The founding member of this group is experimentally characterized and found in a bacterial pathway for the degredation of aromatic hydrocarbons. It exists as a hexamer of 62 amino acid subunits and converts 2-hydroxymuconate to 2-oxo-3-hexenedioate.
Lorenzo S., et. al.
4-Oxalocrotonate Tautomerase, an Enzyme Composed of 62 Amino Acid Residues per Monomer
▸ Abstract
ThexylHgene encoding 4-oxalocrotonate tautomer- ase (4-OT) has been located on a subclonoef the Pseu- domonasputida mt-2TOL plasmid pWWO and inserted into an Escherichia coli expression vector. Severalof the genes of the metafission pathway encoded by pWWO have beencloned in E. coli,but the overexpres- sion of their gene products has met with limited suc- cess. By utilizing theE. coli alkaline phosphatase pro- moter (phoA)coupled with the properpositioning of a ribosome-binding region, we areable to express func- tional 4-OT inyields of at least 10 mg of pure enzyme/ liter of culture. 4-OT has been previously character- ized and shown to be an extremely efficient catalyst (Whitman, C. P.,Aird, B. A., Gillespie, W. R., and Stolowich, N. J. (1991)J.Am. Chem. SOC.113,3154- 3162). Kinetic and physical characterizationof the E. coli-expressed protein show that it is identicalwith that of the 4-OT isolated from P.putida. The func- tional unit is apparently a pentamer of identical sub- units, each consistingof only 62 amino acid residues. This is the smallest enzyme subunit reported to date. The aminoacidsequence,determinedin part from automated Edman degradation and alsodeduced from the primarysequence of xylH, did notshow homology with anyof the sequences inthe current datbaases nor with any of the sequences of enzymes that catalyze similar reactions. We propose that theactive siteof 4- OT may be established by an overlapof subunits and comprisedofamino acid residues belongintgo several, if not all, of the subunits.
