Abstract
Formylglutamate amidohydrolase (FGase) catalyzes the terminal reaction in the five-step pathway for histidine utilization in Pseudomonas putida. By this action, N-formyl-L-glutamate (FG) is hydrolyzed to produce L-glutamate plus formate. Urocanate, the first product in the pathway, induced all five enzymes, but FG was able to induce FGase alone, although less efficiently than urocanate did. This induction by FG resulted in the formation of an FGase with electrophoretic mobility identical to that of the FGase induced by urocanate. A 9.6-kilobase-pair HindIII DNA fragment containing the P. putida FGase gene was cloned into the corresponding site on plasmid pBEU1 maintained in Escherichia coli. Insertion of the fragment in either orientation on the vector resulted in expression, but a higher level was noted in one direction, suggesting that the FGase gene can be expressed from either of two vector promoters with different efficiencies or from a single vector promoter in addition to a less efficient Pseudomonas promoter. FGase was purified 1,110-fold from the higher-expression clone in a yield of 10% through six steps. Divalent metal ions stimulated activity, and among those tested (Co, Fe, Zn, Ca, Ni, Cd, Mn, and Mg), Co(II) was the best activator, followed by Fe(II). FGase exhibited a Km of 14 mM for FG and a specific activity of 100 mumol/min per mg of protein in the presence of 5 mM substrate and 0.8 mM CoCl2 at 30 degrees C. The enzyme was maximally active in the range of pH 7 to 8. FGase was found to be a monomer of molecular weight 50,000. N-Acetyl-L-glutamate was not a substrate for the enzyme, but both it and N-formyl-L-aspartate were competitive inhibitors of formylglutamate hydrolysis, exhibiting Ki values of 6 and 9 mM, respectively. The absence of FGase activity as an integral part of histidine breakdown in most other organisms and the somewhat uncoordinated regulation of FGase synthesis with that of the other hut enzymes in Pseudomonas suggest that the gene encoding its synthesis may have evolved separately from the remaining hut genes.
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