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

Abstract4-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 substrate to the C-5 position of 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. In order to characterize † This research was supported by the National Institutes of Health Grants GM-41239 (CPW) and AI-60915 (SDP), the Robert A.Welch Foundation grant F-1334 (CPW), and the Department of Defense Grant W81XWH0710445 USAMRAA (SDP). Use of the Advanced Photon Source is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract N. W-31-109-Eng-38. The Analytical Instrumentation Facility Core (College of Pharmacy, The University of Texas at Austin) is supported by an NIH Center grant ES07784. * Corresponding authors (CPW) Tel: 512-471-6198; Fax: 512-232-2606; whitman@mail.utexas.edu. (SDP) Tel: 303-871-2533; Fax: 303-871-2254; scott.pegan@du.edu. The atomic coordinates and structure factors have been deposited with the Brookhaven Protein Data Bank (PDB codes 3MB2). 1 Abbreviations: Ap, ampicillin; BSA, bovine serum albumin; dNTPs, deoxyribose nucleotide triphosphates; CHMI, 5-(carboxymethyl)-2-hydroxymuconate isomerase; CaaD, trans-3-chloroacrylic acid dehalogenase; cis-CaaD, cis-3-chloroacrylic acid dehalogenase; DSC, differential scanning calorimetry; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethane sulfonate; hh4-OT, heterohexamer 4-oxalocrotonate tautomerase; IPTG, isopropyl-β-D-thiogalactoside; Kn, kanamycin; LB, Luria-Bertani; MIF, macrophage migration inhibitory factor; MSAD, malonate semialdehyde decarboxylase; NCBI, National Center for Biotechnology Information; NMR, nuclear magnetic resonance; 4-OT, 4-oxalocrotonate tautomerase; PEG, polyethylene glycol; PPT, phenylpyruvate tautomerase; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SUPPORTING INFORMATION AVAILABLEThe expression, overproduction, and purification protocols for the hh4-OT are provided in the Supporting Information. The experimental procedures used for the construction, expression, overproduction, purification, and mass spectral analysis of the hh4-OT mutants and the construction and expression of the separate subunits of the hh4-OT are also provided in the Supporting Information. Finally, the molecular modeling studies are described. This material is available free of charge via the Internet at http://pubs.acs.org....

Kinetic and structural characterization of a cis -3-Chloroacrylic acid dehalogenase homologue in Pseudomonas sp. UW4: A potential step between subgroups in the tautomerase superfamily

“…The kinetic parameters are comparable to those obtained for 4-OT and greater than those obtained for cis -CaaD with a 400-fold greater k cat , 2-fold greater K m , and 194-fold greater k cat / K m . 36,37 …”

“… a The kinetic parameters were measured using the assay described in the text in 20 mM Na 2 HPO 4 buffer (pH 9) at 22 °C. b The kinetic parameters are from reference 36. c The kinetic parameters are from reference 37. …”

Identification and characterization of new family members in the tautomerase superfamily: Analysis and implications

“…In the course of ongoing database searches for new TSF members, a heterohexamer 4-OT (hh4-OT) was identified and characterized by mechanistic and structural studies [36]. Sequence analysis identified two “tautomerases” in the thermophile Chloroflexus aurantiacus J-10-fl.…”

“…The puzzle was solved when co-expression of the two genes produced a stable heterohexamer (verified by the crystal structure) where each dimer consists of an α and β-subunit [36]. Kinetic, mutagenesis, and crystallographic studies identified βPro-1, αArg-12, and αArg-40 as critical residues, and indicated that the hh4-OT has comparable kinetic parameters to those of 4-OT (using 1 ) and an analogous mechanism [36]. The change in oligomeric structure (homohexamer vs heterohexamer) does not appear to affect catalysis, but might be related to thermostability.…”

“…One curious difference between the two enzymes is that the hh4-OT lacks CaaD activity [36]. An examination of the active site regions of 4-OT and hh4-OT shows no significant differences except for the replacement of Phe-50 (in 4-OT) (Figure 2A) with αTrp-51 (in hh4-OT) (Figure 2B).…”