Abstract
L-aspartate aminotransferase is a pyridoxal 5ʹ-phosphate-dependent transaminase that catalyzes reversible transfer of an α-amino group from aspartate to α-ketoglutarate or from glutamate to oxaloacetate. L-aspartate aminotransferase not only mediates amino acid and carbohydrate metabolism but also regulates the cellular level of amino acids by catalyzing amino acid degradation and biosynthesis. To expand our structural information, we determined the crystal structure of L-aspartate aminotransferase from Schizosaccharomyces pombe at 2.1 Å resolution. A structural comparison between two yeast L-aspartate aminotransferases revealed conserved enzymatic mechanism mediated by the open–closed conformational change. Compared with higher eukaryotic species, L-aspartate aminotransferases showed distinguishable inter-subunit interaction between the N-terminal arm and a large domain of the opposite subunit. Interestingly, structural homology search showed varied conformation of the N-terminal arm among 71 structures of the family. Therefore, we classified pyridoxal 5ʹ-phosphate-dependent enzymes into eight subclasses based on the structural feature of N-terminal arms. In addition, structure and sequence comparisons showed strong relationships among the eight subclasses. Our results may provide insights into structure-based evolutionary aspects of pyridoxal 5ʹ-phosphate-dependent enzymes.
Highlights
L-aspartate aminotransferase (AST, EC 2.6.1.1) is a key metabolic enzyme that links amino acid metabolism to carbohydrate metabolism through reversible transamination reaction and an enzyme that regulates the cellular level of amino acid by catalyzing amino acid degradation and biosynthesis [1, 2]
The supernatant was incubated with Ni+-NTA resin for 90 min, and the C-terminal His6-tagged SpAST protein was eluted by 250 mM imidazole after being washed with 5 mM imidazole
Fifteen amino acids present in the N-terminal extended from α1 of the small domain and
Summary
L-aspartate aminotransferase (AST, EC 2.6.1.1) is a key metabolic enzyme that links amino acid metabolism to carbohydrate metabolism through reversible transamination reaction and an enzyme that regulates the cellular level of amino acid by catalyzing amino acid degradation and biosynthesis [1, 2]. ASTs are found from bacterial to eukaryotic species, with sequence identity. AST from Saccharomyces pombe shares relatively high sequence identity with those of chicken cytosolic (46%), Saccharomyces cerevisiae (49%), and Escherichia coli (37%). S. pombe AST shares relatively low sequence identity with those of Pyrococcus horikoshii (16%), Thermus thermophilus (17%), and Thermotoga maritima (15%). The structure of cytosolic or mitochondrial AST from chicken [4,5,6,7], pig heart [8], Saccharomyces cerevisiae [9], and Escherichia coli [10, 11] have been reported in its apo form and as a complex with its cofactor PLP, pyridoxamine-5’-phosphate (PMP), or dicarboxylic inhibitor maleate [12,13,14]. A detailed enzymatic mechanism was proposed based on previously reported structures [5, 12, 13]
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