title: Kinetic and structural characterization of a heterohexamer 4-oxalocrotonate tautomerase from Chloroflexus aurantiacus J-10-fl: implications for functional and structural diversity in the tautomerase superfamily .
abstract: 4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of the substrate to the C-5 position of the product. 4-OT, a homohexamer from Pseudomonas putida mt-2, is the most extensively studied 4-OT isozyme and the founding member of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a coded amino acid sequence in each that had been annotated as a tautomerase-like protein but lacked Pro-1. However, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. To characterize this group of proteins, two genes from Chloroflexus aurantiacus J-10-fl were cloned, and the corresponding proteins were expressed. Kinetic, biochemical, and X-ray structural analyses show that the two expressed proteins form a functional heterohexamer 4-OT (hh4-OT), composed of three alphabeta dimers. Like the P. putida enzyme, hh4-OT requires the amino-terminal proline and two arginines for the conversion of 2-hydroxymuconate to the product, implicating an analogous mechanism. In contrast to 4-OT, hh4-OT does not exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-chloroacrylic acid dehalogenase (CaaD). Characterization of hh4-OT enables functional assignment of the related enzymes, highlights the diverse ways the beta-alpha-beta building block can be assembled into an active enzyme, and provides further insight into the molecular basis of the low-level CaaD activity in 4-OT.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Burks
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Fleming
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mesecar
authors.forename: Christian P
authors.initials: CP
authors.lastname: Whitman
authors.forename: Scott D
authors.initials: SD
authors.lastname: Pegan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D041861:Chloroflexus; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D007527:Isoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases
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