title: Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics.
abstract: Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine, can be genetically encoded using engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows a dramatic loss in the enzyme's catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme.
authors.affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Vangmayee
authors.initials: V
authors.lastname: Sharma
authors.affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Yane-Shih
authors.initials: YS
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Wenshe R
authors.identifier: 0000-0002-7078-6534
authors.initials: WR
authors.lastname: Liu
mesh_terms: D000411:Alanine Racemase / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000031:analogs & derivatives / Q000235:genetics*; D006860:Hydrogen Bonding; D008239:Lysine / Q000031:analogs & derivatives / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
chemical_list: D006639:Histidine; D000411:Alanine Racemase; C456839:pyrrolysine; D008239:Lysine