The 4-oxalocrotonate tautomerase family of enzymes: how nature makes new enzymes using a beta-alpha-beta structural motif.
4-Oxalocrotonate tautomerase (4-OT) catalyzes the isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers. The enzyme is part of a plasmid-encoded pathway, which enables bacteria harboring the plasmid to use various aromatic hydrocarbons as their sole sources of carbon and energy. Among isomerases and enzymes in general, 4-OT is unusual for two reasons: it has one of the smallest known monomer sizes (62 amino acids) and the amino-terminal proline functions as the catalytic base. In addition to Pro-1, three other residues (Arg-11, Arg-39, and Phe-50) have been identified as critical catalytic residues by kinetic analysis, site-directed mutagenesis, chemical synthesis, NMR, and crystallographic studies. Arginine-39 functions as the general acid catalyst (assisted by an ordered water molecule) in the reaction while Arg-11 plays a role in substrate binding and facilitates catalysis by acting as an electron sink. Finally, the hydrophobic nature of the active site, which lowers the pK(a) of Pro-1 to approximately 6.4 and provides a favorable environment for catalysis, is largely maintained by Phe-50. 4-OT is also the title enzyme of the 4-OT family of enzymes. The chromosomal homologues in this family are composed of monomers ranging in size from 61 to 79 amino acids, which code a beta-alpha-beta structural motif. The homologues all retain Pro-1 and generally have an aromatic or hydrophobic amino acid at the Phe-50 position. Characterization of representative members has uncovered mechanistic and structural diversity. A new activity, a trans-3-chloroacrylic acid dehalogenase, has been identified in addition to the previously known tautomerase and isomerase activities. Two new structures have also been found, along with the 4-OT hexamer. The dehalogenase functions as a heterohexamer while the Escherichia coli homologue, designated YdcE, functions as a dimer. Moreover, both 4-OT and the Bacillus subtilis homologue, designated YwhB, exhibit low-level dehalogenase activity. Amplification of this activity could have produced the full-fledged dehalogenase. The sum of these observations indicates that Nature uses the beta-alpha-beta structural motif as a building block in a variety of manners to create new enzymes.
Whitman
Christian P
CP
Medicinal Chemistry Division, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712-1074, USA. whitman@mail.utexas.edu
D001412:Bacillus subtilis / Q000201:enzymology; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D006867:Hydrolases / Q000378:metabolism; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D010957:Plasmids; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016958:Pseudomonas putida / Q000201:enzymology
D006867:Hydrolases; C077721:trans-3-chloroacrylic acid dehalogenase; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases