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

Jiang, Xuejun; Wang, Jia; Chang, Haiyang; Zhou, Yong

doi:10.5582/bst.2015.01150

Cited by 16 publications

(22 citation statements)

References 29 publications

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“…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 [87]. Beyond its canonical function, mitochondrial AATM also functions as kynurenine aminotransferase as well as a transporter for long chain fatty acids [87,88].…”

Section: -Oxoglutarate Enzymesmentioning

confidence: 99%

“…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 [87]. Beyond its canonical function, mitochondrial AATM also functions as kynurenine aminotransferase as well as a transporter for long chain fatty acids [87,88]. AATM also interacts directly with the cardiolipin containing liposomes, the inner-mitochondrial membrane as well as the 2-OGDHC [89,90].…”

Section: -Oxoglutarate Enzymesmentioning

confidence: 99%

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Mitochondrial protein interaction landscape of SS-31

Chavez

Tang

Campbell

et al. 2019

Preprint

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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 StatementSS-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.

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Section: -Oxoglutarate Enzymesmentioning

confidence: 99%

Section: -Oxoglutarate Enzymesmentioning

confidence: 99%

Mitochondrial protein interaction landscape of SS-31

Chavez

Tang

Campbell

et al. 2019

Preprint

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“…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.…”

Section: Sequence Analysismentioning

confidence: 96%

“…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.…”

Section: Introductionmentioning

confidence: 99%

Cloning, expression and characterization of an aspartate aminotransferase gene from Lactobacillus brevis CGMCC 1306

Zhang

et al. 2017

Biotechnology & Biotechnological Equipment

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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 k cat / K m was determined to be 25.5 (mmol/L) ¡1 s ¡1 for L-Asp and 207.8 m(mol/L) ¡1 s ¡1 for a-ketoglutarate. With optimum temperature as 25 8C, the AATase may be a novel and special psychrophilic enzyme which exhibited a good thermal stability below 55 8C. The conserved active site residue of AATase was identified as Lys237 by phylogenetic analysis. Secondary structure of the enzyme includes a-helix (39.2%), b-sheet (5.5%), b-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.

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“…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 ].…”

Section: Introductionmentioning

confidence: 99%

Kynurenine Aminotransferase Isozyme Inhibitors: A Review

Nematollahi

Sun

Jayawickrama

et al. 2016

IJMS

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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.

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

Cited by 16 publications

References 29 publications

Mitochondrial protein interaction landscape of SS-31

Mitochondrial protein interaction landscape of SS-31

Cloning, expression and characterization of an aspartate aminotransferase gene from Lactobacillus brevis CGMCC 1306

Kynurenine Aminotransferase Isozyme Inhibitors: A Review

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