title: Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β-Keto Acid.
abstract: The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD.
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais , Belo Horizonte, 31270-901, Brazil.
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Guimarães
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais , Belo Horizonte, 31270-901, Brazil.
authors.forename: Juliana B
authors.initials: JB
authors.lastname: Coitinho
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais , Belo Horizonte, 31270-901, Brazil.
authors.forename: Débora M A
authors.initials: DM
authors.lastname: Costa
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais , Belo Horizonte, 31270-901, Brazil.
authors.forename: Simara S
authors.initials: SS
authors.lastname: Araújo
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin , Texas 78712-1071, United States.
authors.forename: Christian P
authors.initials: CP
authors.lastname: Whitman
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais , Belo Horizonte, 31270-901, Brazil.
authors.forename: Ronaldo A P
authors.initials: RA
authors.lastname: Nagem
mesh_terms: D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D003653:Decarboxylation; D007651:Keto Acids / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D000072417:Protein Domains; D016958:Pseudomonas putida / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D001426:Bacterial Proteins; D007651:Keto Acids; C021917:4-oxalocrotonate decarboxylase; D002262:Carboxy-Lyases; D008274:Magnesium