AST

Aspartate aminotransferase (EC 2.6.1.1; AspAT) is a pyridoxal-50-phosphate (PLP)-dependent enzyme which catalyzes the reversible transamination between aspartate and 2-oxoglutarate to give oxaloacetate and glutamate. AspAT is widely found in plants, microorganisms and animals and plays a very important role in the process of nitrogen and carbon metabolism in cells. It also plays a role in ureagenesis and is involved in the transfer of reducing equivalents to the mitochondria via the aspartate/malate shuttle. It is mainly responsible for the reversible reaction between acidic amino acids and ketoacids. Transamination of the amino group is accomplished by the conversion of coenzyme between pyridoxal phosphate and pyridoxamine phosphate. With the development of biotechnology, more and more researchers studied its catalytic mechanism from the structure level through molecular simulation and other methods, and guided the modification and reasonable design of enzymes.

Characteristics

In 1984, Kondo. K et al. reported the complete amino acid sequence of AspAT in E. coli and analyze the sequence homology among with the birds and mammals, the results showed that the sequence homology is 40%. In 1987, they further analyzed the amino acid sequence of E. coli AspAT and concluded that E. coli AspAT had 396 amino acid residues with a molecular weight of 4.35 kDa. Also, they found that the AspAT contains 55.8% non-polar residue and 44.2% polar residue, which are similar to isozyme in chicken and pig mitochondria. According to the homology of the sequence, AspATs belong to transaminase Class I. AspATs can also be divided into seven subclasses according to sequence homology. Compared with other subtypes of AspATs and other transaminases with different substrate specificity, AspATs of Iα has been studied extensively and deeply.

Structure

In the early stage, AspAT was studied through fixed-point mutation experiment to infer its structure. In 1986, Smith et al. obtained its three-dimensional structure through X-ray diffraction method. E. coli AspAT is a homologous dimer with two identical subunits, each of which can be divided into a large domain and a small domain. According to the function of each part in the structure, each subunit can be divided into an N terminal arm (used for interconnections between two subunits, the overall structure of a stable enzyme), a large domain (used for binding coenzymes), and a small domain (used for binding subunits). When the enzyme binds to the substrate, the small domain moves toward the larger domain. Therefore, interface composition among structural domains is important, and there are generally highly conserved sequences in these regions. By using the structure of a putative aminotransferase from S. pomeroyi as the search model, the crystals of AspAT diffracted to a resolution limit of 2.50 A˚ (Fig. 1) and its three-dimensional structure was solved by the molecular-replacement method. Analysis of the self-rotation function revealed that the four molecules in the crystal asymmetric unit seem to have 222 molecular symmetry.

Figure 1. A representative diffraction image collected at 1.0 oscillation range from a single AspAT crystal.
(Deepak Chandra Saroj, et al. 2014)

Catalytic mechanism

Aspartate biosynthesis is an essential pathway required for the growth and survival of mycobacterium tuberculosis. Structure analysis of aspartate aminotransferase (AspAT) from Mtb will not only provide a platform for the structure-based design of small-molecule inhibitors, but also aid in elucidating the detailed mechanism of its catalysis. In physiological conditions, AspAT used ketoglutarate and oxaloacetic acid as amino receptors, while Glu and Asp as amino donors. It can interact with two types of substrate in different ways. The most important coenzyme in the reaction process is pyridoxal-5’-phosphate (PLP), whose ɛ-4 carbon atom covalently binds to the amino group of Lysine, the enzyme activity center, to form aldimine.

Figure 2. Reaction mechanism for aspartate aminotransferase.

AspAT has two identical subunits, and the active center is located at the subunit interface. The residue of the active center is highly conserved in different isozymes. Each subunit has a binding site with substrates and coenzymes. The main principle is the interaction between the guanidine with the substrate via the positive arginine band and the carboxyl group of the substrate. In AspAT, the carboxyl group interacts with the terminal carboxyl group. AspAT was covalently bound to Lysine residue in the form of Schiff base with PIP as coenzyme. Through the conversion of coenzyme between PLP and PMP, the process of transamination was promoted.

References

1. CONSTANCE J. EFFERY, et al. cytosolic aspartate aminotransferase. Prorein Science (1998). 71380-1387.
2. Deepak Chandra Saroj, et al. Overexpression, purification, crystallization and structure determination of AspB, a putative aspartate aminotransferase from Mycobacterium tuberculosis. Acta Crystallographica Section F. (2014). 70, 928–932