Crystal structure of Escherichia coli phosphoenolpyruvate carboxylase in complex with substrate analogue

Abstract

Phosphoenolpyruvate carboxylase (PEPC) plays an important role in catalyzing the first step in the fixation of atmospheric CO2 during C4 photosynthesis and crassulacean acid metabolism (CAM). Here we report the crystal structure of Escherichia coli PEPC (EcPEPC) in complex with Mn2+-PEP analogue (3,3-dichloro -2-dihydroxyphosphinoylmethy l-2-propenoate; DCDP) and allosteric inhibitor L-aspartate at 2.35 Å resolution. In the structure of the complex EcPEPC, Mn2+ and DCDP are tightly bound to the active site. In the active site of EcPEPC, Mn2+-DCDP is bound to three positively charged residues (Arg396, Arg699 and Arg713) and two negatively charged residues (Glu506 and Asp543), which are strictly conserved in all known PEPCs. Comparison between the Mn2+-DCDP-aspartate complex of EcPEPC and aspartate complex of EcPEPC shows no significant structural change except the mobile loop at the top of the b barrel. Due to the ion pairs between the phosphoryl group of DCDP and the side chain of Arg699 in the flexible loop, the structure of the loop is partially fixed. However, the loop partially exists in multiple conformations similar to that in the L-aspartate complex EcPEPC. The result is consistent with our conclusion in the previous reports (Kai et al. (1999) Proc. Natl. Acad. Sci. USA, 96, 823-828; Matsumura et al. (1999) FEBS Lett., 458, 93-96).