4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer

• Published in: The Journal of biological chemistry Vol. 267; no. 25; pp. 17716 - 17721
• Main Authors: CHEN, L. H, KENYON, G. L, CURTIN, F, HARAYAMA, S, BEMBENEK, M. E, GHOLAMHOSSEIN HAJIPOUR, WHITMAN, C. P
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05-09-1992
• Subjects: 4-oxalocrotonate tautomerase; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Chromatography, Ion Exchange; Cloning, Molecular; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; genes; Genes, Bacterial; Genetic Vectors; Isomerases; Isomerases - genetics; Isomerases - isolation & purification; Isomerases - metabolism; Kinetics; Molecular Sequence Data; nucleotide sequence; Oligodeoxyribonucleotides; Plasmids; Polymerase Chain Reaction; prediction; Pseudomonas putida; Pseudomonas putida - enzymology; Pseudomonas putida - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; xylH gene; Pseudomonadales; Nucleotide sequence; Enzyme; Gene product; Bacteria; Pseudomonadaceae; Isolation; Molecular cloning; Gene expression; Pseudomonas putida; Aminoacid sequence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)37101-7

The xylH gene encoding 4-oxalocrotonate tautomerase (4-OT) has been located on a subclone of the Pseudomonas putida mt-2 TOL
plasmid pWW0 and inserted into an Escherichia coli expression vector. Several of the genes of the metafission pathway encoded
by pWW0 have been cloned in E. coli, but the overexpression of their gene products has met with limited success. By utilizing
the E. coli alkaline phosphatase promoter (phoA) coupled with the proper positioning of a ribosome-binding region, we are
able to express functional 4-OT in yields of at least 10 mg of pure enzyme/liter of culture. 4-OT has been previously characterized
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 characterization of the E. coli-expressed protein show that it is
identical with that of the 4-OT isolated from P. putida. The functional unit is apparently a pentamer of identical subunits,
each consisting of only 62 amino acid residues. This is the smallest enzyme subunit reported to date. The amino acid sequence,
determined in part from automated Edman degradation and also deduced from the primary sequence of xylH, did not show homology
with any of the sequences in the current data bases nor with any of the sequences of enzymes that catalyze similar reactions.
We propose that the active site of 4-OT may be established by an overlap of subunits and comprised of amino acid residues
belonging to several, if not all, of the subunits.