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Journal of Biochemistry Advance Access published online on May 28, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn069
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© 2008 The Japanese Biochemical Society

Crystal Structure of Pyruvate Kinase from Geobacillus stearothermophilus

Kenichiro Suzuki, Sohei Ito, Akiko Shimizu-Ibuka and Hiroshi Sakai

Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Yada 52-1, Shizuoka 422-8526, Japan

Correspondence to: Hiroshi Sakai, Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Yada 52-1, Shizuoka 422-8526, Japan., Phone: +81-54-264-5576, FAX: +81-54-264-5099, E-mail: gp1314{at}mail.f.u-shizuoka-ken.ac.jp

Received March 14, 2008; Accepted April 21, 2008


  Abstract

SUMMARY

The pyruvate kinase from a moderate thermophile, Geobacillus stearothermophilus, is an allosteric enzyme activated by AMP and ribose 5-phosphate but not fructose 1, 6-bisphosphate, which is a common activator of pyruvate kinases. It has an extra C-terminal sequence, which contains a highly conserved phosphoenolpyruvate binding motif, but its function and structure remain unclear. To elucidate the structural characteristics of the effector binding site and the extra C-terminal sequence, 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, c=118.03 Å. The enzyme was a homotetramer and its overall domain structure was similar to the previously solved structures except that the extra C terminal sequence formed a new domain (C' domain). The structure of the C’ domain closely resembled that of the phosphoenolpyruvate binding domain of maize pyruvate phosphate dikinase. A sulfate ion was found in a pocket in the effector-binding C domain. This site corresponds to the 6-phosphate group binding site in yeast pyruvate kinase bound fructose 1, 6-bisphosphate and seems to be the effector binding site. Through comparison of the structure of the putative effector binding site to that of the fructose 1, 6-bisphosphate binding site of the yeast enzyme, the structural basis of the effector specificity of the Geobacillus stearothermophilus pyruvate kinase is discussed.

Key Words: allosteric, AMP, crystal structure, pyruvate kinase, ribose 5-phosphate


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