title: Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.
abstract: Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode.
authors: Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
authors: Tri Duc
authors: TD
authors: Ngo
authors: Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
authors: Binh
authors: B
authors: Van Le
authors: Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
authors: Vinod Kumar
authors: VK
authors: Subramani
authors: Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
authors: Chi My
authors: CM
authors: Thi Nguyen
authors: Korea Institute of Ocean Science and Technology, Ansan 426-744, Republic of Korea.
authors: Hyun Sook
authors: HS
authors: Lee
authors: Korea Institute of Ocean Science and Technology, Ansan 426-744, Republic of Korea.
authors: Yona
authors: Y
authors: Cho
authors: Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea. Electronic address: kyeongkyu@skku.edu.
authors: Kyeong Kyu
authors: KK
authors: Kim
authors: Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea. Electronic address: hyhwang3@gmail.com.
authors: Hye-Yeon
authors: HY
authors: Hwang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004789:Enzyme Activation; D004795:Enzyme Stability; D006867:Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure*; D008956:Models, Chemical; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure*; D011485:Protein Binding; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019712:Thermococcus / Q000145:classification / Q000201:enzymology*
chemical_list: D006867:Hydrolases; D010744:Phosphoric Monoester Hydrolases; C113022:haloalcohol dehalogenase
keywords: Crystal structure; HAD; Phosphatase; Substrate recognition motif