Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

• DOI: 10.1016/s0021-9258(18)45905-4
• Publication Date: 2021-01-05T09:33:13Z
• Authors: G S Zhao; T H Xia; R S Fischer; R A Jensen
• Keywords: Cloning (programming); Porphobilinogen synthase

The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph Escherichia coli. highly expressed in E. coli due to the use high-copy number vector pUC18. P. purified electrophoretic homogeneity. latter enzyme exhibited identical physical and biochemical properties as those obtained for aeruginosa. activity ratios prephenate arogenate remained constant (about 3.3-fold) throughout purification, thus demonstrating single protein having broad substrate specificity. Km values 0.42 mM 0.22 L-arogenate, respectively. pheC 807 base pairs length, with calculated molecular mass 30,480 daltons. This compares value 29.5 kDa determined sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since native filtration 72 kDa, probably is homodimer. Comparison deduced amino acid sequence dehydratases Corynebacterium glutamicum, Bacillus subtilis, coli, stutzeri standard pairwise alignments did not establish obvious homology. However, more detailed analysis revealed conserved motif (containing threonine residue known be essential catalysis) that shared all proteins.