Preparation of the Crystal Complex of Phosphopantetheine Adenylyltransferase from Mycobacterium tuberculosis with Coenzy
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CTURAL STUDIES Dedicated to the memory of B.N. Grechushnikov
Preparation of the Crystal Complex of Phosphopantetheine Adenylyltransferase from Mycobacterium tuberculosis with Coenzyme A and Investigation of Its ThreeDimensional Structure at 2.1Å Resolution 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, Leninskiі 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 March 9, 2010
Abstract—Recombinant phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis (PPAT Mt), which was produced by a highproducing strain and purified to 99%, was used for the crystal growth of the complex of the enzyme with coenzyme A (CoA). Crystals suitable for Xray diffraction study were obtained by cocrystallization. The crystals belong to sp. gr. R32 and have the unitcell parameters a = b = 98.840 Å, c = 112.880 Å, α = β = 90.00°, and γ = 120.00°. The threedimensional structure of the complex was determined based on Xray diffraction data collected from the crystals to 2.1 Å resolution and refined to Rf = 22.7% and Rfree = 25.93%. Activesite bound coenzyme A was found, and its nearest environment was described. The conformational changes of the enzyme due to ligand binding were revealed. The binding of CoA by tuberculosis phosphopantetheine adenylyltransferase was characterized by comparing the structures of the title complex to a similar complex of PPAT from E. coli (PPAT Ec). DOI: 10.1134/S1063774510060234
INTRODUCTION Bacterial phosphopantetheine adenylyltransferases (PPAT) are involved in the fivestep coenzyme A (CoA) biosynthesis from pantothenate (vitamin B5), cysteine, and ATP [1]. Phosphopantetheine adenylyl transferases catalyze the penultimate step of this pro cess (the reversible transfer of an adenylyl group from ATP to 4’phosphopantetheine [2]). The two last steps of the biosynthesis are presented in Fig. 1. The phos phate group of enzymebound 4'phosphopantetheine acts as a nucleophile and attacks the αphosphate group of ATP. The nucleophilic attack results in the release of pyrophosphate, which is a good leaving group, whereas the adenylyl group binds to phospho pantetheine to produce 3'dephosphocoenzyme A. Another enzyme—dephosphocoenzyme A kinase— catalyzes the final step in CoA biosynthesis, the ATP dependent phosphorylation of the deoxyribose ring of 3'dephosphocoenzyme A at position 3' to produce CoA. It was hypothesized that PPAT catalyzes the reaction by binding substrates in a conformation favorable for the reaction and stabilizing the transi tion state. The functional groups of the enzyme are not directly involved in the acidbase or covalent catalysis [3].
Coenzyme A is the main acylgroup carrier in all living cells. This coenzyme is involved in numerous reactions of the intermediate metabolism, being a cofactor of more th
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