Three-dimensional structure of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis in the apo form an
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CTURE OF MACROMOLECULAR COMPOUNDS
ThreeDimensional Structure of Phosphopantetheine Adenylyltransferase from Mycobacterium Tuberculosis in the Apo Form and in Complexes with Coenzyme A and Dephosphocoenzyme A V. I. Timofeeva, E. A. Smirnovaa, L. A. Chupovab, R. S. Esipovb, and I. P. Kuranovaa a
Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninskii pr. 59, Moscow, 119333 Russia email: [email protected] b Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. MiklukhoMaklaya 16/10, Moscow, 117871 Russia Received September 15, 2011
Abstract—Crystals of phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium tuberculosis in the apo form and in complexes with coenzyme A (PPAT/CoA) and dephosphocoenzyme A (PPAT/dPCoA) were grown in microgravity by the capillary counterdiffusion method. The structures of PPAT Mt in the apo form and in complexes with ligands were solved based on the Xray diffraction data collected from the grown crystals. The crystal structures were refined at 1.76, 1.59, and 1.59 Å resolution to Rf factors of 0.175, 0.159, and 0.157 and Rfree of 0.224, 0.208, and 0.206 for PPAT, PPAT/CoA, and PPAT/dPCoA, respectively. The atomic coordinates of the structures were deposited in the Protein Data Bank (PDB ID: 3RFF, 3RHS, and 3RBA). In these structures, the ligandbinding sites were determined, the environment of these sites was characterized, and the conformational changes accompanying the ligand binding were analyzed. DOI: 10.1134/S1063774512010142
INTRODUCTION
tion to the understanding of the mechanism of action of PPAT.
Bacterial phosphopantetheine adenylyltrans ferase (PPAT) is involved in coenzyme A biosyn thesis by catalyzing the penultimate and fourth step of this process, resulting in the formation of the dephosphocoenzyme A (dPCoA) from 4’phos phopantetheine and ATP (Fig. 1). This is the key step in the synthesis, because this is the step in which the rate of the formation of the end product (coenzyme A) is controlled. The change in the rate is due to the formation of a PPAT complex with coenzyme A as the end product of the biosynthesis, when the latter is formed in an excess amount.
In the present study, the threedimensional struc ture of the PPAT complex with the reaction product— dephosphocoenzyme A (dPCoA)—was determined for the first time (at 1.59 Å resolution) based on the Xray diffraction data collected at the SPring8 syn chrotron radiation facility from crystals grown in microgravity by the counterdiffusion method [1]. The threedimensional structures of the apo form of PPAT and PPAT Mt in the complex with a natural inhibitor (coenzyme A, CoA), which were determined earlier at 1.99 and 2.1 Å resolution, were refined at 1.76 and 1.59 Å, respectively. The structural details were found and described at higher resolution. The positions of the ligands in the enzyme molecule were located, their environment was described, the func tional groups of the enzyme and the atomic groups of the ligands directly invol
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