Cloning, DNA sequence analysis, and expression in Escherichia coli of the gene for mandelate racemase from Pseudomonas putida

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Abstract
The gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) was cloned in Pseudomonas aeruginosa (ATCC 15692). The selection for the cloned gene was based upon the inability of P. aeruginoas to grow on (R)-mandelate as sole carbon source by irtue of the absence of mandelate racemase in its mandelate pathway. Fragments of P. putida DNA obtained by digestion of chromosomal DNA with Sau3A were ligated into the BamHI site of the Gram-negative vector pKT230 and transformed into the P. aeruginosa host. A transformant able to utilize (R)-mandelate as sole carbon source was characterized, and the plasmid was found to contain approximately five kilobase pairs of P. putida DNA. Subcloning of this DNA revealed the position of the gene for the racemase within the cloned DNA from P. putida. The dideoxy-DNA sequencing procedure was used to determine the sequence of the gene and its translated sequence. The amino acid sequence and molar weight for mandelate racemase deduced from the gene sequence (38,570) are in excellent agreement with amino acid composition and molecular weight data for the polypeptide recently determined with enzyme isolated from P. putida; these recent determinations of the polypeptide molecualr weight differ significantly from the originally reported value of 69,500 [Fee, Judith A., Heeman, G. D. and Kenyon, G. L.(1974) Biochemistry 13, 2528], which was used to demonstrate that .alpha.-phenylglycidate, an active site directed irreversible inhibitor, binds to the enzyme with a stoichiometry of 1:1. The 5''-flanking squence is homologous to the consensus sequences for Escherichia coli promoters as well as portions of promoter sequences reported for some Pseudomonas genes, suggesting that the gene for mandelate racemase is the first in the mandelate operon. Placement of the gene behind the trc promoter permitted expression of soluble and catalytically active mandelate racemase in E. coli, with the levels of expression being approximately 1% of the total cellular protein in induced cells; homogeneous, catalytically active enzyme has been isolated from extracts of these induced cells. Comparison of the mandelate racemase encoding DNA sequence and the deduced amino acid sequence with available DNA and amino acid sequence data bases revealed no significant sequence homologies.