Cloning, expression and mutational analysis of the urocanase gene (hutU) from Pseudomonas putida.
The histidine-utilizing hutU gene was isolated from a lambda-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.
Lenz
M
M
Department of Biochemistry, University of Karlsruhe, Germany.
Rétey
J
J
D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine; D004252:DNA Mutational Analysis; D004793:Enzyme Reactivators; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D013058:Mass Spectrometry; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D017510:Protein Folding; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic; D014559:Urocanate Hydratase / Q000235:genetics* / Q000378:metabolism
D001051:Apoenzymes; D004793:Enzyme Reactivators; D011994:Recombinant Proteins; D009243:NAD; D014559:Urocanate Hydratase; D003545:Cysteine