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

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 (5)-mandelate as carbon source. However, in the presence of α-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCRl 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 α-phenylglycidate. When (R)-mandelate is used as carbon source and α-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCRl grow slowly and appear on plates after several days. The plasmia isolated from these cells confers resistance to α-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 α-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. This mutation is within a sequence that is homologous to the consensus sequence observed for promoters in Escherichia coli; in accord with the presumed presence of a promoter sequence, nuclease S₁ mapping reveals that initiation of transcription begins 11 ± 1 bp downstream of the position of the mutation. However, nuclease S₁ mapping of mRNA isolated from P. putida induced for synthesis of the enzymes of mandelate pathway does not reveal significant amounts of a transcript starting at this potential promoter sequence. Thus, in P. putida the promoter for the mandelate racemase gene lies further upstream, suggesting that mandelate racemase is not the first gene in the putative mandelate operon. Large amounts of homogeneous mandelate racemase having the same N-terminal sequence as enzyme isolated from P. putida can be easily isolated from the cells of P. aeruginosa transformed with the plasmid containing the promoter mutation.