UNCORRECTED PROOF 1 2 Direct contacts between conserved motifs of different subunits provide major 3 contribution to active site organization in human and mycobacterial dUTPases 4 Enik } o Takács a,1 , Gergely Nagy a,1 , Ibolya Leveles a , Veronika Harmat b , Anna Lopata a , 5 Judit Tóth a, * , Beáta G. Vértessy a,c, ** 6 a Institute of Enzymology, BRC, Hungarian Academy of Sciences, Budapest, Hungary 7 b Hungarian Academy of Sciences-Eötvös Loránd University, Protein Modeling Research Group, and Eötvös Loránd University, Institute of Chemistry, Budapest, Hungary 8 c Department of Applied Biotechnology, Budapest University of Technology and Economics, Budapest, Hungary 9 11 article info 12 Article history: 13 Received 5 April 2010 14 Revised 12 May 2010 15 Accepted 13 May 2010 16 Available online xxxx 17 18 Edited by Hans Eklund 19 Keywords: 20 dUTPase 21 Mycobacterium tuberculosis 22 DNA repair 23 Nucleotide hydrolysis 24 Aspartate 25 Asparagine 26 Oligomer 27 28 abstract 29 dUTP pyrophosphatases (dUTPases) are essential for genome integrity. Recent results allowed char- 30 acterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within con- 31 served Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously 32 implicated in Mg 2+ -coordination. Our results on transient/steady-state kinetics, ligand binding 33 and a 1.80 Å resolution structure of the mutant mycobacterial enzyme, in comparison with wild type 34 and C-terminally truncated structures, argue that this residue has a major role in providing intra- 35 and intersubunit contacts, but is not essential for Mg 2+ accommodation. We conclude that in addi- 36 tion to the role of conserved motifs in substrate accommodation, direct subunit interaction between 37 protein atoms of active site residues from different conserved motifs are crucial for enzyme 38 function. 39 Ó 2010 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. 40 41 42 43 1. Introduction 44 Mechanisms responsible for DNA integrity, such as controlled 45 biosynthesis of building block nucleotides and DNA damage repair 46 are of vital importance. The enzyme dUTP pyrophosphatase (dUT- 47 Pase) plays a key role in these processes by catalyzing the pyro- 48 phosphate hydrolysis of 2 0 -deoxyuridine triphosphate (dUTP) 49 thereby eliminating the possibility of thymine-replacing uracil 50 incorporation into DNA and providing dUMP as the immediate pre- 51 cursor for de novo TMP biosynthesis [1]. The dUTPase activity re- 52 sides in different protein families that form monomeric, dimeric 53 or trimeric folds. In trimeric dUTPases, the homotrimeric oligomer- 54 ization is essential for adequate enzymatic function [2–6]. These 55 enzymes are indispensable for viability in pro- and eukaryotes 56 alike [7,8] and have been proposed as promising novel targets 57 against cancer cells, Mycobacteria and Plasmodia [1,9–11]. 58 Homotrimeric dUTPases provide an accommodation network 59 for the substrates (dUTP + water) and the phosphate chain chelat- 60 ing co-factor Mg 2+ by a rare structural solution. Within the homo- 61 trimer, three active sites are formed and conserved sequence 62 motifs (Motifs I through V) from all the three subunits contribute 63 to each active site architecture. The dUTP binding pockets are sit- 64 uated at clefts between two neighboring subunits and are covered 65 by the C-terminal segment of the third subunit. In 3D structures of 66 apoenzymes (1Q5U, 1MQ7 [4,10]) and the recently published en- 67 zyme–substrate (dUTPase:Mg:a,b-imido-dUTP (dUPNPP)) com- 68 plexes (2HQU, 2PY4 [12,13]), this interesting architecture could 69 be analyzed in detail for human (hDUT) and Mycobacterium tuber- 70 culosis (mtDUT) dUTPases that showed ordering of the full-length 71 C-termini upon substrate binding (cf. also relevant conclusions 72 based on solution phase experiments [14]). Importantly, these 73 structures represent relevant complexes closely mimicking that 74 of the enzyme-substrate, as (i) the dUPNPP substrate analogue is 0014-5793/$36.00 Ó 2010 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2010.05.018 Abbreviations: M. tuberculosis, Mycobacterium tuberculosis; dUTP, 2 0 -deoxyuri- dine triphosphate; dUTPase, dUTP pyrophosphatase; dUPNPP, a,b-imido-dUTP; hDUT, human dUTPase; mtDUT, Mycobacterium tuberculosis dUTPase; hDUT D49N,F158W , Asp49Asn/Phe158Trp double mutant construct of human dUTPase; mtDUT D28N , mtDUT T138STOP , and mtDUT D28N,H145W , Asp28Asn, Thr138STOP and Asp28Asn/His145Trp mutant constructs of Mycobacterium tuberculosis dUTPase * Corresponding author. Fax: +361 466 5465. ** Corresponding author at: Institute of Enzymology, Karolina út 29, H-1113, Budapest, Hungary. Fax: +361 466 5465. E-mail addresses: tothj@enzim.hu (J. Tóth), vertessy@enzim.hu (B.G. Vértessy). 1 Both authors contributed equally to this work. FEBS Letters xxx (2010) xxx–xxx journal homepage: www.FEBSLetters.org FEBS 34051 No. of Pages 8, Model 5G 23 May 2010 ARTICLE IN PRESS Please cite this article in press as: Takács, E., et al. Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases. FEBS Lett. (2010), doi:10.1016/j.febslet.2010.05.018