Genetic and biochemical characterization of an Escherichia coli K-12 mutant deficient in acyl-coenzyme A thioesterase II

Abstract

Mutants of Escherichia coli deficient in thioesterase II activity were isolated by taking advantage of the fact that thioesterase I specifically hydrolyzes long-chain (C12 to C18) acyl coenzyme A (CoA) esters but is unable to cleave the short-chain substrate decanoyl-CoA. One of these lesions (designated tesBi) reduces thioesterase II activity to about 10% of the normal level. The mutant enzyme activity was abnormally labile to temperature, but It was normal in all the other characteristics examined (pH optimum, Km for decanoyl-CoA, molecular weight). The level of thioesterase I activity was unaffected by the tesBI lesion. The tesB locus was mapped with a closely linked Tn1O insertion. tesB was mapped to minute 10 of the E. coli linkage map, close to the lon locus. The clockwise gene order is Ion tesB acrA dnaZ. The tesB mutation is recessive. We found no phenotype for the mutation. The fatty acid compositions of the phospholipids, lipid A, and lipoprotein components are normal in thioesterase II mutants. These data show that thioesterases I and II of E. coli are encoded by different genetic loci and strongly suggest that tesB is the structural gene for thioesterase II.

Identification of a Hotdog Fold Thioesterase Involved in the Biosynthesis of Menaquinone in Escherichia coli

“…The remaining hotdog fold proteins, namely, PaaI, TesB, YbaW, YbdB (or EntH), YbgC, YciA, YdiI, YigI, and YiiD, are all thioesterases. Among them, YdiI, YigI, and YiiD do not have a known physiological role, whereas YciA and TesB are suggested to be involved in lipid metabolism (36)(37)(38) and YbgC is involved in the Tol-Pal system without a clearly defined role. The physiological functions of other thioesterases are more certain: YbdB (EntH) has been shown to serve as a type II thioesterase in enterobactin biosynthesis (26,33), PaaI is involved in the phenylacetate degradation pathway (39,40), and YbaW has recently been shown to be the third thioesterase (thioesterase III) in fatty acid ␤-oxidation (41).…”

Thioesterase II of Escherichia coli Plays an Important Role in 3-Hydroxydecanoic Acid Production

“…Surprisingly, two thioesterases whose activities are similar to those of E. coli were found in Rhodopseudomonas sphaeroides (4). Similar thioesterase complementation occurring in dissimilar bacteria suggests that these enzymes play important roles (22). However, the physiological function of thioesterase II was not fully understood, as no obvious physiological or biochemical defect was observed in E. coli with tesB overexpression or deletion (21,22).…”

“…Reduction of DTNB by CoA liberated in the thioesterase reaction was measured at 412 nm (22). The initial rates were measured using a recording spectrophotometer (Specord 200; Analytik Jena AG, Jena, Germany) (22). A unit of enzyme activity was defined as the amount of the enzyme catalyzing cleavage of 1 mol of decanoyl-CoA per min under the above-mentioned conditions.…”

“…The assay for thioesterase II contained (per milliliter) 0.1 mmol Tris-hydrochloride buffer (pH 8.0), 100 nmol 5,5Ј-dithiobis(2-nitrobenzoic acid) (DTNB; Sigma), 20 nmol decanoyl-CoA (Sigma), and 100 l of crude cell extract. Reduction of DTNB by CoA liberated in the thioesterase reaction was measured at 412 nm (22). The initial rates were measured using a recording spectrophotometer (Specord 200; Analytik Jena AG, Jena, Germany) (22).…”

“…A unit of enzyme activity was defined as the amount of the enzyme catalyzing cleavage of 1 mol of decanoyl-CoA per min under the above-mentioned conditions. The molar extinction coefficient of reduced DTNB was taken as 13,600 (22).…”