Structural Framework for Pyridoxal 5'-Phosphate Binding to the Human Glutamate-Oxaloacetate Transaminase Variants

Abstract

The active form of Vitamin B6, pyridoxal 5'-phosphate (PLP), plays an essential role in the catalytic mechanism for various proteins, including the human glutamate-oxaloacetate transaminase (GOT1), an enzyme in amino acid metabolism. Recent molecular and cellular data showed that E266K, R267H and P300L substitutions in aspartate aminotransferase, the Arabidopsis analog of GOT1, genetically suppress a developmentally arrested Arabidopsis rus1 mutant. Furthermore, exogenously adding PLP can also suppress rus1 mutant and the mass spectrometry analyses suggest that the variants exist as apoproteins. In this work, we assessed the stability of PLP at the GOT1 PLP-binding pocket for all 3 variants and wild-type (WT) using combined 1.2 μs molecular dynamics (MD) simulations. The position of PLP in the WT binding site was stabilized by a unique hydrogen bond network, including the phosphate group binding cup, which positions the coenzyme within 3A from K259. In the catalytic mechanism an internal PLP-K259 aldimine is formed by a covalent Schiff base linkage to the e-amino group of K259. For all 3 variants and the WT in the holo form, the MD simulations reproduced the “close-to-open” transition needed for substrate binding. This conformational transition was associated with the rearrangement of the P15-R32 small domain loop providing a substrate access to the R387/R293 binding motif. Nevertheless, amino acid substitutions at positions 266 and 300 significantly reduced the hydrogen bonds occupancy at the PLP-binding site and displaced the cofactor from the conformation observed in the WT. The R267H variant showed less displacement of the PLP but still a much weaker h-bond network as compared to the WT. The weaker affinity of the PLP in the GOT1 variants observed in our simulations provides structural rational for both the apoprotein and the Vitamin B6 in regulating metabolic pathways.