title: Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase.
abstract: 4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, The University of Texas, Austin, Texas 78712-1074, USA.
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Poelarends
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Almrud
authors.forename: Hector
authors.initials: H
authors.lastname: Serrano
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Darty
authors.forename: William H
authors.initials: WH
authors.lastname: Johnson
authors.forename: Marvin L
authors.initials: ML
authors.lastname: Hackert
authors.forename: Christian P
authors.initials: CP
authors.lastname: Whitman
mesh_terms: D001665:Binding Sites; D003460:Crystallization; D019143:Evolution, Molecular; D006867:Hydrolases / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D016958:Pseudomonas putida / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D006867:Hydrolases; C077721:trans-3-chloroacrylic acid dehalogenase; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases