Escherichia coli thioesterase I, molecular cloning and sequencing of the structural gene and identification as a periplasmic enzyme.

The structural gene for Escherichia coli thioesterase I (called tesA) has been cloned by use of sequence data obtained from the purified protein. The tesA gene was mapped at 530 kilobase pair of the E. coli physical map (minute 11.6 of E. coli genetic map). The DNA sequence of the tesA gene was obtained and the deduced protein sequence showed that thioesterase I consists of 182 amino acids and has a molecular mass of 20.5 kDa. Comparison of the DNA and protein sequence data suggested that a leader sequence of 26 amino acid residues is cleaved from the primary translation product, and this processing was confirmed by NH2-terminal sequencing of the primary translation product synthesized in vitro. These data predicted that thioesterase I (long believed to be a cytoplasmic protein) is exported to the cell periplasm, a prediction supported by release of the enzyme from cells upon osmotic shock. The TesA protein sequence does not exhibit any significant overall sequence similarity with other known proteins, although the sequence does contain two small sequence elements found in several other thioesterases. One of these elements is a sequence similar to the serine esterase active sites found in serine proteases and four other thioesterases. A serine residue within this TesA element was shown to be covalently labeled with [3H] diisopropyl fluorophosphate, a potent inhibitor of TesA activity. The second sequence element is a histidine-containing sequence found close to the carboxyl terminus that is also found in the carboxyl termini of the four known active serine thioesterases. The physiological role of thioesterase I is unclear. A strain carrying a null mutation of the tesA gene was constructed and found to have no growth phenotype. Moreover, a strain carrying a plasmid that gave massive overproduction of TesA (approximately 100-fold higher than that of the wild type) also grew normally. In addition a strain containing double null mutations in both tesA and tesB (the structural gene for E. coli thioesterase II) also failed to display any growth phenotype. Analysis of the fatty acid compositions of phospholipid, lipid A, and lipoprotein of the above strains showed no significant changes from a wild type strain.