Crystallization and preliminary crystallographic analysis of 2-keto-3-deoxygluconate kinase fromThermus thermophilus

Abstract

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phosphogluconate. Two crystal forms of KDGK from Thermus thermophilus were obtained by vapour-diffusion and microbatch methods. Crystals in the form of triangular plates (TtKDGK-1) were obtained that belong to space group P3, with unit-cell parameters a = b = 145.83, c = 74.63 A, and diffract to 3.2 A. These crystals exhibited nearly perfect hemihedral twinning. Assigning six subunits of TtKDGK to the asymmetric unit of the crystal corresponds to a 46.2% solvent content. A single plate-like crystal (TtKDGK-2) belonged to space group P6(3), with unit-cell parameters a = b = 84.83, c = 168.49 A, and diffracts to 2.25 A. This crystal exhibits only partial hemihedral twinning, with a twin fraction of 24.4%. Diffraction-quality crystals of TtKDGK with bound ATP (TtKDGK-ATP), a = b = 84.72, c = 321.61 A and with bound KDG plus the ATP analogue AMP-PNP (TtKDGK-ATP-KDG), with unit-cell parameters a = b = 84.32, c = 168.7 A, were also prepared and characterized.

Structure of Thermus thermophilus 2-Keto-3-deoxygluconate Kinase: Evidence for Recognition of an Open Chain Substrate

“…Dynamic light-scattering measurements 20 and gel filtration show that Tt KDGK is monodisperse with a high molecular mass of 160 kDa, suggesting that Tt KDGK may exist as hexamers also in solution. Additional support for hexameric structure of Tt KDGK molecules came from similarities of Tt KDGK quaternary structures in two different crystal forms, Tt KDGK-1 and Tt KDGK-2 (Table 2), as well as the crystal structure of Tm KDGK (PDB accession code 1j5v), which also shows a very similar hexameric structure of the molecule.…”

“…The position of cesium is occupied by water or possibly by NH 4 þ in current structures of Tt KDGK, because the NH 4 þ was abundant in the crystallization solution. 20 Although the positions of cation-interacting main-chain carbonyl oxygen and the side-chain of Ec RK Asp249 were retained in Tt KDGK, the Val247 -Gly248 peptide bond exhibits some fluctuations and occupies different conformations in the A and B subunits of Tt KDGK-2. The conformation in the B subunit was the same as in Ec RK, while the conformation in the A subunit was different and unfavorable for binding of monovalent cations and the g-phosphate of ATP (Figure 8(b)).…”

“…20 For the structure determination we first used the diffraction data from the crystal of Tt KDGK-2. The backbone atoms of the ligand-bound Ec RK monomer structure, PDB accession code 1rkd, 10 for which the protruding loops and the terminal parts were truncated was used as a search model for the molecular replacement search with CNS.…”