Mitochondrial aspartate aminotransferase (3PDB) from Mus musculus
Created by: Yiqing Wang
Mammalian mitochondrial aspartate aminotransferase from Mus musculus (PDB ID: 3PDB) is a type of aspartate aminotransferase (AspAT). Mitochondrial aspartate aminotransferase (mAspAT) has kynurenine aminotransferase (KAT) activity. KAT I, II, III and IV are involved in kynurenic acid (KYNA) biosynthesis in the central nervous system. KYNA is an antagonist of the α7-nicotinic acetylcholine receptor and the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. These glutamate receptors are responsible for excitatory synaptic transmission and synaptic plasticity in mammalian brains. If the activity of glutamate receptors is increased uncontrollably, neurons may die, thus causing neurological and psychiatric diseases (1). The variations in brain KYNA levels can protect neurons by controlling these over-stimulations through glutamatergic and cholinergic neurotransmission, but unbalanced KYNA level may contribute to the progress of several neurological and psychiatric diseases, such as schizophrenia, Alzheimer’s and Huntington’s disease (2, 4). mAspAT from rat and human brains is essential in endogenous synthesis and regulation of KYNA by catalyzing the irreversible transamination of the L-tryptophan L-kynurenine to KYNA. Studying the function and structure of mAspAT may contribute to the medical treatment of diseases related to KYNA level abnormality (1, 3).
Mus musculus mAspAT is crystalized using the hanging-drop vapor diffusion method. It is co-crystalized with kynurenine and with oxaloactetate, a substrate and a co-substrate of the mAspAT, respectively (1). Mus musculus mAspAT contains two identical homo-dimers. Subunit A and C form one homo-dimer, and subunit B and D form the other. The Protein Data Bank shows that the four subunits A, B, C and D are identical. Each subunit contains 401 amino acid residues, fifteen α-helices, two 3/10 α-helices and thirteen β-sheets. The rest of the protein is random coils (5). The extensive hydrogen bondings in the secondary structure of the protein stabilize the protein structure. The results from ExPASY compute theoretical isoelectric point (pl)/ molecular weight (Mw) tool show that mAspAT has a Mw of 47411.38 Da and an isoelectric point of 9.13, indicating that the protein has a net positive charge in neutral pH (6). The hydrophilic and ionic interaction abilities of basic amino acid residues that bring the net positive charge to the protein enhance the protein’s ability to bind to substrates. At the active sites of the protein, basic residues like lysine and arginie contribute to the binding of substrates.
mAspAT is a pyridoxal-5'-phosphate (PLP) dependent enzyme that primarily catalyzes the reversible transamination of L-aspartate and 2-oxoglutarate to oxaloacetate and L-glutamate (10, 13). The PLP molecule shown here is from Gallus gallus mAspAT (PDB ID: 7AAT), because the crystalized PLP ligand in Mus musculus mAspAT is in amino form, PMP. The oxaloacetate product is an important intermediate in metabolic processes like gluconeogenesis. The PLP serves as a coenzyme in this catalytic cycle by alternating between PLP and PMP form, which is the amino form of PLP (9). The reactions involved in the catalytic cycle is shown below:
L-Aspartate
+ PLP-enzyme ↔ Oxalacetate + PMP-enzyme;
2-Oxoglutarate + PMP-enzyme ↔ L-glutamate +
PLP-enzyme
Each
subunit of the protein consists of an N-terminal arm (residues 30-42), a large domain (residues 76-348) and a small domain (residues 43-75, 349-430). The active site is
located at the interface between the large and small domains. Each interface binds one molecule of PLP with a total of two molecules of PLP bind to the mAspAT. The PLP shown in the molecular document is
in its amino form PMP. In one subunit of mAspAT, Tyr-246 and Asp-243 form hydrogen bonding with the O3 and N1 molecules of the PLP and Ala-245 and Trp-162 form hydrophobic interaction with the PLP pyridine ring.
The NZ atom of Lys-279 forms covalent bond with
the C4A atom of PLP, resulting in a new residue LLP-279 containing an Schiff base, an internal aldimine. The phosphate group of
PLP that involves in hydrophilic interaction with Thr-135, Ser-133, Ser-276, Arg-287 and Tyr-96 from the other subunit fixes the position of the PLP coenzyme (1, 10). The incoming kynurenine substrate that
contains amine group displaces the lysine e-amino group from the Schiff base
and forms a new external aldimine with lysine (12). The strong interactions between the large number of amino acid residues in the active site and PLP molecule indicate strong binding of PLP molecule to the protein. The binding of the cofactor
PLP in mAspAT facilitates the binding of substrate kynurenine and the KAT
activity of the protein. If mutation occurred at important residue in the
active site of PLP, the enzymatic activity of mAspAT would be affected
significantly.
Studies have shown that a single substitution of active-site residues in mAspAT could alter its activity and impede substrate specificity significantly. One experiment shows that after the Trp-140 residue is changed to histidine in the AspAT of Escherichia coli (eAspAT), PLP dissociates 50 times more rapidly from the mutant protein than from the wild-type protein, because unlike the aromatic ring in tryptophan side chain, the imidazole ring of His-140 does not form a π-π stacking interaction with the pyridine ring of PLP. Another experiment shows that substituting Trp-140, Ile-17 and Val-37 residues by histidine in Gallus gallus mAspAT results in decreased aminotransferase activity toward aromatic amino acids by a fraction of 10 to 100 (11). Similarly, if mutation occurred at Trp-162 in Mus musculus mAspAT, the binding of PMP would be impeded due to the lack of π-π stacking interaction between the PMP molecule and the tryptophan residue. As a result, the KAT activity of mAspAT would decrease.
The binding site for the substrate kynurenine of mAspAT lies near the cofactor PMP and is defined by residues from two subunits. Ile-44, Thr-135, Trp-162, Asn-215, Arg-287 and Arg-407 from one subunit, and Tyr-96, Arg-313, Ser-317 and Asn-318 from the other subunit interact with kynurenine. The guanidinium group of Arg-407 forms salt bridge with the carboxylic group of the kynurenine. The bridge is fixed by hydrogen bonding with side chains of Asn-215 and Gly-65 at both sides of the bridge. The kynurenine molecule shown in the molecular document is from human kynurenine aminotransferase II (KAT II, PDB ID: 2R2N). The crystalized structure of kynurenine for Mus muculus mAspAT is not available. mAspAT contacts the co-substrate oxaloacetate in a similarly way. Kynureneing and oxaloacetate interact with mAspAT at the same binding site. However, because oxaloacetate molecule is smaller than kynurening, the interaction between oxaloacetate and mAspAT involves fewer amino acid residues and is weaker than the interaction between kynurenine and mAspAT (1). This weaker interaction between oxaloacetate and mAspAT indicates that oxaloacetate needs to be transfered from mitochondria to cytosol to take part in the gluconeogenesis pathway, wherease the kynurenine is transformed to KYNA at the active site.
The binding of ligand and substrate is facilitated by small domain conformational change of the mAspAT. Upon substrate binding, the mAspAT can change conformation from unliganded open form to liganded closed form. In Mus muculus mAspAT, upon substrate binding, small domain would undergo domain closure. In closed conformation, residues in active site, such as Ile-44 and Arg-407, would move and interact with substrate kynurenine and oxaloacetate. This conformation change not only facilitates the substrate binding, but also effectively shields bulky molecules from the substrate-binding site.
A well-studied AspATs, the cytosolic aspartate aminotransferase (cAspAT) of Sus scrofa (PDB ID: 1AJS), has similar primary and tertiary structures to Mus musculus mAspAT. The E value between the Mus musculus mAspAT and Sus scrofa cAspAT obtained from PSI-BLAST, a server that could identify proteins that have similar primary structure to the protein query, is 6e-136, indicating that the two proteins have similar primary structures, because an E-value larger than 0.5 is considered significant (7). While each chain of mAspAT contains 401 amino acid residues, each chain of cAspAT contains 412 amino acid residues (5). The secondary structures of these two proteins are similar. Each domain of cAspAT and mAspAT contains fifteen α-helices, thirteen β-sheets and two 3/10 α-helices. The Z-score for structural similarities between the sus scrofa cAspAT and Mus musculus mAspAT obtained from Dali server, which could identify proteins that have similar tertiary structural to a protein query, is 58.4, indicating that the two proteins have similar tertiary structures, because a Z-score greater than 2 is considered significant (8). Like mAspAT, Sus scrofa cAspAT also contains small domains (residues12-49 and 327-412) and large domains. The substrate binding of cAspAT also cause small domain conformation change. The active site is at the interface betweem the two domains (10). However, unlike Mus musculus mAspAT, Sus scrofa cAspAT is a homo-dimer with two identical chains, allowing it to bind only one molecule of substrate, instead of two in Mus musculus mAspAT. These two proteins perform similar biological functions. They are both PLP dependant enzyme that mainly catalyze the reversible transamination of L-aspartate and 2-oxoglutarate to oxaloacetate and L-glutamate. Wherease the oxaloacetate produced by cAspAT is directly converted to glucose via gluconeogenesis pathway in cytosol, the oxaloacetate produced by mAspAT in mitochondrial needs to be transportated to cytosol. The similarities between the structures and fundctions of these proteins make it possible to understand the properties of Mus musculus mAspAT by studying the structure and function of Sus scrofa cAspAT.
Although
the Mus muculus mAspAT has not been
crystalized in both open and closed conformations, Gallus gallus mAspAT (PDB ID: 7AAT, 8AAT, 9AAT), eAspAT (PDB ID: 1ASM, 1ASN) and Sus scrofa cAspAT (PDB ID: 1AJR, 1AJS) that have a similar
open-to-closed transition have been observed and published (5, 9, 10). The two
identical subunits of the Sus scrofa cAspAT
adopt the open conformation in the absence of substrate. During substrate
binding, the negative charges of incoming substrate neutralizes two arginine
residues in the active site and the backbone torsion angles of two glycine
residues change greatly in one subunit, inducing the small domain to shift
toward the active site. As a result, a closed conformation is formed. However, only one subunit shows a conformational change after binding
to substrate, because the other subunit is locked by the crystal lattice in the
open conformation. The driving force of the conformation change is the increase
in entropy of the solution caused by the burial of hydrophobic amino acid
residues during domain closure (10). The mAspAT changes conformation in a similar way. The two positively charged arginine residues, Arg-407 and Arg-313, at the active site would be neutralized by the negative charge of the incoming substrate, kynurenine or oxaloacetate. The neutralized arginine residues become hydrophobic and fold into the protein, thus inducing small domain shifts and resulting in a closed conformation.
In
conclusion, the enzymatic activities of the mAspAT depend on its structure. The hydrophilic and hydrophobic interactions between the amino acid residues and substrates at the active stie allow strong binding of the substrates; the hydrogen bonding in secondary structure stabilize the protein; the conformation change of the tertiary structure of the small domain make the substrate binding more sufficient. The
protein structure determines how well the mAspAT binds to the coenzyme PLP, the substrate kynurenine and the co-substrate oxaloacetate.