Thermodynamics of a protein acylation: activation of Escherichia coli hemolysin toxin.

HlyC, hemolysin-activating lysine-acyltransferase, catalyses the acylation (from acyl-acyl carrier protein [ACP]) of Escherichia coli prohemolysin (proHlyA) on the epsilon-amino groups of specific lysine residues, 564 and 690 of the 1024 amino acid primary structure, to form hemolysin (HlyA). Isothermal titration calorimetry was used to measure the thermodynamic properties of the protein acylation of proHlyA-derived structures, altered by substantial deletions and separation of the acylation sites into two different peptides and site directed mutation analyses of acylation sites. Acylation of proHlyA-derived proteins catalyzed by HlyC was overall an exothermic reaction driven by a negative enthalpy.

Activation of hemolysin toxin: relationship between two internal protein sites of acylation.

HlyC, hemolysin-activating lysine acyltransferase, catalyzes the acylation (from acyl-ACP) of Escherichia coli prohemolysin (proHlyA) on the epsilon-amino groups of specific lysine residues, Lys564 and Lys690 of the 1024-amino acid primary structure, to form hemolysin (HlyA). The amino acid sequences flanking the two acylation sites are not homologous except that each has a glycine residue immediately preceding the lysine which is acylated; there are, however, numerous GK sequences throughout proHlyA that are not acylation sites. The substrate specificity of acylation was examined.

Amino acid residues of Escherichia coli acyl carrier protein involved in heterologous protein interactions.

Acyl carrier protein (ACP) is a small, highly conserved protein with an essential role in a myriad of reactions throughout lipid metabolism in plants and bacteria where it interacts with a remarkable diversity of proteins. The nature of the proper recognition and precise alignment between the protein moieties of ACP and its many interactive proteins is not understood. Residues conserved among ACPs from numerous plants and bacteria were considered as possibly being crucial to ACP's function, including protein-protein interaction, and a method of identifying amino acid residue clusters of high hydrophobicity on ACP's surface was used to estimate residues possibly involved in specific ACP-protein interactions.

Insights into the catalytic mechanism of HlyC, the internal protein acyltransferase that activates Escherichia coli hemolysin toxin.

Hemolysin, a toxic protein secreted by pathogenic Escherichia coli, is converted from nontoxic prohemolysin, proHlyA, to toxic hemolysin, HlyA, by an internal protein acyltransferase, HlyC. Acyl-acyl carrier protein (ACP) is the essential acyl donor. The acyltransferase reaction proceeds through two partial reactions and entails formation of a reactive acyl-HlyC intermediate [Trent, M.

HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis.

Hemolysin, a toxic protein produced by pathogenic Escherichia coli, is one of a family of homologous toxins and toxin-processing proteins produced by Gram-negative bacteria. HlyC, an internal protein acyltransferase, converts it from nontoxic prohemolysin to toxic hemolysin. Acyl-acyl carrier protein is the essential acyl donor.

HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.

Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis.

HlyC, the internal protein acyltransferase that activates hemolysin toxin: role of conserved histidine, serine, and cysteine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.

HlyC is an internal protein acyltransferase that activates hemolysin, a toxic protein produced by pathogenic Escherichia coli. Acyl-acyl carrier protein (ACP) is the essential acyl donor. Separately subcloned, expressed, and purified prohemolysin A (proHlyA), HlyC, and [1-14C]myristoyl-ACP have been used to study the conversion of proHlyA to HlyA [Trent, M.

The biochemistry of hemolysin toxin activation: characterization of HlyC, an internal protein acyltransferase.

Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster, CABD. The A gene product, nontoxic hemolysin (proHlyA), is made toxic by posttranslational fatty acylation of two internal lysine residues.

Early catalytic steps of Euglena gracilis chloroplast type II fatty acid synthase.

Euglena gracilis is a very ancient eukaryote whose chloroplast acquisition and evolution has been independent of higher plants. The organism in unique in possessing two de novo fatty acid synthases, a true multienzyme complex of great size in the cytosol and a plastid-localized type II fatty acid synthase composed of discrete enzymes and acyl carrier protein (ACP). The enzymology of the early steps of fatty acid biosynthesis differed in the Euglena type II fatty acid synthase compared to those of Escherichia coli and plants.

Comparison of acyl-carrier protein and other protein structures in aqueous solutions by Fourier-transform infrared spectroscopy.

Protein solution structures were analyzed by horizontal attenuated total reflectance (ATR) FTIR spectroscopy. Secondary structure compositions determined from analyses of amide-I and II region and amide-III region difference spectra were compared. Data for proteins of known solution structure, cytochrome c, concanavalin A and lysozyme, were compared with those reported in the literature.

Nucleotide cofactor-binding-domain-specific antibodies show immunologic relatedness among unrelated proteins that bind phosphoryl compounds.

The immunologic relatedness of various cofactor-binding sites of enzymes requiring different nucleotide cofactors was examined. Chicken antibodies specific for NADPH- or CoA-binding domains were raised using an NADPH- or CoA-requiring enzyme as an immunogen. Antibodies specific for either NADPH- or CoA-binding domains were isolated by immunoaffinity chromatography of the respective antisera using unrelated NADPH- or CoA-requiring enzymes as affinity ligands.

Purification and partial characterization of acyl carrier protein from Euglena gracilis variety bacillaris.

Acyl carrier protein (ACP) was purified from Euglena gracilis variety bacillaris in yields of about 1 mg/100 g (wet wt) of cells. Antibodies against the purified protein were raised in hens and isolated from eggs. Antibodies raised against Euglena ACP inhibited the Euglena chloroplast nonaggregated fatty acid synthetase using either Euglena or Escherichia coli ACP as a substrate.

Acyl carrier protein interacts with melittin.

Acyl carrier protein (ACP) from Escherichia coli has been shown to form complexes with melittin, a cationic peptide from bee venom. ACP is a small (Mr 8847), acidic, Ca2(+)-binding protein, which possesses some characteristics resembling those of regulatory Ca2(+)-binding proteins including interaction with melittin. Complexing between melittin and ACP which occurred both in the presence and absence of Ca2+ was evident by chemical cross-linking the two peptides, fluorescence changes (including anisotropy measurements), and inhibition by melittin of the activity of a nonaggregated fatty acid synthetase from Euglena.

Behavior of acyl carrier proteins on western blots.

Acyl carrier proteins (ACPs) from Escherichia coli and Euglena were analyzed on Western blots using rabbit antibodies raised against E. coli ACP. Euglena ACP, unlike that from E.

Chemical cross-linking and its effect on fatty acid synthetase activity in intact chloroplasts from Euglena gracilis.

Intact chloroplasts were isolated from Euglena gracilis variety bacillaris, aliquots were exposed to several different chemical cross-linking reagents. The reagents penetrated the triple membrane of Euglena chloroplasts. This was shown by gradient acrylamide gel electrophoresis under denaturing conditions.

The amphiphilicity of ACP helices: a means of macromolecular interaction?

ACP interacts with diverse proteins in an unknown way. Possibly there is a similar mode of interaction between ACP and all ACP-binding proteins, the amphiphilic helix. The hydrophobicities of helices from 4 different ACPs were compared.

An NADH-Dependent Acetoacetyl-CoA Reductase from Euglena gracilis: Purification and Characterization, Including Inhibition by Acyl Carrier Protein.

An NADH-dependent acetoacetyl-CoA reductase from Euglena gracilis variety bacillaris was extensively purified and characterized. Two different isoelectric forms of the reductase with identical characteristics otherwise were found. The reductase was noncompetitively inhibited by acyl carrier protein, K(i) 5.

Comparison of 1-acylglycerophosphate and glycerophosphate acyltransferases from Euglena microsomes.

Both glycerophosphate and monoacylglycerophosphate acyltransferases from Euglena microsomes were inhibited by N-ethylmaleimide, but their responses to heat inactivation and sn-glyceraldehyde-3-phosphate differed. Glycerophosphate acyltransferase had a higher V with palmitoyl-CoA compared to oleoyl-CoA; the reverse was true for monoacylglycerophosphate acyltransferase. Km's (microM) for the glycerophosphate acyltransferase were: palmitoyl-CoA, 21; oleoyl-CoA, 14; and sn-glycerol-3-phosphate, 2900.

Comparison of glycerophosphate acyltransferases from Euglena chloroplasts and microsomes.

The acylation of sn-glycerol 3-phosphate is a common reaction in the pathways leading to the biosynthesis of glycerol-derived phospholipids, galactolipids, and sulfolipids. Enzymes catalyzing this reaction have been solubilized from Euglena chloroplasts, microsomes, and mitochondria (B. A.

Relatedness of acyl carrier proteins shown by amino acid compositions.

1. Relatedness among the following carrier proteins was assessed on the basis of amino acid compositions: eight acyl carrier proteins (ACP's) associated with fatty acid synthesis, ACP's associated with citrate lyase and citramalate lyase, a biotin carboxyl carrier protein and cytochrome 552. Two independent indices of amino acid composition were used.

Kinetic studies of the fatty acid synthetase multienzyme complex from Euglena gracilis variety bacillaris.

A fatty acid synthetase multienzyme complex was purified from Euglena gracilis variety bacillaris. The fatty acid synthetase activity is specifically inhibited by antibodies against Escherichia coli acyl-carrier protein. The Euglena enzyme system requires both NADPH and NADH for maximal activity.