Crystal structure of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila.

Phosphotransacetylase (Pta) [EC 2.3.1.8] is ubiquitous in the carbon assimilation and energy-yielding pathways in anaerobic prokaryotes where it catalyzes the reversible transfer of the acetyl group from acetyl phosphate to CoA forming acetyl CoA and inorganic phosphate. The crystal structure of Pta from the methane-producing archaeon Methanosarcina thermophila, representing the first crystal structure of any Pta, was determined by multiwavelength anomalous diffraction at 2.7 A resolution. In solution and in the crystal, the enzyme forms a homodimer. Each monomer consists of two alpha/beta domains with a cleft along the domain boundary, which presumably contains the substrate binding sites. Comparison of the four monomers present in the asymmetric unit indicates substantial variations in the relative orientation of the two domains and the structure of the putative active site cleft. A search for structural homologs revealed the NADP(+)-dependent isocitrate and isopropylmalate dehydrogenases as the only homologs with a similar two-domain architecture.

Expression, purification, crystallization and preliminary X-ray analysis of phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase (Pta) from the anaerobic archaeon Methanosarcina thermophila has been heterologously expressed in a soluble form which facilitated crystallization using the hanging-drop vapor-diffusion method with ammonium sulfate as a precipitant. This is the first report of the crystallization of any Pta. While the M. thermophila Pta has high sequence identity to Ptas from other organisms, it has no homology to any previously crystallized proteins. The protein crystallized in space group I4(1), with unit-cell parameters a = b = 114.8, c = 127.8 A, alpha = beta = gamma = 90 degrees. The crystals diffracted to 2.5 A resolution using Cu Kalpha radiation. The enzyme had previously been reported to exist as a monomer; however, the self-rotation function showed the presence of a non-crystallographic symmetry axis at psi = 90, phi = 90, kappa = 180 degrees, suggesting oligomerization. Dynamic light-scattering analysis supported a dimeric state for Pta in solution.