Cloning, expression and mutational analysis of the urocanase gene (hutU) from Pseudomonas putida

Abstract

The histidine‐utilizing hutU gene was isolated from a λ‐EMBL3 phage of a genomic library from Pseudomonas putida nicII and subcloned into the expression vector pT7‐7. Escherichia coli BL21 cells were transformed with the recombinant plasmid and produced a catalytically active protein, amounting to approximately 30% of the total protein in the crude cell‐free extract. The addition of NAD+ to the growth medium ensured the full occupation of active sites by the cofactor. This requires a mechanism for the transport of NAD+ into E. coli cells. Using the overproducing mutant a new, fast and efficient isolation procedure is described which yields electrophoretically homogeneous urocanase within two days. The yield of pure enzyme, based on the culture volume, has been improved 50–80‐fold compared with the traditional method. To investigate the possible role of cysteine residues in the catalysis or in the tight binding of the cofactor NAD+, six different mutants were prepared. In each mutant protein, one conserved cysteine was exchanged for alanine. The resulting clones were tested for the expression of urocanase with catalytic activity; the Km and Vmax values were determined. Only Cys410 was essential for catalysis. There was no detectable reconstitution or increase of activity after the addition of NAD+, either in the essential Cys/Ala mutant or the other mutant proteins. Electrospray‐mass spectroscopy of the wild‐type enzyme revealed that the coenzyme is not covalently bound to the protein and computational analysis showed no typical sequence for a mononucleotide‐binding domain like the Rossman fold. To obtain urocanase apoenzyme, P. putida nicII was transformed with pGP1‐2 and pTET7‐U and grown in nicotinate‐depleted medium. Like the mutant proteins, no activation of the apoform occurred after the addition of NAD+. These observations led us to postulate a new model for the non‐covalent but tight binding of NAD+ to the enzyme by ‘trapping’ the cofactor while folding the nascent protein. Copyright © 1993, Wiley Blackwell. All rights reserved