| Glutamate oxaloacetate transaminases (GOTs) mainly catalyze the reversible reaction of L-aspartate and ?-ketoglutarate into oxaloacetate and L-glutamate and play a key role in carbon and nitrogen metabolism in all organisms. In human tissues, GOTs are pyridoxal 5'-phosphate-dependent (PLP) enzymes which exist in cytoplasm and mitochondrial forms, GOT1 and GOT2, respectively. GOTl was demonstrated to be required to sustain pancreatic ductal adenocasinoma (PDAC) cell growth through the metabolism of the glutamine carbon skeleton and maintenance of the redox balances in PDACs. Therefore, GOT1 represents a valid molecular target for the development of anti-neoplastic agents. Elucidation of the structure of GOT1 will aid in the design of drugs to selectively inhibit cancer cell growth.1. High purity recombinant expression and characterization analysis of GOT1.By constructing an expression vector pET22b-gotl, recombinant enzyme was expressed in soluble form in Escherichia coli cells. The protein was purified by using HisTrapTM FF crude column chromatography and HiTrapTM Q XL column anion exchange chromatography. The specific activity, the purification fold and the activity recovery rate was 70.83 U/mg, 133.6 times and 18.3% respectively. The gel size exclusion chromatography showed that recombinant GOT1 aggregated as a dimer and the molecular weight of the dimer was 66 kDa. The enzyme had optimal activity at 37? and pH 4.8. It was stable at 35?45? and showed high activity in alkaline conditions (pH 7.5?10.0). The activity of purified GOT1 was slightly suppressed by Na+, K+ and Mg2+, and was remarkably inhibited by Zn2+, Mn2+, Cu2+, Ni2+, Co2+ and Ca2+.2. Screening out crystallization conditions and crystal data processing of GOT1.The initial screening concentration of GOT1 protein crystals was 5 mg/ml and three screening conditions were identified, namely Crystal Screen kit No.9 (0.2 M ammonium acetate,0.1 M Na citrate tribasic dihydrate pH 5.6,30% w/v Polyethylene glycol 4000), Index kit No.42 (0.1 M BIS-Tris pH 5.5,20% w/v PEG 3350) and Index kit No.43 (0.1 M BIS-Tris pH 6.5,25% w/v PEG 3350). After the optimized conditions, the best crystallization condition of recombinant GOT1 was 0.1 M BIS-Tris pH 6.0,21% w/v PEG 3350. GOT1 crystals (PDB ID:3WZF) were obtained by vapor diffusion hanging drop method and structural data were collected by X- ray diffraction.The crystal diffracted to 2.99 A resolution and belonged to space group P43212 with the unit cell parameters ?=93.4 A,b=93.4 A, c=107.4 A, ?=?=?=90°.3. Bioinformatics analysis of glutamate oxaloacetate transaminasesAlthough two kinds of human cytoplasmic GOTs used different protein expression vectors and crystallization conditions, there were up to 95% on overall sequence similarity. The 100% residues of crystal 3110 located in favored region, and the 98.5% residues of crystal 3WZF located in favored region including six residues which (?-? angles were abnormal. The amino acid sequence similarity of GOT1 and GOT2 was less than 50%, but the identity of tertiary structures was high. The key amino acids in activity pocket of GOT1 were Gly 39, Trp 141, Asn 195, Arg 387, while in GOT2 were Gly 65, Trp 162, Asn 215, Arg 407. The catalytic amino acids are identical in two isozymes but located differently. GOT1's amino acid sequence was similar to pig-derived and chicken-derived GOTs in evolution by phylogenetic tree analysis. Molecular docking analysis indicated that the interaction among PLP and GOT1 was strong. Substrate amino acids,such as Asp, Cys and Glu, could form hydrogen bonds with key residues within 3 A and binded tightly with GOT1. |
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