title: Crystal structure and biochemical studies of Brucella melitensis 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase.
abstract: The prokaryotic 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), a process that plays a key role in several metabolic pathways. Its absence in all mammalian species has implicated this enzyme as a promising target for antimicrobial drug design. Here, we report the crystal structure of BmMTAN in complex with its product adenine at a resolution of 2.6 Å determined by single-wavelength anomalous dispersion method. 11 key residues were mutated for kinetic characterization. Mutations of Tyr134 and Met144 resulted in the largest overall increase in Km, whereas mutagenesis of residues Glu18, Glu145 and Asp168 completely abolished activity. Glu145 and Asp168 were identified as active site residues essential for catalysis. The catalytic mechanism and implications of this structure for broad-based antibiotic design are discussed.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Xusheng
authors.initials: X
authors.lastname: Kang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Daohua
authors.initials: D
authors.lastname: Jiang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Li
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xianping
authors.initials: X
authors.lastname: Wang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Food Science and Engineering College, Beijing University of Agriculture, Beijing 102206, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Zeliang
authors.initials: Z
authors.lastname: Chen
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: zhangc@ibp.ac.cn.
authors.forename: Xuejun C
authors.initials: XC
authors.lastname: Zhang
mesh_terms: D000225:Adenine / Q000737:chemistry / Q000378:metabolism; D017347:Brucella melitensis / Q000737:chemistry / Q000201:enzymology*; D002006:Brucellosis / Q000382:microbiology; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003839:Deoxyadenosines / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D013872:Thionucleosides / Q000378:metabolism
chemical_list: D003839:Deoxyadenosines; D013872:Thionucleosides; C008500:5'-methylthioadenosine; D009699:N-Glycosyl Hydrolases; C019606:adenosylhomocysteine nucleosidase; D000225:Adenine
keywords: Brucella; Crystal structure; MTAN