2FM7

Evolution of Enzymatic Activity in the Tautomerase Superfamily: Mechanistic and Structural Consequences of the L8R Mutation in 4-Oxalocrotonate Tautomerase

Summary for 2FM7

• Entry DOI: 10.2210/pdb2fm7/pdb
• Descriptor: 4-Oxalocrotonate Tautomerase, CHLORIDE ION (3 entities in total)
• Functional Keywords: 4-oxalocrotonate; tautomerase; 4-ot; homo-hexamer; dehalogenase; mutant; l8r, transferase
• Biological source: Pseudomonas putida
• Total number of polymer chains: 6
• Total formula weight: 40629.85

Authors

Almrud, J.J.,Hackert, M.L. (deposition date: 2006-01-08, release date: 2006-09-26, Last modification date: 2023-08-30)

Primary citation

Poelarends, G.J.,Almrud, J.J.,Serrano, H.,Darty, J.E.,Johnson, W.H.,Hackert, M.L.,Whitman, C.P.
Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase
Biochemistry, 45:7700-7708, 2006

PubMed 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.

PubMed: 16784221
DOI: 10.1021/bi0600603

Experimental method

X-RAY DIFFRACTION (2.8 Å)