Enzymatic synthesis of acyl‐acyl carrier protein and assay of acyl carrier protein

Spencer, A. K.; Greenspan, A. D.; Cronan, John E.

doi:10.1016/0014-5793(79)81019-4

Cited by 26 publications

(21 citation statements)

References 7 publications

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“…However, this value lies in the same range as the values for thioesterases of bacteria and of plant or animal tissues . With respect to its high substrate specificity citrate lyase deacetylase of RlioL~o~'.~e"~omoiius gclutimsu is distinct from the acyl-CoA hydrolases present in EscIicv+cliitr coli [30,33,34].…”

Section: Disc Ltssionmentioning

confidence: 99%

Citrate Lyase Deacetylase of Rhodopseudomonas gelatinosa. Isolation of the Enzyme and Studies on the Inhibition by l‐Glutamate

Giffhorn

Rode

Kuhn

et al. 1980

European Journal of Biochemistry

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Citrate lyase deacetylase or acetyl-5'-(acyl-carrier protein) enzyme thioester hydrolase (acetate) (EC 3.1.2. -), was purified 3100-fold wilh a yield of 3.8 0;; from cell extracts of Rlzoc~~pseu~~jn~oizus gelurinosu. The final enzyme preparation gave a single protein band upon polyacrylamide-gel electrophoresis in the absence or in the presence of sodium dodecylsulfate. The molecular weight of the native enzyme was estimated by gel filtration to be 14300 1000. Sodium dodecylsulfate/ polyacrylamide gel electrophoresis yielded a molecular weight of 7300 & 600 indicating that the enzyme consisted of two subunits.Citrate lyase deacetylase acted as an S-acetyl cnzyme thioesterhydrolase because it catalyzed the conversion of citrate lyase (S-acetyl form) into citrate lyase (sulfhydryl form) and acetate.Citrate lyase deacetylase was strongly inhibited by L-glutamate. The half-maximal inhibitor concentration was 7.5 x M, and a Ki-value of 1.2 x lop4 M was determined. The mode of inhibition appeared to be of the linear mixed type. L-Glutamate was bound by citrate lyase deacetylase but not by citrate lyase.The pool concentrations of r2-glutamate in R. gdrrinosu were 10 niM when citrate was present in substrate amounts and 2.7 mM after total consumption of citrate. Simulation of conditions in vivo using homogeneous enzyme preparations of citrate lyase and citrate lyase deacetylase, and glutamate concentrations of 10 mM and 2.7 mM respectively, revealed that the rate of citrate lyase inactivation increased from 8 ?fA within 15 min during growth on citrate to 50 06 within 15 min in the absence of citrate.The activity of citrate lyase in Rho~op~rurlo~zonus gel(i/inosci is regulated by covalent modification. Two enzymes are involved in this regulatory process. They catalyze the acetylation and deacetylation of citrate lyase [1,2] . In E. wrogenes the citrate lyase is also inactivated by deacetylation under certain conditions but this process is energy-dependent and is not catalyzed by a soluble deacetylase [6]. In this publication the purification of citrate lyase deacetylase, some of its kinetic properties and its regulation by 1.-glutamate are reported.

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Section: Disc Ltssionmentioning

confidence: 99%

Citrate Lyase Deacetylase of Rhodopseudomonas gelatinosa. Isolation of the Enzyme and Studies on the Inhibition by l‐Glutamate

Giffhorn

Rode

Kuhn

et al. 1980

European Journal of Biochemistry

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“…However, it remains to be determined whether the thioesterase fraction used in this study, referred to as thioesterase II, contained only thioesterase II (27) or perhaps contained more than one thioesterase. Several attempts have been made to elucidate the function of thioesterase II (26,28,29). So far, no specific function has been assigned to this enzyme, because the growth properties of E. coli cells seem to be unaffected when thioesterase II is overexpressed or its gene (tesB) is silenced (29).…”

Section: ␤-Oxidation Of Oleic Acid In E Colimentioning

confidence: 99%

An Alternative Pathway of Oleate β-Oxidation in Escherichia coli Involving the Hydrolysis of a Dead End Intermediate by a Thioesterase

Ren

Aguirre²,

Ntamack

et al. 2004

Journal of Biological Chemistry

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The degradation of 2-trans,5-cis-tetradecadienoylCoA, a metabolite of oleic acid, by the purified complex of fatty acid oxidation from Escherichia coli was studied to determine how much of the metabolite is converted to 3,5-cis-tetradecadienoyl-CoA and thereby diverted from the classical, isomerase-dependent pathway of oleate ␤-oxidation. Approximately 10% of the 2,5-intermediate was converted to the 3,5-isomer. When the latter compound was allowed to accumulate, it strongly inhibited the flux through the main pathway. Since ⌬ 3,5 ,⌬ 2,4 -dienoyl-CoA isomerase was not detected in E. coli cells grown on oleate, the 3,5-intermediate cannot be metabolized via the reductase-dependent pathway. However, it was hydrolyzed by a thioesterase, which was most active with 3,5-cis-tetradecadienoyl-CoA as substrate and which was induced by growth of E. coli on oleate. An analysis of fatty acids present in the medium after growth of E. coli on oleate revealed the presence of 3,5-tetradecadienoate, which was not detected after cells were grown on palmitate or glucose. Altogether, these data prompt the conclusion that oleate is mostly degraded via the classical, isomerase-dependent pathway in E. coli but that a small amount of 2-trans,5-cistetradecadienoyl-CoA is diverted from the pathway via conversion to 3,5-cis-tetradecadienoyl-CoA by ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase. The 3,5-intermediate, which would strongly inhibit ␤-oxidation if allowed to accumulate, is hydrolyzed, and the resultant 3,5-tetradecadienoate is excreted into the growth medium. This study provides evidence for the novel function of a thioesterase in ␤-oxidation.

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“…Lauroyl-and palmitoleoyl-acyl carrier proteins (ACP) were synthesized from commercial ACP using recombinant E. coli acyl-ACP synthase (30). Previous procedures (31) were modified by including an additional purification step (an octyl-Sepharose column) (32) to remove residual non-acylated ACP, which may be present at higher amounts with unsaturated fatty acids because of the reduced activity of acyl-ACP synthase with palmitoleate as the substrate (33).…”

Section: Materials-[␥-mentioning

confidence: 99%

An Escherichia coli Mutant Lacking the Cold Shock-induced Palmitoleoyltransferase of Lipid A Biosynthesis

Vorachek-Warren¹,

Carty²,

Lin³

et al. 2002

Journal of Biological Chemistry

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An acyltransferase induced by cold shock in Escherichia coli, designated LpxP, incorporates a palmitoleoyl moiety into nascent lipid A in place of the secondary laurate chain normally added by LpxL(HtrB) (Carty, S. M., Sreekumar, K. R., and Raetz, C. R. H. (1999) J. Biol. Chem. 274, 9677-9685). To determine whether the palmitoleoyl residue alters the properties of the outer membrane and imparts physiological benefits at low growth temperatures, we constructed a chromosomal insertion mutation in lpxP, the structural gene for the transferase. Membranes from the lpxP mutant MKV11 grown at 12°C lacked the cold-induced palmitoleoyltransferase present in membranes of cold-shocked wild type cells but retained normal levels of the constitutive lauroyltransferase encoded by lpxL. When examined by mass spectrometry, about two-thirds of the lipid A molecules isolated from wild type E. coli grown at 12°C contained palmitoleate in place of laurate, whereas the lipid A of cold-adapted MKV11 contained only laurate in amounts comparable with those seen in wild type cells grown at 30°C or above. To probe the integrity of the outer membrane, MKV11 and an isogenic wild type strain were grown at 30 or 12°C and then tested for their susceptibility to antibiotics. MKV11 exhibited a 10-fold increase in sensitivity to rifampicin and vancomycin at 12°C compared with wild type cells but showed identical resistance when grown at 30°C. We suggest that the palmitoleoyltransferase may confer a selective advantage upon E. coli cells growing at lower temperatures by making the outer membrane a more effective barrier to harmful chemicals.A unique glycolipid known as lipopolysaccharide is the major component of the outer surface of the outer membranes of Gram-negative bacteria and forms a barrier around the cell (1-3). Compounds with molecular weights of less than 600 penetrate the outer membrane via porins, but larger, potentially harmful agents, including many antibiotics, are excluded (2). Lipid A is the hydrophobic anchor of the lipopolysaccharide molecule (1, 4 -6). In wild type Escherichia coli grown at 30°C or above, lipid A consists of a ␤,1Ј-6-linked disaccharide of glucosamine that is phosphorylated at the 1-and 4Ј-positions ( Fig. 1) (1, 4 -6). E. coli lipid A usually contains six acyl chains (1, 4). (R)-3-Hydroxymyristoyl groups are located at positions 2, 3, 2Ј, and 3Ј of the glucosamine disaccharide, and two short saturated acyl chains are attached to the (R)-3-hydroxymyristoyl groups of the distal unit, forming acyloxyacyl moieties (Fig. 1, "Normal" lipid A) (1, 4 -6). Laurate is linked to the (R)-3-hydroxymyristoyl residue at position 2Ј, and myristate is similarly attached at position 3Ј (1, 4 -6). These so-called "secondary" acyl chains are the same in wild type E. coli and Salmonella.Two inner membrane enzymes, LpxL(HtrB) and LpxM(MsbB), which act late in the lipid A pathway (Fig. 1), are responsible for the incorporation of laurate and myristate, respectively (7-9). These enzymes require the presence of a 3-deoxy-D-manno-oct...

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Enzymatic synthesis of acyl‐acyl carrier protein and assay of acyl carrier protein

Cited by 26 publications

References 7 publications

Citrate Lyase Deacetylase of Rhodopseudomonas gelatinosa. Isolation of the Enzyme and Studies on the Inhibition by l‐Glutamate

Citrate Lyase Deacetylase of Rhodopseudomonas gelatinosa. Isolation of the Enzyme and Studies on the Inhibition by l‐Glutamate

An Alternative Pathway of Oleate β-Oxidation in Escherichia coli Involving the Hydrolysis of a Dead End Intermediate by a Thioesterase

An Escherichia coli Mutant Lacking the Cold Shock-induced Palmitoleoyltransferase of Lipid A Biosynthesis

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