3MB2

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

Summary for 3MB2

• Entry DOI: 10.2210/pdb3mb2/pdb
• Descriptor: 4-oxalocrotonate tautomerase family enzyme - alpha subunit, 4-oxalocrotonate tautomerase family enzyme - beta subunit, SULFATE ION, ... (4 entities in total)
• Functional Keywords: trans-3-chloroacrylic acid dehalogenase, caad, dehalogenase, hydrolase, 4-oxalocrotonate tautomerase, 4-ot, heterohexamer, isomerase
• Biological source: Chloroflexus aurantiacus; More
• Total number of polymer chains: 12
• Total formula weight: 95897.83

Authors

Burks, E.A.,Fleming, C.D.,Mesecar, A.D.,Whitman, C.P.,Pegan, S.D. (deposition date: 2010-03-24, release date: 2010-06-02, Last modification date: 2023-09-06)

Primary citation

Burks, E.A.,Fleming, C.D.,Mesecar, A.D.,Whitman, C.P.,Pegan, S.D.
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
Biochemistry, 49:5016-5027, 2010

PubMed 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.

PubMed: 20465238
DOI: 10.1021/bi100502z

Experimental method

X-RAY DIFFRACTION (2.41 Å)