Crystal structure of P-protein of the glycine cleavage system

Abstract

Precursors for isoprenoid synthesis are essential in all organisms. These compounds are synthesized by one of two known routes: the well characterized mevalonate pathway [1] or a recently discovered non-mevalonate route which is used in many bacteria and human pathogens [2]. Since the second pathway is both vital and unlike any found in humans, enzymes catalysing reactions along this synthetic route are possible drug targets. The structure of one such enzyme from the thermophilic bacterium Thermus thermophilus has been solved to high resolution in the presence of substrate and with a substrate analogue. Enzyme co-crystallized with substrate shows only one product, cytosine monophosphate (CMP), in the active site. At the high resolution of the refinement (1.6 Å) the positions and coordination of the magnesium ions in the active site are clearly seen.Acetyl-CoA carboxylases (ACCs) catalyze the first committed step of fatty acid biosynthesis. Although ACC is an essential enzyme (complex) in every organism, the structure-function relationship of ACC remains to be unclear. As the first step for elucidating the structure-function relationship of ACC, we started the crystallographic analysis of DtsR1. DtsR1 is the -subunit of ACC multisubunit complex in Corynebacterium glutamicum, which catalyzes the transcarboxylation between biotin and acetyl-CoA.DtsR1 was over-expressed in E. coli, purified, and crystallized by the sitting-drop vapor diffusion method using PEG 6000 as a precipitant. The approximate dimensions of the obtained crystals were 0.07x0.07x0.03mm 3 . Diffraction data of the crystals were collected at NW12 of the Photon Factory (Tsukuba), revealing that the crystals belong to the space group R32. The crystal structure of DtsR1 was solved at 3.2Å resolution by the molecular replacement method using single-subunit coordinates of the 12S transcarboxylase (PDB ID: 1ON3) as a search model. The obtained structure suggests that the biological unit of DtsR1 is a ring-shaped hexamer with the 32-point group symmetry. Crystallographic refinement of DtsR1 is in progress at 2.7Å resolution.