title: Crystal structures of vertebrate dihydropyrimidinase and complexes from Tetraodon nigroviridis with lysine carbamylation: metal and structural requirements for post-translational modification and function.
abstract: Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apoenzyme as well as two forms of the holoenzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified for the possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala(69)-Arg(74) and Met(158)-Met(165), located in the tunnel for the substrate entrance. The substrate/product tunnel is in the "open form" in the apo-TnDhp, in the "intermediate state" in the monometal TnDhp, and in the "closed form" in the dimetal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala(69)-Arg(74) dynamic loop. In addition, the structures of the dimetal TnDhp in complexes with hydantoin, N-carbamyl-β-alanine, and N-carbamyl-β-amino isobutyrate as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.
authors.affiliation: From the Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan.
authors.forename: Yin-Cheng
authors.initials: YC
authors.lastname: Hsieh
authors.affiliation: the Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30010, Taiwan.
authors.forename: Mei-Chun
authors.initials: MC
authors.lastname: Chen
authors.affiliation: the Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30010, Taiwan.
authors.forename: Ching-Chen
authors.initials: CC
authors.lastname: Hsu
authors.affiliation: the Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan,; the Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 911525.
authors.forename: Sunney I
authors.initials: SI
authors.lastname: Chan
authors.affiliation: the Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30010, Taiwan,. Electronic address: ysyang@mail.nctu.edu.tw.
authors.forename: Yuh-Shyong
authors.initials: YS
authors.lastname: Yang
authors.affiliation: From the Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan,; the Department of Physics, National Tsing Hua University, Hsinchu 30043, Taiwan, and; the Institute of Biotechnology and; University Center for Bioscience and Biotechnology, National Cheng Kung University, 1 University Road, Tainan City 701, Taiwan. Electronic address: cjchen@nsrrc.org.tw.
authors.forename: Chun-Jung
authors.initials: CJ
authors.lastname: Chen
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D029941:Fish Proteins / Q000737:chemistry* / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D007098:Imino Acids / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D023721:Tetraodontiformes
chemical_list: D029941:Fish Proteins; D020035:Holoenzymes; D007098:Imino Acids; C020184:N-(2-acetamido)iminodiacetic acid; D000581:Amidohydrolases; C020047:dihydropyrimidinase; D008239:Lysine
keywords: Crystal Structure; Crystallography; Metalloenzymes; Post-translational Modification; Protein Carboxylation; Protein Complexes