Cloning, DNA sequence analysis, and expression in Escherichia coli of the gene for mandelate racemase from Pseudomonas putida.
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. aeruginosa to grow on (R)-mandelate as sole carbon source by virtue 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 molecular 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 molecular weight differ significantly from the originally reported value of 69,500 [Fee, Judith A., Hegeman, G.D., & 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Ransom
S C
SC
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143.
Gerlt
J A
JA
Powers
V M
VM
Kenyon
G L
GL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D007535:Isomerases / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D019998:Racemases and Epimerases / Q000235:genetics*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D007535:Isomerases; D019998:Racemases and Epimerases; C021540:mandelate racemase