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Recombinant expression, purification and crystallographic studies of the mature form of human mitochondrial aspartate aminotransferase

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Mitochondrial aspartate aminotransferase (mAspAT) was recognized as a moonlighting enzyme because it has not only aminotransferase activity but also a high-affinity long-chain fatty acids (LCFA) binding site. This enzyme plays a key role in amino acid metabolism, biosynthesis of kynurenic acid and transport of the LCFA. Therefore, it is important to study the structure-function relationships of human mAspAT protein. In this work, the mature form of human mAspAT was expressed to a high level in Escherichia coli periplasmic space using pET-22b vector, purified by a combination of immobilized metal-affinity chromatography and cation exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 47.5ºC and a pH of 8.5. Crystals of human mAspAT were grown using the hanging-drop vapour diffusion method at 277K with 0.1 M HEPES pH 6.8 and 25%(v/v) Jeffamine(®) ED-2001 pH 6.8. The crystals diffracted to 2.99 Å and belonged to the space group P1 with the unit-cell parameters a =56.7, b = 76.1, c = 94.2 Å, α =78.0, β =85.6, γ = 78.4º. Elucidation of mAspAT structure can provide a molecular basis towards understanding catalysis mechanism and substrate binding site of enzyme.
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BioScience Trends Advance Publication P1
Recombinant expression, purification and crystallographic
studies of the mature form of human mitochondrial aspartate
aminotransferase
Xiuping Jiang1,*, Jia Wang1, Haiyang Chang1, Yong Zhou2
1 School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China;
2 School of Software, Dalian University of Technology, Dalian 116620, China.
1. Introduction
Aspartate aminotransferase (AspAT, EC 2.6.1.1) is
present as two homologous, genetically independent
isozymes in animal cells, one located in the cytoplasm
(cAspAT) and the other in mitochondria (mAspAT) (1,2).
mAspAT was recognized as a moonlighting enzyme
because it was found to have two or more different
functions. Similar to cAspAT, mAspAT catalyzes the
reversible reaction of L-aspartate and α-ketoglutarate
(α-KG) into oxaloacetate and L-glutamate via a ping-
pong mechanism, with pyridoxal 5'-phosphate (PLP) as
an essential cofactor (3). Glutamine not only provides a
carbon source to fuel the tricarboxylic acid (TCA) cycle
via α-KG, but also provides nitrogen for the synthesis of
nonessential amino acids and nucleotides, i.e., purines,
pyrimidines, alanine, serine, aspartate, ornithine,
glycine, cysteine, arginine, asparagine, and proline (4,5).
Therefore, mAspAT is one of the key enzymes that links
amino acid metabolism to carbohydrate metabolism
through catalysis of the reversible transamination
reaction.
Different to cAspAT, mammalian mAspAT is also
recognized as kynurenine aminotransferase-IV because
this enzyme is capable of catalyzing the irreversible
transamination of kynurenine to produce kynurenic
acid (KYNA) and plays a role in the biosynthesis of
KYNA in rat, mouse and human brains (6-8). KYNA is
an endogenous antagonist of N-methyl-D-aspartate and
α7-nicotinic acetylcholine receptors (9,10). In addition,
KYNA is identified as an endogenous ligand for an
orphan G-protein-coupled receptor (11 ). Abnormal
concentration of KYNA in brain tissue has been observed
in patients with mental and neurological disorders,
Summary Mitochondrial aspartate aminotransferase (mAspAT) was recognized as a moonlighting
enzyme because it has not only aminotransferase activity but also a high-affinity long-chain
fatty acids (LCFA) binding site. This enzyme plays a key role in amino acid metabolism,
biosynthesis of kynurenic acid and transport of the LCFA. Therefore, it is important to study
the structure-function relationships of human mAspAT protein. In this work, the mature form
of human mAspAT was expressed to a high level in Escherichia coli periplasmic space using
pET-22b vector, purified by a combination of immobilized metal-affinity chromatography and
cation exchange chromatography. Optimal activity of the enzyme occurred at a temperature
of 47.5ºC and a pH of 8.5. Crystals of human mAspAT were grown using the hanging-drop
vapour diffusion method at 277K with 0.1 M HEPES pH 6.8 and 25%(v/v) Jeffamine® ED-
2001 pH 6.8. The crystals diffracted to 2.99 Å and belonged to the space group P1 with the
unit-cell parameters a =56.7, b = 76.1, c = 94.2 Å, α =78.0, β =85.6, γ = 78.4º. Elucidation of
mAspAT structure can provide a molecular basis towards understanding catalysis mechanism
and substrate binding site of enzyme.
Keywords: Aspartate aminotransferase (AspAT), plasma membrane fatty acid binding protein
(FABPpm), kynurenine aminotransferase-IV, crystallization, moonlighting protein
DOI: 10.5582/bst.2015.01150
Brief Report
Released online in J-STAGE as advance publication February
22, 2016.
*Address correspondence to:
Dr. Xiuping Jiang, School of Life Science and Biotechnology,
Dalian University of Technology, Linggong Road No.2,
Dalian116024, China.
E-mail: xpjiang@dlut.edu.cn
Advance Publication
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BioScience Trends Advance Publication
including the Huntington's disease, Alzheimer's
disease, and schizophrenia (12). Therefore, mAspAT
can be envisioned to be a valid molecular target for the
treatment of these neurological diseases.
Additionally, mAspAT is recognized as plasma
membrane fatty acid binding protein (FABPpm)
because this enzyme has a high affinity for long-chain
fatty acids (LCFA) and is a key enzyme involved in
the transport of the saturated LCFA and unsaturated
LCFA (13,14). Accordingly, it is important to study the
structure-function relationships of mAspAT protein
because it possesses both enzymatic catalytic activity
and LCFA binding activity. To date, AspATs have been
purified from many sources and X-ray structures have
been determined for those from Escherichia coli (15),
cytosolic yeast cytoplasm (16), pig cytoplasm (17),
chicken mitochondria (18), and chicken cytoplasm (19).
However, the crystal structure of the human mAspAT
has not been solved.
Human mAspAT contains 4 cysteine residues forming
two disulfide bonds. In addition, the overexpression of
mAspAT is toxic to the growth of host cells as mAspAT
is a key metabolic enzyme in amino acid metabolism.
This study aimed to generate and purify recombinant
human mAspAT protein in a high level that is sufficient
for characterization analysis and further structural
studies. We report here the expression, purification and
characterization of human mAspAT expression in E. coli
periplasmic space using the plasmid pET-22b. Moreover,
the crystallization and preliminary X-ray analysis of
mAspAT protein were also performed.
2. Materials and Methods
2.1. Materials
The pET-22b (+) vector and E. coli strain BL21
(DE3) were obtained from Novagen (Beijing, China).
pMD18-T Simple Vector, E. coli strain JM 109, T4
DNA ligase, Ex Taq DNA polymerase, Noc I and Xho
I restriction enzymes were purchased from Takara
Biotechnology (Dalian, China). The Amicon Ultra
centrifugal filter (3 kDa) used for filtration of the
cell culture medium was obtained from Millipore
Corporation (Bedford, MA, USA). The AST (SGOT)
Reagent Kit used for monitoring the activity of
mAspAT was purchased from BIO QUANT (San
Diego, CA, USA). Crystallization Screening kits were
purchased from Hampton Research (Oklahoma City,
OK, USA). All primers were synthesized by Takara
Biotechnology.
2.2. Construct of expression vector
The nucleotide sequence encoding for the mature form
of human mAspAT (GenBank accession no. M22632.1)
was artificially synthesized by Taihe Biotechnology
Co., Ltd. (Beijing, China). Nco I and Xho I restriction
sites were added to the N-terminus and C-terminus of
the mAspAT sequence by PCR, respectively, using the
forward primer F1 5'-CATGCCATGGCAGAGCCAG
CTCCTGGTGGA-3' (Nco I site is underlined) and the
reverse primer R1 5'-CCGCTCGAGCTTGGTGACCT
GGTGAATGGCAT-3' (Xho I is underlined). The PCR
product was digested with Nco I-Xho I, and cloned into
the Nco I-Xho I site of the pMD18-T simple vector and
then transformed into E. coli strain JM 109. Nucleic
acid sequences of the cloning DNA fragment were
confirmed by DNA sequencing (BigDye™ Kit, Applied
Biosystems, USA) and ABI PRISM™ 3730XL DNA
Analyzer, according to the recommended protocols.
The target DNA fragment was further subcloned in the
same site of pET-22b (+) vector, resulting in pET22b-
hmAspAT. The resulting vector was then transformed
into chemically competent E. coli strain BL21 (DE3)
by heat shock for protein expression.
2.3. Expression of human mAspAT
The single colony of E. coli BL21 (DE3) harboring
the expression vector in 30 mL of Luria-Bertani (LB)
medium containing 100 μg/mL ampicillin, and then
cultivated at 37ºC until the optical density (OD600)
reached 0.6. The cells were harvested by centrifugation
at 4,000 × g for 10 min, and resuspended in 3 L
fresh LB medium containing 100 μg/mL ampicillin.
Subsequently, protein expression was induced with 1
mM isopropyl β--1-thiogalactopyranoside (IPTG) for
20 h at 16ºC. The cells were harvested by centrifugation
at 8,000 × g for 15 min and washed with buffer A (20
mM NaH2PO4, 0.5 M NaCl, pH 7.4). Approximated 45
g (wet weight) cells were obtained from 3 L culture.
After centrifugation, the cell pellets was resuspended
in 40 mL (for 1 L culture) ice-cold extraction buffer
A, and lysed by ultrasonication at ice-cold temperature
using an UP400S instrument (Dr. Hielscher GmbH,
Stuttgart, Germany). The cell lysis was centrifuged at
12,000 rpm for 15 min to separate soluble (supernatant)
and precipitated (pellet) fractions.
2.4. Purification of human mAspAT
The resulting supernatant was filtered with a 0.22 μm
syringe filter and then loaded onto a 5 mL HisTrapTM
FF crude column (GE Healthcare, Uppsala, Sweden)
pre-equilibrated with buffer A. After washing the
column with buffer A containing 50 mM imidazole,
the target protein was eluted with buffer B (20 mM
NaH2PO4, 0.5 M NaCl, 300 mM imidazole, pH 7.4).
The buffer was exchanged with 20 mM Tris buffer (pH
7.5) containing 20 mM NaCl using a 5 mL HiTrapTM
desalting column (GE Healthcare). The desalted
sample was loaded onto a 1 mL SP SepharoseTM FF
column (GE Healthcare). After washing, the column
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BioScience Trends Advance Publication P3
were then flash-cooled in liquid nitrogen. All X-ray
diffraction data were collected on the ESRF beamline
BL-17U at the Shanghai Synchrotron Radiation
Facility in China and were processed using the HKL-
2000 package (20). Diffraction data were collected at
a wavelength of 0.98 Å, an oscillation angle of 1º, an
exposure time of 1 s per image and a crystal-to-detector
distance of 250 mm.
3. Results and Discussion
Human mAspAT is encoded by nuclear gene and
synthesized in the cytoplasm as precursor protein
containing N-terminal presequences of 30 amino acid
residues in length (21). After completion of translation,
human mAspAT is imported into mitochondria
matrix via several apparently discrete steps including
proteolytic cleavage of the presequence with processing
proteases and assembly into mature protein (22,23).
Therefore, the mature form of human is post-
translationally imported into the mitochondrial matrix
and lack 30 amino acid residues from the N-terminus
compared with the precursor (24). The recombinant
expression results indicated that the precursor form of
mAspAT was found to express as inclusion body in E.
coli expression system, however, the mature could be
found in the supernatant of the bacterial homogenate
(25). Therefore the mature form of human mAspAT
was chosen to study the recombinant expression and
crystallization of protein.
Human mAspAT is a toxic protein to host cell and
also contains 4 cysteine residues forming two disulphide
bonds. In order to obtain the large amounts of soluble
mature mAspAT in E. coli for the crystallization study,
we introduced the use of pET-22b plasmid as the
expression vector. The pET-22b vector possessed an
N-terminal pelB secretion signal under the control of
the strong bacteriophage T7 promoter, which directed
the recombinant protein to the E. coli periplasmic space
(26). The periplasm of E. coli contains the disulfide
oxidoreductases and isomerases, which is an oxidizing
environment and can facilitate formation of disulfide
bonds that are always required for correct protein
folding (27,28). Therefore the periplasm of E. coli was
expected to be an ideal compartment for expression of
human mAspAT.
The plasmid pET22b- hmAspAT was then
transformed into E. coli BL21 (DE3). When the culture
was propagated at 37°C until OD600 reached 0.6, the
expression of the fusion protein was induced with 1.0
mM of IPTG, and then the culture was grown for an
additional 20 h at 16°C. After lysis, the supernatant was
analyzed by Western blotting using anti-his tag mouse
antibody. Western blot analysis confirmed that the
recombinant human mAspAT was present in the soluble
fraction of the cell lysate after induction with IPTG
(Figure 1A).
was eluted with a linear gradient of NaCl from 20 to
500 mM in Tris buffer (pH 7.5) at a flow rate of 1 mL/
min. Fractions with enzyme activity were pooled and
the buffer was exchanged for 20 mM Tris (pH 7.0)
containing 20 mM NaCl by using a 5 mL HiTrapTM
desalting column (GE Healthcare), and then the protein
was concentrated to a final concentration of 5 mg/mL
with a 3 kDa cut-off concentrator (Millipore). During
purification, the activity of mAspAT was monitored
using the AST (SGOT) Reagent Kit (BQ Kits). In this
method, a diazonium salt was used which selectively
reacted with the oxalacetate to produce a color complex
that was measured photometrically. According to the
protocol, reaction mixture including enzyme, substrate,
assay buffer and assay developer was incubated at 37°C
for 60 min, and OD450 was then measured. One unit
of enzyme is defined as the amount of enzyme which
generates 1.0 μmol of glutamate per minute at 37°C.
The purity of the eluted protein was analyzed by SDS–
PAGE and found to be > 95%.
2.5. SDS-PAGE and Western blot analysis
SDS-PAGE analysis was performed using 12%
resolving gel and 5% stacking gel. The protein bands
were visualized by Coomassie brilliant blue R-250 and
then analyzed by image-density analysis software (Gel-
Pro, USA). Soluble fractions of cell lysates after IPTG
induction were subjected to SDS-PAGE and transferred
to polyvinylidene difluoridemembrane (Millipore).
The membranes were blocked with 5% defatted milk
at room temperature for 60 min and then incubated
with anti-his tag mouse monoclonal antibody (1 μg/
mL, Millipore) at 4ºC overnight. The membranes were
washed twice with PBS buffer and incubated with
horseradish peroxidase conjugated goat anti-mouse IgG
(1:5,000, Millipore) at room temperature for 90 min.
Finally, the membranes were washed five times with
PBS buffer, and a 3′-diaminobenzidine kit was used
for color development (Sigma-Aldrich, St. Louis, Mo,
USA).
2.6. Crystallization and X-ray diffraction of human
mAspAT
Initial crystallization screening was carried out in 24-
well tissue-culture plates at 277 K by the hanging-drop
vapor diffusion method using commercially available
Index Screen, Crystal Screen, Crystal Screen 2 and
PEG/Ion Screen (Hampton Research). Crystals were
grown in a mixture containing 1 μL of protein (5 mg/
mL in 20 mM Tris pH 7.0, 20 mM NaCl) and 1 μL of
reservoir solution and were equilibrated against 400
μL reservoir solution. Crystallization conditions were
optimized based on the initial screening.
Single crystals were soaked for several minutes in
a reservoir solution containing 25%(v/v) glycerol and
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Recombinant human mAspAT was purified
by a combination of immobilized metal-affinity
chromatography and cation exchange chromatography.
The purified protein was resolved as a single band in
the SDS-PAGE gel with a molecular mass of about
43 kDa, indicating that these two chromatographic
steps were very effective (Figure 1B, lane 3). The final
yield of pure protein was approximately 10 mg from
1 L of expression culture, allowing the preparation of
approximately 2 mL of a 5.0 mg/mL protein sample for
crystallization assays. After two chromatography steps,
the specific activity and overall recovery of purified
mAspAT were 36.3 U/mg and 19.4%, respectively
(Table 1). The purification of mAspAT protein is
summarized in Table 1.
The optimal pH for enzyme activity was determined
to be pH 8.5 (Figure 2A), similar to that (pH 8.0) of
AspAT from Bacillus subtilis (29). Actually, the enzyme
activity over the pH range 8.0-10.0 was more than
60% of the maximum activity (Figure 2A). Moreover,
human mAspAT showed optimal activity at 47.5°C and
maintained over 80% activity at temperatures from 40
to 60°C, indicating a certain degree of thermostability
in this temperature range (Figure 2B). The recombinant
human mAspAT tended to have relatively high activity
and stability in alkaline environments, similar to that of
the AspATs from B. subtilis and B. circulans (29,30).
Thus the recombinant human mAspAT had optimal
Figure 1. Quality assessment of human mAspAT expression
and purification. (A) Western blot analysis of human mAspAT
expression using an anti-his tag mouse monoclonal antibody.
Lane 1, soluble fraction of the cell lysate after induction with
IPTG. (B) SDS–PAGE analysis of human mAspAT samples
during protein purification. Lane M, protein marker; lane 1,
soluble fraction after cell lysis; lane 2, elution fractions from
the HisTrapTM FF crude column; lane 3, elution fractions from
the SP SepharoseTM FF column.
Table 1. Purification efficiency of recombinant human
mAspAT
Purication step
Lysate supernatant
His Trap
SP Sepharose
Purication
(fold)
1
8.42
26.8
Specic activity
(U/mg)
0.574
12.5
36.3
Yield
(%)
100
21.9
19.4
Figure 3. Crystals and X-ray diffraction image of recombinant human mAspAT. (A) Crystals grown in 0.1 M HEPES pH 6.8
and 25%(v/v) Jeffamine® ED-2001 pH 6.8 at 277 K. (B) X-ray diffraction image of human mAspAT.
Figure 2. Effects of pH and temperature on activity of
purified recombinant human mAspAT. Enzyme activity
was determined at different pHs (A) and temperatures (B)
using the AST (SGOT) Reagent Kit.
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BioScience Trends Advance Publication P5
activity at pH 8.5 and 47.5°C.
Initial crystallization screening yielded a
crystallization hit under the condition No. 39 of the
Index Screen consisting of 0.1 M HEPES pH 7.0 and
30%(v/v) Jeffamine® ED-2001 pH 7.0. This condition
was subsequently optimized by testing 231 new
crystallization combinations with varying pH values from
6.5 to 7.5 using HEPES buffer and Jeffamine® ED-2001
concentrations from 20 to 40%(v/v). Finally, we obtained
an optimal composition for the reservoir solution of 0.1
M HEPES pH 6.8 and 25%(v/v) Jeffamine® ED-2001 pH
6.8 (Figure 3A).
The human mAspAT crystal data were collected to
a resolution of 2.99 Å (Figure 3B) and processed using
the HKL-2000 package (20). The crystals belonged to
the space group P1, with unit cell parameters a =56.7,
b = 76.1, c = 94.2 Å, α =78.0, β =85.6, and γ =78.4º.
The space group of cytosolic AspATs from chicken
(19), pig heart (17), and Saccharomyces cerevisiae (16)
is P212121, while the space group for AspAT crystals
from E. coli is P21 (15). Data collection statistics are
summarized in Table 2.
In conclusion, we constructed the expression
construct for human mAspAT and expressed it as an
active enzyme in E. coli periplasmic space. Moreover,
the recombinant human mAspAT was purified and
its some biochemical properties were also examined.
The crystals of mAspAT protein have been obtained
by the hanging-drop vapor-diffusion method and an
X-ray diffraction data set was collected from a single
crystal to 2.99 Å resolution. This study may provide
a method useful for the recombinant preparation of
other cytotoxic proteins. The structure of mAspAT
protein will provide insight into the structure reactivity
relationships of human mAspAT and its substrates. The
human mAspAT structure will be useful for screening
of its inhibitors and may also have implications for
future therapeutic approaches.
Acknowledgements
This study was supported by grants from the National
Natural Science Foundation of China (81102378,
61202252) and from the Specialized Research Fund for
the Doctoral Program of Higher Education of China
(20120041120052).
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Diraction source
Wavelength (Å)
Temperature (K)
Crystal-detector distance (mm)
Rotation range per image (°)
Total rotation range (°)
Exposure time per image (s)
Space group
a, b, c (Å)
α, β, γ (°)
Resolution range (Å)
Total No. of reections
No. of unique reections
Completeness (%)
Average I/σ (I)
Rmerge (%)*
Rmeas (%)**
BL-17U, ESRF
0.98
100
250
1
180
1
P1
56.7, 76.1, 94.2
78.0, 85.6, 78.4
2.99
578579
30161
97.2 (97.1)
4.3 (2.0)
0.083 (0.265)
0.117 (0.375)
* Rmerge = Σhkl Σi | Ii(hkl) - <I(hkl)> | / Σhkl Σi Ii(hkl).
** Rmeas = Σhkl{N(hkl) / [N(hkl)-1]}½Σi | Ii(hkl)- <I(hkl)> | / Σhkl Σi Ii(hkl),
where <I(hkl)> is the mean intensity of the N(hkl) observations Ii(hkl) of
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(Received November 6, 2015; Revised February 9, 2016;
Accepted February 11, 2016)
... To date, four members of this family have been reported [5]. Among them, Homo sapiens crystal structures of KAT-1 and 2 have been deposited on the Protein Data Bank (PDB) server [6][7][8][9][10][11][12], along with one human model, published in 2016, for KAT-4 [13]. For KAT-3, only a Mus musculus crystallographic model has been reported [14]. ...
... The reported optimum pH of mKAT-3 is nine [14,21]. KAT-4 is the last member of KATs, and though not extensively characterized yet, one crystal structure of hKAT-4 has been deposited in the PDB [13]. The biochemical conditions from rat brain KAT-4 proposed the optimum pH at around eight, which is almost consistent with the data for hKAT-4 with optimum activity at pH 8.5. ...
... The biochemical conditions from rat brain KAT-4 proposed the optimum pH at around eight, which is almost consistent with the data for hKAT-4 with optimum activity at pH 8.5. The range of α-ketoacids used in the studies was on rat and mouse KAT-4, and most of them were suitable; but the group the worked on hKAT-4 used α-ketoglutarate as a co-substrate alone [5,13]. ...
Article
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Kynurenine aminotransferase isozymes (KATs 1-4) are members of the pyridoxal-5'-phosphate (PLP)-dependent enzyme family, which catalyse the permanent conversion of l-kynurenine (l-KYN) to kynurenic acid (KYNA), a known neuroactive agent. As KATs are found in the mammalian brain and have key roles in the kynurenine pathway, involved in different categories of central nervous system (CNS) diseases, the KATs are prominent targets in the quest to treat neurodegenerative and cognitive impairment disorders. Recent studies suggest that inhibiting these enzymes would produce effects beneficial to patients with these conditions, as abnormally high levels of KYNA are observed. KAT-1 and KAT-3 share the highest sequence similarity of the isozymes in this family, and their active site pockets are also similar. Importantly, KAT-2 has the major role of kynurenic acid production (70%) in the human brain, and it is considered therefore that suitable inhibition of this isozyme would be most effective in managing major aspects of CNS diseases. Human KAT-2 inhibitors have been developed, but the most potent of them, chosen for further investigations, did not proceed in clinical studies due to the cross toxicity caused by their irreversible interaction with PLP, the required cofactor of the KAT isozymes, and any other PLP-dependent enzymes. As a consequence of the possibility of extensive undesirable adverse effects, it is also important to pursue KAT inhibitors that reversibly inhibit KATs and to include a strategy that seeks compounds likely to achieve substantial interaction with regions of the active site other than the PLP. The main purpose of this treatise is to review the recent developments with the inhibitors of KAT isozymes. This treatise also includes analyses of their crystallographic structures in complex with this enzyme family, which provides further insight for researchers in this and related studies.
... This interaction network revealed that IL4I1 links methionine and aspartic acid, thereby bridging the gap between cysteine and methionine metabolism and alanine, aspartate, and glutamate metabolism. These proteins mediate the reversible transamination from glutamate to oxaloacetate, generating α-ketoglutarate and aspartate [39]. As shown in Figure 3C, IL4I1 mRNA expression was analyzed by q-PCR, represented in the heatmap, and found at significantly decreased levels in HBL1R (p-value = 0.014) TMD8R (p-value = 0.0052) compared to wild-type HBL1 and TMD8, respectively. ...
... GOT1 and GOT2 are the respective cytoplasmic and mitochondrial varieties of glutamic-oxaloacetic transaminase enzymes ( Figure 6). They participate similarly to aspartate aminotransferases (AST) by catalyzing the reversible transamination of oxaloacetate and glutamate, producing aspartate and α-ketoglutarate [39]. The mitochondrial form of glutamic-oxaloacetic transaminase, GOT2, is a prominent player in the urea cycle and TCA cycle, particularly in the malate-aspartate shuttle [56]. ...
Article
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Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL). B-cell NHLs rely on Bruton’s tyrosine kinase (BTK) mediated B-cell receptor signaling for survival and disease progression. However, they are often resistant to BTK inhibitors or soon acquire resistance after drug exposure resulting in the drug-tolerant form. The drug-tolerant clones proliferate faster, have increased metabolic activity, and shift to oxidative phosphorylation; however, how this metabolic programming occurs in the drug-resistant tumor is poorly understood. In this study, we explored for the first time the metabolic regulators of ibrutinib-resistant activated B-cell (ABC) DLBCL using a multi-omics analysis that integrated metabolomics (using high-resolution mass spectrometry) and transcriptomic (gene expression analysis). Overlay of the unbiased statistical analyses, genetic perturbation, and pharmaceutical inhibition was further used to identify the key players contributing to the metabolic reprogramming of the drug-resistant clone. Gene-metabolite integration revealed interleukin four induced 1 (IL4I1) at the crosstalk of two significantly altered metabolic pathways involved in producing various amino acids. We showed for the first time that drug-resistant clones undergo metabolic reprogramming towards oxidative phosphorylation and are modulated via the BTK-PI3K-AKT-IL4I1 axis. Our report shows how these cells become dependent on PI3K/AKT signaling for survival after acquiring ibrutinib resistance and shift to sustained oxidative phosphorylation; additionally, we outline the compensatory pathway that might regulate this metabolic reprogramming in the drug-resistant cells. These findings from our unbiased analyses highlight the role of metabolic reprogramming during drug resistance development. Our work demonstrates that a multi-omics approach can be a robust and impartial strategy to uncover genes and pathways that drive metabolic deregulation in cancer cells.
... These proteins mediate the reversible transamination from glutamate to oxaloacetate, generating α-ketoglutarate and aspartate. [21] In turn, those metabolites form a direct interaction with 4-aminobutyrate aminotransferase (ABAT), linking alanine, α-ketoglutarate, and gamma-aminobutyric acid (GABA) into this drug-resistance mechanism. Lymphomas are generally considered glycolytic. ...
... They participate similarly to aspartate aminotransferases (AST) by catalyzing the reversible transamination of oxaloacetate and glutamate, producing aspartate and α-ketoglutarate. [21] The mitochondrial form of glutamic-(which was not certified by peer review) is the author/funder. All rights reserved. ...
Preprint
Background: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL). B-cell NHLs rely on Bruton tyrosine kinase (BTK) mediated B-cell receptor signaling for survival and disease progression. However, they are often resistant to BTK inhibitors or soon acquire resistance after drug exposure resulting in the drug-tolerant form. The drug-tolerant clones proliferate faster, have increased metabolic activity, and shift to oxidative phosphorylation; however, how this metabolic programming occurs in the drug-resistant tumor is poorly understood. Methods: In this study, we explored for the first time the metabolic regulators of ibrutinib-resistant activated B-cell (ABC) DLBCL using a multi-omics analysis that integrated metabolomics (using high-resolution mass spectrometry) and transcriptomic (gene expression analysis). Overlay of the unbiased statistical analyses, genetic perturbation, and pharmaceutical inhibition, was further used to identify the key players that contribute to the metabolic reprogramming of the drug-resistant clone. Results: Gene-metabolite integration revealed interleukin 4 induced 1 (IL4I1) at the crosstalk of two significantly altered metabolic pathways involved in the production of various amino acids. We showed for the first time that drug-resistant clones undergo metabolic reprogramming from glycolysis towards oxidative phosphorylation & is activated via the BTK-PI3K-AKT-IL4I1 axis and can be targeted therapeutically. Conclusions: Our report shows how these cells become dependent on PI3K/AKT signaling for survival after acquiring ibrutinib resistance and shift to sustained Oxidative phosphorylation, additionally we outline the compensatory, pathway that regulates this metabolic reprogramming in the drug-resistant cells. These findings from our unbiased analyses highlight the role of metabolic reprogramming during drug resistance development. Furthermore, our work demonstrates that a multi-omics approach can be a powerful and unbiased strategy to uncover genes and pathways that drive metabolic dysregulation in cancer cells.
... Mitochondrial AATM catalyzes the reversible reaction of aspartate and 2-OG to glutamate and oxaloacetate. AATM is a key enzyme, linking amino acid and carbohydrate metabolism by providing a carbon source (2-OG) to the TCA cycle as well as nitrogen for the synthesis of nonessential amino acids and nucleotides (88). Beyond its canonical function, mitochondrial AATM also functions as kynurenine aminotransferase as well as a transporter for long chain fatty acids (88,89). ...
... AATM is a key enzyme, linking amino acid and carbohydrate metabolism by providing a carbon source (2-OG) to the TCA cycle as well as nitrogen for the synthesis of nonessential amino acids and nucleotides (88). Beyond its canonical function, mitochondrial AATM also functions as kynurenine aminotransferase as well as a transporter for long chain fatty acids (88,89). AATM also interacts directly with the cardiolipin-containing liposomes, the inner mitochondrial membrane, as well as 2-OGDHC (90,91). ...
Article
Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin–protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.
... The mitochondrial aspartate aminotransferase (AATM) catalyzes the reversible reaction of aspartate and 2-OG to glutamate and oxaloacetate. AATM is a key enzyme linking amino acid and carbohydrate metabolism by providing a carbon source (2-OG) to the TCA cycle as well as nitrogen for the synthesis of non-essential amino acids and nucleotides [81]. Beyond its canonical function, mitochondrial AATM also functions as kynurenine aminotransferase as well as a transporter for long chain fatty acids [81,82]. ...
... AATM is a key enzyme linking amino acid and carbohydrate metabolism by providing a carbon source (2-OG) to the TCA cycle as well as nitrogen for the synthesis of non-essential amino acids and nucleotides [81]. Beyond its canonical function, mitochondrial AATM also functions as kynurenine aminotransferase as well as a transporter for long chain fatty acids [81,82]. AATM also interacts directly with the cardiolipin containing liposomes, the inner-mitochondrial membrane as well as the 2-OGDHC [83,84]. ...
Preprint
Full-text available
Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer the first glimpse of the protein interaction landscape of SS-31 and provide new mechanistic insight relevant to SS-31 mitochondrial therapy. Significance Statement SS-31 is a synthetic peptide that improves mitochondrial function and is currently undergoing clinical trials for treatments of heart failure, primary mitochondrial myopathy, and other mitochondrial diseases. SS-31 interacts with cardiolipin which is abundant in the inner mitochondrial membrane, but mechanistic details of its pharmacological effects are unknown. Here we apply a novel chemical cross-linking/mass spectrometry method to provide the first direct evidence for specific interactions between SS-31 and mitochondrial proteins. The identified SS-31 interactors are functional components in ATP production and 2-oxoglutarate metabolism and signaling, consistent with improved mitochondrial function resultant from SS-31 treatment. These results offer the first glimpse of the protein interaction landscape of SS-31 and provide new mechanistic insight relevant to SS-31 mitochondrial therapy.
... AATases from many species have been isolated and characterized, and the three-dimensional (3D) structures of AATases from various sources have been determined [6][7][8][9][10]. The structure and active site residues of the enzymes are well conserved. ...
... The AATase domain also possesses a conserved lysine residue (Lys237) that is required for the binding of cofactor PLP to form a Schiff base, via a covalent imino linkage between the e-amino group of the Lys residue and C-4' of the PLP cofactor. The conserved amino acids (Tyr64, Trp125, Asp202, Tyr204, and Arg245) in the active site were found in other AATases [9]. Grand average of hydropathicity value of the AATase was 0.239, and the protein was hydrophobic. ...
Article
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An aspartate aminotransferase (AATase) gene from Lactobacillus brevis CGMCC 1306 was cloned, which contains a 1182-bp open reading frame coding for 393 amino acids (41.43 kDa). When expressed in Escherichia coli BL21 (DE3), the recombinant AATase was purified and subsequently characterized. The recombinant AATase can catalyse the conversion of L-Asp to L-Glu, and the kcat/Km was determined to be 25.5 (mmol/L)⁻¹ s⁻¹ for L-Asp and 207.8 m(mol/L)⁻¹ s⁻¹ for α-ketoglutarate. With optimum temperature as 25 ˚C, the AATase may be a novel and special psychrophilic enzyme which exhibited a good thermal stability below 55 ˚C. The conserved active site residue of AATase was identified as Lys237 by phylogenetic analysis. Secondary structure of the enzyme includes α-helix (39.2%), β-sheet (5.5%), β-turn (8.8%), and random coil (36.5%) by circular dichroism spectral analysis. Phase diagram for the fluorescence data analysis showed that guanidinium chloride-induced unfolding of AATase involved at least one intermediate.
... The mRNA expression of a gene, GOT2, encoding for mitochondrial aspartate aminotransferase isoenzyme 2 was upregulated in the liver tissue of low-RFI steers. Mitochondrial aspartate aminotransferase is involved in several metabolic processes; the enzyme links amino acid metabolism to carbohydrate metabolism by catalyzing the reaction of L-aspartate and a-ketoglutarate to form oxaloacetate and L-glutamate, which both fuel the tricarboxylic acid cycle for ATP synthesis (Jiang et al., 2016). Mitochondrial aspartate aminotransferase also has high affinity for LCFA and is known to facilitate cellular transport of both saturated and unsaturated LCFA, a key step in energygenerating mitochondrial beta-oxidation of fatty acids (Roepstorff et al., 2004), which is in line with upregulation of fatty acid metabolism genes observed in this study. ...
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We evaluated the mRNA expression of genes involved in hepatic fatty acid, amino acid, and mitochondrial energy metabolism in crossbred beef steers with divergent low and high residual feed intake (RFI). Low-RFI beef steers (n = 8; RFI = - 1.93 kg/d) and high-RFI beef steers (n = 8; RFI = + 2.01kg/d) were selected from a group of 56 growing crossbred beef steers (average BW = 261 ± 18.5 kg) fed a high-forage total mixed ration after a 49-d performance testing period. At the end of the 49-d performance testing period, liver biopsies were collected from the low-RFI and high-RFI beef steers for RNA extraction and cDNA synthesis. The mRNA expression of 84 genes each related to fatty acid metabolism, amino acid metabolism, and mitochondrial energy metabolism were analyzed using pathway-focused PCR-based arrays. The mRNA expression of 8 genes (CRAT, SLC27A5, SLC27A2, ACSBG2, ACADL, ACADSB, ACAA1, and ACAA2) involved fatty acid transport and β-oxidation were upregulated (FC > 2.0, P < 0.05) in low-RFI, compared to high-RFI steers. Among those involved in amino acid metabolism, hepatic mRNA expression of a gene encoding for aminoadipate aminotransferase, an enzyme related to lysine degradation, was downregulated (FC = -5.45, P = 0.01) in low-RFI steers, whereas those of methionine adenosyltransferase I and aspartate aminotransferase 2, which both link amino acid and lipid metabolism, were upregulated (FC > 2, P < 0.05). Two mitochondrial energy metabolism genes (UQCRC1 and ATP5G1) involved in ATP synthesis via oxidative phosphorylation were upregulated (FC > 2; P < 0.05) in low-RFI beef steers, compared to high-RFI beef steers. The results of this study demonstrated that low-RFI beef steers exhibit upregulation of molecular mechanisms related to fatty acid transport, fatty acid β-oxidation, and mitochondrial ATP synthesis, which suggest that low-RFI beef steers have enhanced metabolic capacity to maximize capture of energy and nutrients from feeds consumed.
... We modeled the variants based on a template structure of the mature human GOT2 protein, solved as a homodimer complex at 3.0 Å resolution (PDB: 5AX8, chains A and C). 16 ...
Article
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Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.
... The crystal structures of hKAT I (Rossi et al., 2004;Han et al., 2009b;Nadvi et al., 2017), hKAT II (Han et al., 2008a,b;Rossi et al., 2008aRossi et al., , 2010Dounay et al., 2012Dounay et al., , 2013Tuttle et al., 2012;Nematollahi et al., 2016a), mKAT III (Han et al., 2009a;Wlodawer et al., 2018), and mKAT IV (Han et al., 2011) in their holo-forms, in different ligand-bound states and in complex with inhibitors, enormously expanded the ability to identify the structural determinants that are the basis for the common features and unique traits displayed by each KAT. More recently, the structure of the apo-form of mature human mitochondrial aspartate aminotransferase was solved (Jiang et al., 2016), however, considering the high percentage of sequence identity between human and mouse KAT IV (95%) and their structural conservation (root mean square deviation = 0.49 Å), only the murine isozyme will be discussed. ...
Article
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Kynurenic acid (KYNA) is a bioactive compound that is produced along the kynurenine pathway (KP) during tryptophan degradation. In a few decades, KYNA shifted from being regarded a poorly characterized by-product of the KP to being considered a main player in many aspects of mammalian physiology, including the control of glutamatergic and cholinergic synaptic transmission, and the coordination of immunomodulation. The renewed attention being paid to the study of KYNA homeostasis is justified by the discovery of selective and potent inhibitors of kynurenine aminotransferase II, which is considered the main enzyme responsible for KYNA synthesis in the mammalian brain. Since abnormally high KYNA levels in the central nervous system have been associated with schizophrenia and cognitive impairment, these inhibitors promise the development of novel anti-psychotic and pro-cognitive drugs. Here, we summarize the currently available structural information on human and rodent kynurenine aminotransferases (KATs) as the result of global efforts aimed at describing the full complement of mammalian isozymes. These studies highlight peculiar features of KATs that can be exploited for the development of isozyme-specific inhibitors. Together with the optimization of biochemical assays to measure individual KAT activities in complex samples, this wealth of knowledge will continue to foster the identification and rational design of brain penetrant small molecules to attenuate KYNA synthesis, i.e., molecules capable of lowering KYNA levels without exposing the brain to the harmful withdrawal of KYNA-dependent neuroprotective actions.
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Both immune regulation and endocrine systems are great challenges to marine organisms, and effective protocols for determining these adverse outcome pathways are limited, especially in vivo. The increasing usage of graphene nanomaterials can lead to the frequent exposure to marine organisms. Triphenyl phosphate (TPP), an organophosphate flame retardant, is frequently detected in natural environments. In this study, the combined toxic effects of co-exposure to graphene and TPP was investigated in Mytilus galloprovincialis using computational toxicology and multi-omics technology. Noticeably, graphene could disturb the membrane stability and increase the tissue accumulation of TPP. The adsorption behavior of TPP on graphene could inhibit the surface activity of graphene. In the digestive gland, transcriptomics analysis revealed the down-regulated genes in graphene + TPP treatment, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sorbitol dehydrogenase (SORD), glutathione s-transferase mu 3 (GSTM3) and 4-aminobutyrate aminotransferase (ABAT), were mainly associated with oxidative stress and energy metabolism. Moreover, metabolic responses indicated that graphene + TPP could cause disturbances in energy metabolism and osmotic regulation marked by differentially altered ATP, glucose and taurine in mussels. These data underline the need for further knowledge on the potential interactions of nanomaterials with existing contaminants in marine organisms.
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Production of recombinant proteins at high yields in Escherichia coli requires extensive optimization of expression conditions. Production is further complicated for proteins that require specific post-translational modifications for their eventual folding. One common and particularly important post-translational modification is oxidation of the correct pair of cysteines to form a disulfide bond. This unit describes methods to produce disulfide-bonded proteins in E. coli in either the naturally oxidizing periplasm or the cytoplasm of appropriately engineered cells. The focus is on variables key to improving the oxidative folding of disulfide-bonded proteins, with the aim of helping the researcher optimize expression conditions for a protein of interest. Curr. Protoc. Mol. Biol. 108:16.1B.1-16.1B.21. (c) 2014 by John Wiley & Sons, Inc.
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Kynurenic acid (KYNA) is an astrocyte-derived non-competitive antagonist of the α7 nicotinic acetylcholine receptor (α7nAChR) and inhibits the NMDA receptor (NMDAR) competitively. The main aim of the present study was to examine the possible effects of KYNA (30 - 1000 nm), applied locally by reverse dialysis for 2 h, on extracellular GABA levels in the rat striatum. KYNA concentration-dependently reduced GABA levels, with 300 nm KYNA causing a maximal reduction to ~60% of baseline concentrations. The effect of KYNA (100 nm) was prevented by co-application of galantamine (5 μm), an agonist at a site of the α7nAChR that is very similar to that targeted by KYNA. Infusion of 7-chlorokynurenic acid (100 nm), an NMDAR antagonist acting selectively at the glycine(B) site of the receptor, affected neither basal GABA levels nor the KYNA-induced reduction in GABA. Inhibition of endogenous KYNA formation by reverse dialysis of (S)-4-(ethylsulfonyl)benzoylalanine (ESBA; 1 mm) increased extracellular GABA levels, reaching a peak of 156% of baseline levels after 1 h. Co-infusion of 100 nm KYNA abolished the effect of ESBA. Qualitatively and quantitatively similar, bi-directional effects of KYNA on extracellular glutamate were observed in the same microdialysis samples. Taken together, the present findings suggest that fluctuations in endogenous KYNA levels, by modulating α7nAChR function, control extracellular GABA levels in the rat striatum. This effect may be relevant for a number of physiological and pathological processes involving the basal ganglia.
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Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ 2 ) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
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Disulfide bonds are covalent bonds formed post-translationally by the oxidation of a pair of cysteines. A disulfide bond can serve structural, catalytic, and signaling roles. However, there is an inherent problem to the process of disulfide bond formation: mis-pairing of cysteines can cause misfolding, aggregation and ultimately result in low yields during protein production. Recent developments in the understanding of the mechanisms involved in the formation of disulfide bonds have allowed the research community to engineer and develop methods to produce multi-disulfide-bonded proteins to high yields. This review attempts to highlight the mechanisms responsible for disulfide bond formation in Escherichia coli, both in its native periplasmic compartment in wild-type strains and in the genetically modified cytoplasm of engineered strains. The purpose of this review is to familiarize the researcher with the biological principles involved in the formation of disulfide-bonded proteins with the hope of guiding the scientist in choosing the optimum expression system.
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Aspartate aminotransferase (AAT) is a prototypical pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the reversible interconversion of L-aspartate and α-ketoglutarate with oxalacetate and L-glutamate via a ping-pong catalytic cycle in which the pyridoxamine 5'-phosphate enzyme form is an intermediate. There is a bountiful literature on AAT that spans approximately 60 years, and much fundamental mechanistic information on PLP dependent reactions has been gained from its study. Here, we review our recent work on AAT, where we again used it as a test bed for fundamental concepts in PLP chemistry. First, we discuss the role that coenzyme protonation state plays in controlling reaction specificity, then ground state destabilization via hyperconjugation in the external aldimine intermediate is examined. The third topic is light enhancement of catalysis of C-H deprotonation by PLP in solution and in AAT, which occurs through a triplet state of the external aldimine intermediate. Lastly, we consider recent advances in our analyses of enzyme multiple sequence alignments for the purpose of predicting mutations that are required to interconvert structurally similar but catalytically distinct enzymes, and the application of our program JANUS to the conversion of AAT into tyrosine aminotransferase.
Chapter
The kynurenine pathway is the main pathway of tryptophan metabolism. L-kynurenine is a central compound of this pathway since it can change to the neuroprotective agent kynurenic acid or to the neurotoxic agent quinolinic acid. The break-up of these endogenous compounds’ balance can be observable in many disorders. It can be occur in neurodegenerative disorders, such as Parkinson’s disease, Huntington’s and Alzheimer’s disease, in stroke, in epilepsy, in multiple sclerosis, in amyotrophic lateral sclerosis, and in mental failures, such as schizophrenia and depression. The increase of QUIN concentration or decrease of KYNA concentration could enhance the symptoms of several diseases. According to numerous studies, lowered KYNA level was found in patients with Parkinson’s disease. It can be also noticeable that KYNA-treatment prevents against the QUIN-induced lesion of rat striatum in animal experiments. Administrating of KYNA can be appear a promising therapeutic approach, but its use is limited because of its poorly transport across the blood-brain barrier. The solution may be the development of KYNA analogues (e.g. glucoseamine-kynurenic acid) which can pass across this barrier and disengaging in the brain, then KYNA can exert its neuroprotective effects binding at the excitatory glutamate receptors, in particular the NMDA receptors. Furthermore, it seems hopeful to use kynurenine derivatives (e.g. 4-chloro-kynurenine) or enzyme inhibitors (e.g. Ro-61-8048) to ensure an increased kynurenic acid concentration in the central nervous system.
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Molecular interactions are necessary for proteins to perform their functions. The identification of a putative plasma membrane fatty acid transporter as mitochondrial aspartate aminotransferase (mAsp-AT) indicated that the protein must have a fatty acid binding site. Molecular modeling suggests that such a site exists in the form of a 500-Å(3) hydrophobic cleft on the surface of the molecule and identifies specific amino acid residues that are likely to be important for binding. The modeling and comparison with the cytosolic isoform indicated that two residues (Arg201 and Ala219) were likely to be important to the structure and function of the binding site. These residues were mutated to determine if they were essential to that function. Expression constructs with wild-type or mutated cDNAs were produced for bacteria and eukaryotic cells. Proteins expressed in Escherichia coli were tested for oleate binding affinity, which was decreased in the mutant proteins. 3T3 fibroblasts were transfected with expression constructs for both normal and mutated forms. Plasma membrane expression was documented by indirect immunofluorescence before [(3)H]oleic acid uptake kinetics were assayed. The V(max) for uptake was significantly increased by overexpression of the wild-type protein but changed little after transfection with mutated proteins, despite their presence on the plasma membrane. The hydrophobic cleft in mAsp-AT can serve as a fatty acid binding site. Specific residues are essential for normal fatty acid binding, without which fatty acid uptake is compromised. These results confirm the function of this protein as a fatty acid binding protein.