Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme

• Published in: Biochemistry (Easton) Vol. 28; no. 3; pp. 969 - 975
• Main Authors: Tsou, Amy Y, Ransom, Stephen C, Gerlt, John A, Powers, Vincent M, Kenyon, George L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.02.1989
• Subjects: Affinity Labels - metabolism; Cloning, Molecular; Drug Resistance, Microbial - genetics; Genes; Genes, Bacterial; Isomerases - genetics; Mandelic Acids - metabolism; Mutation; Phenylpropionates - metabolism; Phenylpropionates - pharmacology; Pseudomonas - enzymology; Pseudomonas - genetics; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - growth & development; Racemases and Epimerases - genetics; Racemases and Epimerases - isolation & purification; Racemases and Epimerases - metabolism; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00429a008

The plasmid pSCR1 containing the gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) allows Pseudomonas aeruginosa (ATCC 15692) to grow on (R)-mandelate as its sole carbon source [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540]; the chromosome of the P. aeruginosa host apparently does not contain the gene for mandelate racemase but does contain genes for the remaining enzymes in the mandelate pathway and enables growth on (S)-mandelate as carbon source. However, in the presence of alpha-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCR1 can utilize (S)-mandelate but not (R)-mandelate as carbon source. This inhibition of growth on (R)-mandelate provides a metabolic selection for mutants that are resistant to alpha-phenylglycidate. When (R)-mandelate is used as carbon source and alpha-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCR1 grow slowly and appear on plates after several days. The plasmid isolated from these cells confers resistance to alpha-phenylglycidate on newly transformed cells of P. aeruginosa. This resistance to the affinity label is not due to a mutation within the primary structure of the enzyme. A single base change (C---A) located 87 bp upstream of the initiation codon for the gene for mandelate racemase was detected in three independent isolates of alpha-phenylglycidate-resistant colonies and appears responsible for a 30-fold increase in the amount of mandelate racemase encoded by the gene contained in the plasmid.