Crystal structures of D-alanine-D-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides Thanh Thi Ngoc Doan a,1 , Jin-Kwang Kim a,1 , Ho-Phuong-Thuy Ngo b , Huyen-Thi Tran b , Sun-Shin Cha c , Kyung Min Chung d , Kim-Hung Huynh b , Yeh-Jin Ahn e , Lin-Woo Kang b,f,⇑ a Department of Advanced Technology Fusion, Konkuk University, Hwayang dong, Gwangjin-gu, Seoul 143-01, Republic of Korea b Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea c Marine Biotechnology Research Division, Korea Institute of Ocean Science and Technology, Ansan 426-744, Republic of Korea d Department of Microbiology and Immunology, Chonbuk National University Medical School, Jeonju, Jeonbuk 561-756, Republic of Korea e Department of Green Life Science, College of Convergence, Sangmyung University, 7 Hongji-dong, Jongno-gu, Seoul 110-743, Republic of Korea f Institute for Cellular and Structural Bioloy, Sun Yat-Sen University, Guangzhou, People’s Republic of China article info Article history: Received 4 July 2013 and in revised form 4 January 2014 Available online 16 January 2014 Keywords: D-Alanine-D-alanine ligase (DDL) Drug target Bacterial cell wall synthesis Xanthomonas oryzae pv. oryzae (Xoo) X-ray crystallography abstract D-Alanine-D-alanine ligase (DDL) catalyzes the biosynthesis of D-alanyl-D-alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We deter- mined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.3 and 2.0 Å. Similarly with other DDLs, the active site of XoDDL is formed by three loops from three domains at the center of enzyme. Compared with D-alanyl-D-alanine and ATP-bound TtDDL structure, the c-phosphate of ATP in XoDDL structure was shifted outside toward solution. We swapped the x-loop (loop3) of XoDDL with those of Escherichia coli and Helicobacter pylori DDLs, and measured the enzymatic kinetics of wild-type XoDDL and two mutant XoDDLs with the swapped x-loops. Results showed that the direct interactions between x-loop and other two loops are essential for the active ATP conformation for D-ala-phosphate formation. Ó 2014 Elsevier Inc. All rights reserved. Introduction Xanthomonas oryzae pv. oryzae (Xoo) 2 is a phytopathogen that causes Bacterial Blight (BB), one of the most destructive diseases affecting rice in most rice-cultivating countries. In 2006, BB resulted in a production loss worth 100 million dollars in South Korea alone. Unfortunately, there are no effective antibacterial agents against Xoo. In 2005, the complete genome sequence of Xoo was determined [1], providing valuable information for the selection of antibacterial target proteins. D-Alanine-D-alanine ligase (DDL) is one such drug target based on its essential role in the synthesis of the bacterial cell wall, and many efforts have been made to find potent inhibitors against DDL [2]. DDL is an enzyme that catalyzes the formation of the dipeptide D-alanyl-D-alanine (D-ala-D-ala) before it is added to the peptide chain of peptidoglycan, which is a vital component of bacterial cell walls. The dimerization of D-alanine begins with the nucleophilic attack of the first D-alanine (D-ala1) on the c-phosphate of ATP to yield a phosphorylated D-alanine intermediate [3]. This is followed by the attack of the amino group of the second D-alanine (D-ala2), which eliminates the phosphate and yields the product D-ala-D-ala [4]. DDLs possess three domains and two D-alanines and ATP bind to the crevice between the domains [5–7]. The ATP-binding site is located in the cleft between C-terminal and central domains, and two D-alanine binding sites are located in the intersection between N-terminal and C-terminal domains. Recently, DDL structures from Thermus thermophilus (TtDDL) [8] were reported, explaining that the catalytic mechanism of DDL involves conformational changes of the three domains and three loops during substrate binding. Especially, x-loop (loop3) donated two residues (Tyr and Lys) for substrate binding, and showed big conformation changes depend- ing on bound substrates or ligands. In the structure of Escherichia coli DDL (EcDDL), the x-loop was shown only in complex with both D-ala-D-ala phosphinate and ADP [9], and in Helicobacter pylori DLL (HpDDL) structure the x-loop had a completely open conformation pushed outward to the solution [5]. In terms of catalytic activity, EcDDL showed high activity and HpDDL possessed much lower activity [5,10]. In order to elucidate a new target structure for BB and to study the catalytic role of x-loop, we determined the crystal 0003-9861/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2014.01.009 ⇑ Corresponding author. Fax: +82 2 444 6707. E-mail address: lkang@konkuk.ac.kr (L.-W. Kang). 1 These authors contributed equally to this work. 2 Abbreviations used: Xoo, Xanthomonas oryzae pv. oryzae; BB, Bacteria Blight; DDL, D-alanine-D-alanine ligase; AMPPNP, Adenylyl-imidodiphosphate; D-ala-D-ala, D-ala- nyl-D-alanine; XoDDL, DDL from Xanthomonas oryzae pv. oryza; TtDDL, DDL from Thermus thermophilus; EcDDL, DDL from Escherichia coli; HpDDL, DLL from Helicobac- ter pylori; BaDDL, DDL from Bacillus anthracis; XoDDL-xEc, XoDDL that carried the x- loop of EcDDL; XoDDL-xHp, XoDDL carrying the x-loop of HpDDL. Archives of Biochemistry and Biophysics 545 (2014) 92–99 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi