of the AeKAT
PLP form was solved using its PMP structure as an ini-tial refinement model by shelx-97 and x-plor at a reso-lution of 10 to 1.55 A˚ A homodimer is present in the asymmetric unit Excellent electron density allowed the modeling of 418 of 429 residues, 2 PLPs and 441 sol-vent molecules in the PLP form structure Both forms have the same structure except that there is no bond formed between the cofactor and Lys255 in the PMP form The stereochemistry of the model was assessed using procheck [41] In both the PLP and PMP forms, 87% of the residues were in the most favored regions of the Ramachandran plot Although Tyr286 (A) and (B)
in both forms fall within a disallowed region of the Ramachandran plot of the solved structures, the excel-lent electron density allowed us to unambiguously assign the observed conformation
The protein architecture revealed by AeKAT con-sists of the prototypical fold of aminotransferases sub-group I [42,43], characterized by an N-terminal arm, and a small and a large domain (Fig 2) The N-ter-minal arm consists of a random coiled stretch made up
of residues 12–26, the small domain (residues 27–52 and 310–429) folds into a five-stranded parallel and antiparallel b sheet surrounded by four a helices The large domain (residues 53–309) adopts an a⁄ b structure that resembles the Rossmann fold, which shows con-served a⁄ b topology, in which a sharply twisted seven-stranded b-sheet inner core is nested into a conserved array of nine a helices which are contributed by both the interior and external of the molecule
As observed in other subgroup I aminotransferases, the functional unit of AeKAT consists of a homodimer with subunits related by a dyad axis to its two active sites located at the domain interface in each subunit, and at the subunit interface in the dimer (Fig 2) Many hydrogen bonds are formed between two sub-units, i.e Lys17–Gln119 (2.63, 2.68 A˚), Lys17–Val122 (2.69, 2.89 A˚), Asn71–Trp262 (2.89, 2.95 A˚), Asn71– Gly261 (2.87, 2.95 A˚), Trp72–Thr260 (2.52, 3 A˚),
Fig 1 Line ribbon representation of initial SAD model, homology model and final refined model (A) Initial SAD model (B) Homology model (C) Final refined model of the PMP form The molecules are viewed down the molecular twofold axis The two subunits are colored blue and green, respectively.
Table 1 Crystal parameters, data collection and refinement
statis-tics of AeKAT.
Unit cell (a, b, c) 55.28, 94.98,
167.60
55.34, 95.32, 167.67
R merge (%) 5.7 (29.1) 6.3 (19.6)
Refinement
All-atom RMS fit for
the two chains (A ˚ )
Ca-only RMS fit
for the two chains (A ˚ )
No of solvent molecules (water) 441 440
Average B factor main chain (A˚2 ) 18.43 18.26
Average B side chain (A˚2 ) 23.24 23.78
Average B over all (A ˚ 2 ) 20.82 20.99
Average B factor PLP ⁄ PMP (A˚ 2 ) 15.57 12.41
Average B factor
solvet (water) (A˚2)
Trang 4Trp72–Ser258 (2.84, 2.95 A˚), Tyr73–Thr250 (2.87,
3.9 A˚), Tyr111–Gln282 (2.79, 2.95 A˚), two Tyr115
(2.57 A˚), and Gly261–Thr290 (2.78, 2.85 A˚) of the
opposite subunits in PLP form Several hydrophobic
interactions participate in the stabilization of the
homodimer, in particular, the interactions between
Phe46 and Leu69 (3.57, 4.39 A˚) and the two Val108
(4 A˚) of the opposite subunits Moreover, the
N-ter-minal arms also contribute towards the stability of
the AeKAT dimer Two salt bridges are established
between Asp112 and Lys6 (3.96, 4.15 A˚) and Asp112
and Arg7 (3.48, 3.55 A˚) of the opposite subunits In
AeKAT structures, Cys284 (A) and Cys284 (B) with
distances of 3.46 A˚ (PLP form) and 3.57 A˚ (PMP
form) form a thiol–thiolate hydrogen bond, which is
unique in AeKAT (Fig 5B)
The AeKAT active site
Similar to human KAT-I, AeKAT possesses two active
sites located around the local dyad axis Each active
site contains one PLP molecule and is hosted in a deep
cleft at the domain interface made up of residues from
both subunits (Fig 2) Both PLP and PMP can be
identified with confidence, with clear electronic density
maps of the structures (Fig 3) Each PLP cofactor sits
within a binding pocket defined by two regions
contri-buted by residues from the large domains of both
sub-units (Fig 2) The bottom of the PLP-binding pocket
is entirely defined by residues from the large domain
of the corresponding monomer With the exception of
Lys255 and Gly110, all these residues are at, or close
to, the edge of the inner core b sheet, pointing toward the domain interface and facing the re-face of the PLP ring Distinct arrays of residues form the lateral walls
Fig 2 Stereo ribbon representation of the
AeKAT molecule The small and large
doma-ins of one subunit are given in green and
blue, respectively Both N-terminal arms are
grey, and other subunit is red The cleft
hos-ting the enzyme active site can be seen at
the domain interface where the PLP
cofac-tor is shown as a ball-and-stick The
C-termi-nus and N-termiC-termi-nus are indicated as C-ter
and N-ter, respectively.
Fig 3 Diagrams of 2Fo– Fc electron density maps for the active sites of the PMP and PLP forms The map contoured at 2.0 sigma
is calculated using data between 10.0 and 1.90 A ˚ and 10.0 and 1.55 A ˚ resolution for the PMP and PLP forms of AeKAT, respect-ively (A) PLP form; (B) PMP form.
Trang 5of the PLP-binding pocket In particular, the stretch
43–46, together with the side chains of Arg405,
Asn189 and Asn193 of the large domain, make up one
pocket wall The opposite wall consists of residues
Tyr111 and Tyr138 of the same monomer and Phe286,
His287 and Tyr73 of the other subunit
In the PLP form, AeKAT carries the PLP molecule
covalently bound in the active site by Schiff base
link-age to the catalytic Lys255 (Figs 3B and 4A), resulting
in the formation of an internal aldimine bond (C4a¼
Nz) with an angle of 105.5 at the pyridine ring of
PLP Several other residues contact the PLP molecule
and participate in its recognition and binding The
phosphate group of the cofactor is engaged in a
num-ber of interactions with residues making up the strictly
conserved ‘PLP-phosphate binding cup’, featuring
PLP-dependent enzymes [44] In particular, its OP1
oxygen forms a set of hydrogen bonds with Ser252
(2.97 A˚), Ala110 (with its backbone nitrogen atom at
2.78 A˚) and the solvent molecule W120 at a distance
of 2.82 A˚ PLP OP2 forms a hydrogen bond with
Tyr73 (B) (in other subunit, 2.55 A˚) and the solvent
molecule W20 (2.73 A˚) Finally, the PLP OP3 atom
interacts with Lys263 (at 2.73 A˚ from the Nz atom)
and with the backbone nitrogen atom of Tyr111 (at
2.87 A˚) The PLP phenolic oxygen is held in place by
interactions with Tyr224 (at 2.56 A˚ from its OH atom)
and Asn193 (at a distance of 2.62 A˚ with its OD1
atom) Moreover, the N1 atom of the PLP pyridine moiety forms hydrogen bonds with the carboxylic oxy-gen atoms of Asp221 (at 2.66 A˚) Several hydrophobic interactions further stabilize PLP In particular, Phe135 and Val223 surround the anthranilic moiety
of the cofactor, on its si- and re-face, respectively (Fig 4A) In the PMP form, there is no internal aldi-mine bond between Lys255 and PMP and the distance between PMP amine and Lys255 Nz is 3.45 A˚ (Fig 3B) The interactions in the active site are similar
to the PLP form (Figs 3A and 4B)
It is interesting to find a thiol–thiolate hydrogen bond formed between two subunits in AeKAT, which might be a target for understanding AeKAT regula-tion Changes in the sulfur oxidation state of cysteine residues influence the activity of many proteins [45,46] Reversible disulfide bond formation and the associated conformation changes are likely to play an important role in cellular redox regulation In particular, disulfide bond formation between distant cysteines may be an effective mechanism for the induction of conformatio-nal changes that lead to switches in protein activity [47–49] In human mitochondrial branched chain aminotransferase, the redox-active dithiol⁄ disulfide Cys315-Xaa-Xaa-Cys318 center has been proposed in the regulation of enzyme activity Cys315 appears to
be the sensor for redox regulation of the enzyme activ-ity, whereas Cys318 acts as the ‘resolving cysteine’, allowing for reversible formation of a disulfide bond [50,51] AeKAT has a similar sequence, Cys284-Xaa-Xaa-Xaa-Cys288 (Fig 5A), but there is no
thiol–thio-Fig 5 Putative cysteine regulation site of AeKAT (A) Partial align-ment result of human KAT-I and AeKAT (B) Diagrams of 2Fo) F c
electron density maps for Cys284 (A) and Cys284 (B) and surround-ing residues The map was contoured at 2.0 sigma.
Fig 4 Schematic diagram showing active site interactions in AeKAT.
Hydrogen bonds are shown by dotted lines Phe135 and Val223
sandwich the pyridine ring of PLP (A) PLP form; (B) PMP form.
Trang 6late hydrogen bond between the two cysteine residues
(9.4 A˚) in the AeKAT structure To understand the
mode of enzyme regulation by cysteine, we
comparat-ively analyzed human KAT-I and AeKAT First,
AeKAT activity can be stimulated by cysteine [1],
whereas the human homolog is not positively affected
by cysteine [3] Second, sequence alignment showed
that AeKAT had two additional cysteine residues
com-pared with human KAT-I, as indicated in Fig 5A
(green background), the equivalent of Cys284 in
AeKAT is Ser276 in the human enzyme Third, there
are no thiol–thiolate hydrogen bonds or disulfide
bonds in the human KAT-I structure [39], whereas in
the AeKAT structure, Cys284 is located in the large
domain of the homodimer, the structure shows
evi-dence of a thiol–thiolate hydrogen bond between
Cys284 (A) and Cys284 (B) (Fig 5B), which is close to
the active center Under oxidizing conditions, these
cysteine residues in AeKAT can reasonably form a
disulfide bond because of the short distance between
the sulfur atoms (3.46 A˚ in the PLP form and 3.57 A˚
in the PMP form), requiring a decrease of only
1.5–1.6 A˚ Thus, residue Cys284 is most likely the
cys-teine-regulation target of AeKAT Further mutation
experiments of this residue along with biochemical and
structural analyses are needed to eventually understand
the mechanism of the enzyme modulation by cysteine
Experimental procedures
Expression and purification of recombinant
AeKAT
AeKAT lacking the N-terminal mitochondrial leader
sequence (amino acids 1–48) was expressed in a
baculo-virus⁄ insect cell protein expression system, and purified by
DEAE Sepharose, phenyl Sepharose, hydroxyapatite, and
native PAGE separation (PMP form) or gel filtration
(PLP⁄ native form) [36] To begin with, we did not pay
attention to the enzyme forms, and obtained only the PMP
form of AeKAT, because the running buffer for native
PAGE has 192 mm glycine Later, when we used gel
filtra-tion as the last step of purificafiltra-tion, we obtained the native
form of AeKAT The proposed reaction for
aminotrans-ferases is shown in Scheme 1 The purity of the protein was
assessed by SDS⁄ PAGE analysis Protein concentration was determined by a BioRad protein assay kit using bovine serum albumin as a standard The purified recombinant AeKAT was concentrated to 10 mgÆmL)1 protein in 5 mm phosphate buffer, pH 7.5 using a Centricon YM-30 concen-trator (Millipore, Billerica, MA, USA)
AeKAT crystallization
Initial crystallization screening was performed using Hamp-ton Research Crystal Screens (HampHamp-ton Research, Laguna Niguel, CA, USA) with sitting-drop and hanging-drop vapor diffusion methods with the volume of reservoir solu-tion at 500 lL and the drop volume at 5 lL, containing 2.5 lL of protein sample and 2.5 lL of reservoir solution AeKAT crystals were obtained in a solution containing
10 mgÆmL)1of protein, 30% (w⁄ v) PEG 1000, and 0.1 m of Tris⁄ HCl at pH 8.5 Refinement of preliminary crystalliza-tion condicrystalliza-tions resulted in the growth of quality crystals in
a solution containing 5 mgÆmL)1 protein, 15% (w⁄ v) PEG 1000, and 0.1 m Tris⁄ HCl at pH 8.5 Single crystals for suitable X-ray analysis appeared in 4 days and grew to maximum sizes of 0.5· 0.3 · 0.2 mm3
in 3 weeks at 4C
Data collection and processing
To solve the AeKAT structure through multiwavelength anomalous diffusion (MAD) [52] and single-wavelength anomalous diffusion (SAD) [53], we first tried l-seleno-methionine-labeled AeKAT, but failed to obtain quality diffraction data from its crystals Subsequently, the Br-AeKAT derivative was generated by soaking AeKAT crystals in 1 m NaBr for 5–10 s followed by transferr of the NaBr-AeKAT crystals to a cryoprotectant solution containing mother liquid [1 m NaBr, and 25% (v⁄ v) glyc-erin in Tris⁄ HCl pH 8.5] for 30–60 s [54] For X-ray ana-lyses, oscillation diffraction images of Br-AeKAT were obtained using a Bruker General Area Detector Diffrac-tion System (Madison, WI, USA) equipped with a four-circle diffractometer and a HiStar multiwire area detector Individual AeKAT crystals were frozen using 10% sucrose plus 30% PEG 400 as a cryoprotectant solution
in order to prevent the appearance of ice diffraction dur-ing data collection at cryogenic temperatures Diffraction data for Br-AeKAT crystals were collected at the Brook-haven National Synchrotron Light Source X12C beamline (wavelengths k¼ 0.97 A˚) Single crystals were exposed to
a cold nitrogen stream during data collection using a MAR research 165 mm CCD detector The PEG 400, sucrose, or glycerin provided sufficient cryoprotection dur-ing data collection All data were auto-indexed and integ-rated using hkl software [55], scaling and merging of diffraction data was performed using scalepack [56] The parameters of crystal and data collection are listed in Table 1
Trang 7Structure determination
Initial phases for the PMP crystal form were obtained from
a SAD experiment The structure of the PMP form was
determined by the molecular replacement method with the
homology module in insight ii (Accelrys) using the partial
SAD model and two search models, Thermus thermophylus
aspartate aminotransferase (Protein Data Bank code 1gck)
[57] and glutamine aminotransferase (Protein Data Bank
code 1v2d) [38] as template structures The program amore
[58] was used to calculate both cross-rotation and
transla-tion functransla-tions in the 10–3.0 A˚ resolutransla-tion range The initial
model was subjected to iterative cycles of crystallographic
refinement with the programs x-plor [59], shelx-97 [60]
and with graphic sessions for model building using the
pro-gram o [61] A random sample containing 1000 reflections
was set apart to calculate the free R-factor [62] Solvent
molecules were manually added at positions with density
> 1.5 sigma in the 2Fo) Fc map, considering only peaks
engaged in at least one hydrogen bond with a protein atom
or a solvent atom The procedure converged to an R-factor
and free R-factor of 0.218 and 0.264, respectively, with
ideal geometry Residues of the two subunits in AeKAT
are numbered 12 (A) to 429 (A) and 12 (B) to 429 (B),
respectively The results of refinement are summarized in
Table 1
Acknowledgements
We thank Dr John M Sanders, Department of
Chem-istry, University of Illinois at Urbana-Champaign, for
his help in our homology model building using Insight
II This work was supported by Grant AI 44399 from
the National Institutes of Health and the work was
carried out in part at the National Synchrotron Light
Source, Brookhaven National Laboratory
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