Purification, crystallization and preliminary X-ray crystallographic analysis of branched-chain aminotransferase from Deinococcus radiodurans

Abstract

The branched-chain amino-acid aminotransferase (BCAT), which requires pyridoxal 5'-phosphate (PLP) as a cofactor, is a key enzyme in the biosynthetic pathway of the hydrophobic amino acids leucine, isoleucine and valine. DrBCAT from Deinococcus radiodurans, which has a molecular weight of 40.9 kDa, was crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction data to 2.50 A resolution from a DrBCAT crystal, the crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.37, b = 90.70, c = 155.47 A. Preliminary analysis indicates the presence of two DrBCAT molecules in the asymmetric unit, with a solvent content of 47.52%.

Figure 1

Coomassie blue stained 12.5% Tricine SDS–PAGE of pools from DrBCAT purification. Purification fractions containing BCAT activity were collected at each step. All samples were boiled at 373 K for 10 min in 4× sample buffer. Lane 1, pool from total cell lysate in 0.3 M NaCl, 10 mM imidazole and 50 mM NaH₂PO₄ pH 8.0; lane 2, pool from supernatant after centrifugation; lane 3, pool from pellet after centrifugation; lane 4, pool from the first His-tag Ni²⁺–NTA column; lane 5, pool from the second His-tag Ni²⁺–NTA column; lane M, molecular-weight markers (kDa).

Figure 2

Single needle crystals of DrBCAT grown by the hanging-drop method.