Crystal Structure of Pyruvate Kinase from Geobacillus

The pyruvate kinase (PK) from a moderate thermophile, Geobacillus stearothermo-philus, is an allosteric enzyme activated by AMP and ribose 5-phosphate but not fructose 1, 6-bisphosphate (FBP), which is a common activator of PKs. It has an extra C-terminal sequence (ECTS), which contains a highly conserved phosphoenolpyr-uvate (PEP) binding motif, but its function and structure remain unclear. To elucidate the structural characteristics of the effector-binding site and the ECTS, the crystal structure of the C9S/C268S mutant of the enzyme was determined at 2.4 Å resolution. The crystal belonged to space group P6222, with unit cell parameters a, b = 145.97 Å, c = 118.03 Å. The enzyme was a homotetramer and its overall domain structure was similar to the previously solved structures except that the ECTS formed a new domain (C0 domain). The structure of the C0 domain closely resembled that of the PEP binding domain of maize pyruvate phosphate dikinase. A sulphate ion was found in a pocket in the effector-binding C domain. This site corresponds to the 6-phosphate group-binding site in yeast PK bound FBP and seems to be the effector-binding site. Through comparison of the structure of the putative effector-binding site to that of the FBP binding site of the yeast enzyme, the structural basis of the effector specificity of the G. stearothermophilus PK is discussed