Dimerization misalignment in human glutamate-oxaloacetate transaminase variants is the primary factor for PLP release

Abstract

The active form of vitamin B6, pyridoxal 5’-phosphate (PLP), plays an essential role in the catalytic mechanism of various proteins, including human glutamate-oxaloacetate transaminase (hGOT1), an important enzyme in amino acid metabolism. A recent molecular and genetic study showed that the E266K, R267H, and P300L substitutions in aspartate aminotransferase, the Arabidopsis analog of hGOT1, genetically suppress a developmentally arrested Arabidopsis RUS mutant. Furthermore, CD analyses suggested that the variants exist as apo proteins and implicated a possible role of PLP in the regulation of PLP homeostasis and metabolic pathways. In this work, we assessed the stability of PLP bound to hGOT1 for the three variant and wildtype (WT) proteins using a combined 6 μs of molecular dynamics (MD) simulation. For the variants and WT in the holo form, the MD simulations reproduced the “closed-open” transition needed for substrate binding. This conformational transition was associated with the rearrangement of the P15-R32 small domain loop providing substrate access to the R387/R293 binding motif. We also showed that formation of the dimer interface is essential for PLP affinity to the active site. The position of PLP in the WT binding site was stabilized by a unique hydrogen bond network of the phosphate binding cup, which placed the cofactor for formation of the covalent Schiff base linkage with K259 for catalysis. The amino acid substitutions at positions 266, 267, and 300 reduced the structural correlation between PLP and the protein active site and/or integrity of the dimer interface. Principal component analysis and energy decomposition clearly suggested dimer misalignment and dissociation for the three variants tested in our work. The low affinity of PLP in the hGOT1 variants observed in our computational work provided structural rationale for the possible role of vitamin B6 in regulating metabolic pathways.

The role of glutamate oxaloacetate transaminases in sulfite biosynthesis and H2S metabolism

“…While aspartate is highly abundant [ 63 ], intracellular concentrations of CSA could not be determined [ 2 ], which would suggest lower levels. Our data therefore confirm that deamination of aspartate would be favored under physiological conditions, which is in line with the numerous reports regarding the importance of aspartate synthesis and metabolism [ 56 , 62 , 64 , 65 ].…”

Insight into Structural Characteristics of Protein-Substrate Interaction in Pimaricin Thioesterase

“…Due to the limitation of experimental instruments, present computational strategies combining homology modeling, molecular docking, MD simulation, and QM/MM calculation have been extensively utilized to provide insight into atomistic details in protein-substrate recognition and catalytic mechanism. Over recent years, packages and software [ 30 , 31 , 32 ] to study protein-substrate interaction have sprung up relentlessly, and MD simulation has become a regular routine herein [ 33 , 34 , 35 ].…”

Cysteine Aminotransferase (CAT): A Pivotal Sponsor in Metabolic Remodeling and an Ally of 3-Mercaptopyruvate Sulfurtransferase (MST) in Cancer

“…Two isoforms of CAT, classified according to their cellular localization as cytoplasmic (cCAT) and mitochondrial CAT (mCAT), are encoded respectively by GOT1 (glutamic-oxaloacetic transaminase 1) and GOT2 (glutamic-oxaloacetic transaminase 2) [ 14 ], which are extremely well conserved across species [ 15 , 16 ]. CAT is a homodimeric pyridoxal phosphate (PLP)-dependent aminotransferase [ 17 , 18 ], being considered the best studied PLP-dependent enzyme, given its easy large-scale purification and its stability [ 19 , 20 ]. The mCAT was in fact the first PLP-dependent enzyme to have its X-ray structure determined [ 21 ], leading to key insights on its catalytic mechanism, holding to this day [ 19 ].…”

“…Each cCAT monomer consists of two domains: A small domain composed of four α-helices and three β-strands, and a large domain comprising a 7-stranded β-sheet and several short α-helices. Protein dimerization is ensured by the large domains, with two PLP-binding sites which are stabilized by the surrounding residues [ 20 ]. PLP has a dual physiological effect: While it is vital for normal cellular metabolism, excessive levels of free PLP can non-specifically and covalently bind to thiol and amino groups [ 22 , 23 ].…”

“…Indeed, spontaneous formation of hydrogen sulfide (H 2 S) by free PLP and cysteine has been reported [ 24 ]. Mutations in cCAT impact on its structure and prevent effective PLP binding by a dimer interface misalignment, which can lead to its dissociation and PLP release [ 20 ].…”