Expression, purification, and characterization of the Mycobacterium tuberculosis acyl carrier protein, AcpM. (1/22)

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: the multifunctional polypeptide, FASI, in which the acyl carrier protein (ACP) domain forms an integral part of the polypeptide, and the dissociated FASII system, which is composed of monofunctional enzymes and a discrete ACP (AcpM). In order to characterize enzymes of the FASII system, large amounts of AcpM are required to generate substrates such as holo-AcpM, malonyl-AcpM and acyl-AcpM. The M. tuberculosis acpM gene was overexpressed in Escherichia coli and AcpM purified, yielding approximately 15-20 mg/l of culture. Analysis of AcpM by mass spectrometry, N-terminal sequencing, amino acid analysis, and gas chromatography indicated the presence of three species, apo-, holo-, and acyl-AcpM, the former comprising up to 65% of the total pool. The apo-AcpM was purified away from the in vivo generated holo- and acyl-forms, which were inseparable and heterogeneous with respect to acyl chain lengths. Once purified, we were able to convert apo-AcpM into holo- and acyl-forms. These procedures provide the means for the preparation of the large quantities of AcpM and derivatives needed for characterization of the purified enzymes of the mycobacterial FASII system.  (+info)

Site-directed mutagenesis of acyl carrier protein (ACP) reveals amino acid residues involved in ACP structure and acyl-ACP synthetase activity. (2/22)

Acyl carrier protein (ACP) interacts with many different enzymes during the synthesis of fatty acids, phospholipids, and other specialized products in bacteria. To examine the structural and functional roles of amino acids previously implicated in interactions between the ACP polypeptide and fatty acids attached to the phosphopantetheine prosthetic group, recombinant Vibrio harveyi ACP and mutant derivatives of conserved residues Phe-50, Ile-54, Ala-59, and Tyr-71 were prepared from glutathione S-transferase fusion proteins. Circular dichroism revealed that, unlike Escherichia coli ACP, V. harveyi-derived ACPs are unfolded at neutral pH in the absence of divalent cations; all except F50A and I54A recovered native conformation upon addition of MgCl(2). Mutant I54A was not processed to the holo form by ACP synthase. Some mutations significantly decreased catalytic efficiency of ACP fatty acylation by V. harveyi acyl-ACP synthetase relative to recombinant ACP, e.g. F50A (4%), I54L (20%), and I54V (31%), whereas others (V12G, Y71A, and A59G) had less effect. By contrast, all myristoylated ACPs examined were effective substrates for the luminescence-specific V. harveyi myristoyl-ACP thioesterase. Conformationally sensitive gel electrophoresis at pH 9 indicated that fatty acid attachment stabilizes mutant ACPs in a chain length-dependent manner, although stabilization was decreased for mutants F50A and A59G. Our results indicate that (i) residues Ile-54 and Phe-50 are important in maintaining native ACP conformation, (ii) residue Ala-59 may be directly involved in stabilization of ACP structure by acyl chain binding, and (iii) acyl-ACP synthetase requires native ACP conformation and involves interaction with fatty acid binding pocket residues, whereas myristoyl-ACP thioesterase is insensitive to acyl donor structure.  (+info)

Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis. (3/22)

Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10 S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [(32)P]azido-ATP showed labelling of a 35 kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35 kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The K(m) values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7 microM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10 S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.  (+info)

A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy. (4/22)

The formation of S-hydroxymethylglutathione from formaldehyde and glutathione is a central reaction in the consumption of the cytotoxin formaldehyde in some methylotrophic bacteria as well as in many other organisms. We describe here the discovery of an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction. The rates of S-hydroxymethylglutathione formation and cleavage were determined under equilibrium conditions via two-dimensional proton exchange NMR spectroscopy. The pseudo first order rate constants k(1)* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k(1)* was found to be 0.02 s(-1) for the spontaneous reaction. A 10-fold increase of the rate constant was observed upon addition of cell extract from P. denitrificans grown in the presence of methanol corresponding to a specific activity of 35 units mg(-1). Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 units mg(-1). The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene gfa is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.  (+info)

Functional role of fatty acyl-coenzyme A synthetase in the transmembrane movement and activation of exogenous long-chain fatty acids. Amino acid residues within the ATP/AMP signature motif of Escherichia coli FadD are required for enzyme activity and fatty acid transport. (5/22)

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP forming; EC ) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif ((213)YTGGTTGVAKGA(224) and (356)GYGLTE(361)) placing it in the superfamily of adenylate-forming enzymes. A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45% wild type activity (fadD(G216A), fadD(T217A), fadD(G219A), and fadD(K222A)) and 2) those with 10% or less wild-type activity (fadD(Y213A), fadD(T214A), and fadD(E361A)). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.  (+info)

Isolation and expression pattern of two putative acyl-ACP desaturase cDNAs from Bassia scoparia. (6/22)

The seed lipids of some higher plants contain unusual fatty acids with potentially valuable non-food uses. Seeds of Bassia scoparia contain one such monounsaturated fatty acid, 16:1Delta5. This fatty acid can be used for the production of an insect oviposition pheromone, which is potentially valuable in the control of the mosquito Culex quinquefasciatus, a vector of West Nile virus. Previous work has established that a number of unusual monounsaturated fatty acids are produced by variant forms of the ubiquitous acyl-ACP desaturases. The isolation and initial characterization of two putative acyl-ACP desaturases from B. scoparia, one of which is seed-specific, suggests that such a variant enzyme occurs in this species.  (+info)

A dynamic zinc redox switch. (7/22)

The crystal structures of glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans, which catalyzes the formation of S-hydroxymethylglutathione from formaldehyde and glutathione, and its complex with glutathione (Gfa-GTT) have been determined. Gfa has a new fold with two zinc-sulfur centers, one that is structural (zinc tetracoordinated) and one catalytic (zinc apparently tricoordinated). In Gfa-GTT, the catalytic zinc is displaced due to disulfide bond formation of glutathione with one of the zinc-coordinating cysteines. Soaking crystals of Gfa-GTT with formaldehyde restores the holoenzyme. Accordingly, the displaced zinc forms a complex by scavenging formaldehyde and glutathione. The activation of formaldehyde and of glutathione in this zinc complex favors the final nucleophilic addition, followed by relocation of zinc in the catalytic site. Therefore, the structures of Gfa and Gfa-GTT draw the critical association between a dynamic zinc redox switch and a nucleophilic addition as a new facet of the redox activity of zinc-sulfur sites.  (+info)

Lysophospholipid flipping across the Escherichia coli inner membrane catalyzed by a transporter (LplT) belonging to the major facilitator superfamily. (8/22)

The transfer of phospholipids across membrane bilayers is protein-mediated, and most of the established transporters catalyze the energy-dependent efflux of phospholipids from cells. This work identifies and characterizes a lysophospholipid transporter gene (lplT, formally ygeD) in Escherichia coli that is an integral component in the 2-acylglycerophosphoethanolamine (2-acyl-GPE) metabolic cycle for membrane protein acylation. The lplT gene is adjacent to and in the same operon as the aas gene, which encodes the bifunctional enzyme 2-acyl-GPE acyltransferase/acyl-acyl carrier protein synthetase. In some bacteria, acyltransferase/acyl-ACP synthetase (Aas) and LplT homologues are fused in a single polypeptide chain. 2-Acyl-GPE transport to the inside of the cell was assessed by measuring the Aas-dependent formation of phosphatidylethanolamine. The Aas-dependent incorporation of [3H]palmitate into phosphatidylethanolamine was significantly diminished in deltalplT mutants, and the LplT-Aas transport/acylation activity was independent of the proton motive force. The deltalplT mutants accumulated acyl-GPE in vivo and had a diminished capacity to transport exogenous 2-acylglycerophosphocholine into the cell. Spheroplasts prepared from wild-type E. coli transported and acylated fluorescent 2-acyl-GPE with an apparent K(d) of 7.5 microM, whereas this high-affinity process was absent in deltalplT mutants. Thus, LplT catalyzes the transbilayer movement of lysophospholipids and is the first example of a phospholipid flippase that belongs to the major facilitator superfamily.  (+info)

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