In silico analysis and molecular dissection of the PPDK:PDRP interaction

Abstract

Pyruvate phosphate dikinase (PPDK) is a key bidirectional enzyme of the C4/CAM photosynthetic pathway that rejuvenates PEP by transferring a phosphate from ATP to a pyruvate. This enzyme is reversibly phosphorylated at Thr456 by the PPDK regulatory protein (PDRP); mediating light/dark regulation of C4/CAM PPDK activity. Attempts to elucidate the tertiary structure of PDRP have been wholly disappointing although previous research has established the mechanism by which PDRP regulates PPDK. Since nothing is known about the 3-D structure of PDRP, we utilized an in silico analysis of residues surrounding the highly conserved 455–462 region of PPDK to identify potentially interacting residues. Based on this analysis, point mutations of PPDK were performed in vivo and mutant enzyme with functional PPDK activity but inhibited PDRP regulation were recovered. Mutant PPDK was compared to wild-type PPDK for enzymatic efficacy and then assayed for the degree of PPDK phosphorylation by PDRP. Using this approach, we will provide novel insights into the structural basis of PDRP catalysis. This work was supported by U.S. National Science Foundation Grant No. IOS-0642190.