Abstract
Aspartate aminotransferase from Corynebacterium glutamicum (CgAspAT) is a PLP-dependent enzyme that catalyzes the production of L-aspartate and α-ketoglutarate from L-glutamate and oxaloacetate in L-lysine biosynthesis. In order to understand the molecular mechanism of CgAspAT and compare it with those of other aspartate aminotransferases (AspATs) from the aminotransferase class I, we determined the crystal structure of CgAspAT. CgAspAT functions as a dimer, and the CgAspAT monomer consists of two domains, the core domain and the auxiliary domain. The PLP cofactor is found to be bound to CgAspAT and stabilized through unique residues. In our current structure, a citrate molecule is bound at the active site of one molecule and mimics binding of the glutamate substrate. The residues involved in binding of the PLP cofactor and the glutamate substrate were confirmed by site-directed mutagenesis. Interestingly, compared with other AspATs from aminotransferase subgroup Ia and Ib, CgAspAT exhibited unique binding sites for both cofactor and substrate; moreover, it was found to have unusual structural features in the auxiliary domain. Based on these structural differences, we propose that CgAspAT does not belong to either subgroup Ia or Ib, and can be categorized into a subgroup Ic. The phylogenetic tree and RMSD analysis also indicates that CgAspAT is located in an independent AspAT subgroup.
Highlights
Corynebacterium glutamicum is an aerobic, gram-positive, short and rod-shaped bacterium, and is known to be involved in the fermentation of various amino acids, such as L-lysine and L-glutamate [1]
The polymerase chain reaction (PCR) products were subcloned into pET30a (Novagen), and the resulting expression vector pET30a:CgAspAT was transformed into a E. coli BL21(DE3)-T1R strain, which was grown in 1 L of LB medium containing 100 μM kanamycin at 310 K
In order to elucidate the molecular mechanism of CgAspAT, we determined the crystal structure of the protein at 2.0 Å
Summary
Corynebacterium glutamicum (initially reported as Micrococcus glutamicus) is an aerobic, gram-positive, short and rod-shaped bacterium, and is known to be involved in the fermentation of various amino acids, such as L-lysine and L-glutamate [1]. Various C. glutamicum mutants have been isolated, which produce significant amounts of different Lamino acids [2]. In 2003, the genome of C. glutamicum ATCC 13032 was sequenced, providing an abundant source of data on the metabolic pathways used for the biosynthesis of industrially important products [2, 3]. During last 10 years, the market size of amino acid for livestock has been continuously increasing. L-methionine, L-lysine, L-threonine, L-tryptophan, and L-valine are essential amino acids manufactured industrially. L-methionine, L-lysine, L-threonine, L-tryptophan, and L-valine are essential amino acids manufactured industrially. [2, 4, 5].
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