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. The enzymes of Euglena participating in both priming and elongation reactions to form a new carbon-carbon bond were acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I. The effects of inhibitors on the three different enzymes were noted. All carbon-carbon bond formation was inhibited by cerulenin. Although neither fatty acid biosynthesis nor any of the isolated enzymes were sensitive to diisopropylphosphofluoridate, the three Euglena enzymes studied were sensitive to different sulfhydryl-alkylating agents. Acetyl-ACP supported fatty acid biosynthesis as effectively as did comparable amounts of ACPSH and acetyl-CoA. There was no evidence for a beta-ketoacyl-ACP synthase III for priming such as has been reported in type II fatty acid synthase of higher plants and bacteria. The roles of the acetyl-CoA-ACP transacylase and beta-ketoacyl-ACP synthase I appear to be unique in the type II fatty acid synthase of Euglena. Acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I were separated from one another and shown to have different molecular weights.

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. The reactivities of the NADPH- and CoA-binding-site-specific antibodies with a variety of enzymes that required different cofactors was shown on Western blots of SDS-PAGE of the enzymes. Variable cross-reactivities were observed among all nucleotide-cofactor requiring enzymes with each specific cofactor-domain-antibody population. Numerous proteins not physiologically associated with nucleotide cofactors, including acyl carrier protein, were completely unreactive. Proteins that bound phosphoryl compounds either as substrates or cofactors showed varying degrees of reactivity with each population of specific antibodies. These included aldolase, ribulose-1,5-bisphosphate carboxylase/oxygenase, ribonuclease A, carbonic anhydrase and triosephosphate isomerase. The immunologic cross-reactivity suggested that these proteins share a common structural feature, probably a primary structure epitope, since the proteins had been subjected to denaturing polyacrylamide gel electrophoresis. A candidate for this common structural feature is a glycine-rich sequence comprising a phosphate binding loop.

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. Melittin, a peptide from bee venom whose secondary structural configuration varies depending upon solution conditions was also examined. Acyl-carrier protein (ACP) is a small protein of recognized dynamic structure that in its diverse physiologic roles interacts specifically with numerous different proteins. Horizontal ATR FTIR analysis of ACP's secondary structure indicated a predominantly helical structure best defined as a combination of ordered and disordered helices. The FTIR-derived structural composition agreed with those determined for ACP by other techniques. Comparison of independent analyses of the amide-I and III regions to determine protein configuration compositions was a useful method of verifying the internal consistency of the calculated structural compositions of dynamically-structured proteins.

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 activity of the nonaggregated fatty acid synthetase of Euglena was located within the chloroplast stroma, and the effects of dimethylsuberimidate cross-linking on the activity of the enzyme system were examined. The acyl-carrier protein concentration in the chloroplast was measured at about 0.24 mM.

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. Also, anti-Apis mellifera antibodies which contained antibodies against melittin specifically inhibited the same enzyme system activity relative to non-immune IgG.

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. coli, behaves upon electrophoresis under denaturing conditions as its size would predict. Oligomeric forms of both ACPs were evident on Western blots, but the bacterial ACP had more tendency to aggregate. That the oligomeric forms were not due to impurities was shown by their regeneration from low-Mr protein, reaction with antibodies isolated from low-Mr protein, and by molecular weight determination of the ACP by low-angle laser light scattering.

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. Hydrophobic moment plots were prepared for ACP helices and those of many EF hand calcium-binding proteins. Both groups of proteins occupied the same region of the plot.

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) 5.6 micromolar at pH 5.4; this inhibition decreased with increasing pH or ionic strength. Coenzyme A was a competitive inhibitor, K(i) 230 micromolar. Kinetic parameters with respect to acetoacetyl-CoA and NADH were sensitive to changes in pH and ionic strength.

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. Km's (microM) for monoacylglycerophosphate acyltransferase were: palmitoyl-CoA, 7; oleoyl-CoA, 4; and 1-palmitoyl-sn-glycerol-3-phosphate, 48.

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. 2. The fatty acid synthesis-associated ACP's of many organisms and the lyase-associated ACP's show a high degree of relatedness among one another. 3. The ACP's show no relatedness to biotin carboxyl carrier protein or cytochrome 552.

A multienzyme complex for CO2 fixation.

Acetyl-coenzyme A carboxylase from Euglena gracilis strain Z was isolated as a component of a multienzyme complex which includes phosphoenolpyruvate carboxylase and malate dehydrogenase. The multienzyme complex was shown to exist in crude extracts and was purified to a homogeneous protein with a molecular weight of 360,000 by gel filtration. The ratio of the activities of the constituent enzymes was acetyl-CoA carboxylase:phosphoenolpyruvate carboxylase:malate dehydrogenase, 1:25:500. The complex is proposed to operate in conjunction with malic enzyme, which is present in Euglena, to facilitate the formation of substrates, malonyl-CoA, and NADPH, for fatty acid biosynthesis. The interaction of the enzymes may represent a means of control of acetyl-CoA carboxylase activity in organisms which do not possess an enzyme subject to allosteric regulation. The acetyl-CoA carboxylase activity from Euglena is unaffected by citrate and isocitrate.

Dissociation and characterization of enzymes from a multienzyme complex involved in CO2 fixation.

A multienzyme complex from Euglena, molecular weight about 360,000, containing phosphoenolpyruvate carboxylase, malate dehydrogenase, and acetyl-coenzyme A carboxylase has been dissociated into active constituent enzymes. The respective molecular weights are 183,000, 67,000, and 127,000. The malate dehydrogenase contained in the complex is electrophoretically distinct from other malate dehydrogenase isozymes found in Euglena. The K-m for HCO3minus of the free and complexed acetyl-CoA carboxylase is 4.2-5.4 mM, and the substrate dependency for acetyl-CoA describes a sigmoidal relationship. The HCO3minus K-m for the free phosphoenolpyruvate carboxylase is 7.3-5.4 mM while that for the same enzyme contained in the complex is 0.7-1.3 mM. Both the free and complexed forms ofphosphoenolpyruvate carboxylase have a K-m for phosphoenolpyruvate of 0.9-1.7 mM. The latter enzyme in both the complex and free forms is stimulated by NADH, acetyl-CoA, and ATP. In the free phosphoenolpyruvate carboxylase, the stimulation passes through a maximum depending on effector concentration. The effect of NADH is to increase V-max while K-m values remain unmodified.

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. Comparisons with other ACPs included the following items: biologic activity, acidic pI, size, behavior in size exclusion media, and amino acid sequence of the N-terminal portion of the molecule.

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. An analysis was done of the steady-state kinetics of the reaction catalysed by the fatty acid synthetase multienzyme complex. Initial-velocity studies were done in which the concentrations of the following pairs of substrates were varied: malonyl-CoA and acetyl-CoA, NADPH and acetyl-CoA, malonyl-CoA and NADPH. In all three cases patterns of the Ping Pong type were obtained. Product-inhibition studies were done with NADP+ and CoA. NADP+ is a competitive inhibitor with respect to NADPH, and uncompetitive with respect to malonyl-CoA and acetyl-CoA. CoA is uncompetitive with respect to NADPH and competitive with respect to malonyl-CoA and acetyl-CoA. When the concentrations of acetyl-CoA and malonyl-CoA were varied over a wide range, mutual competitive substrate inhibition was observed. When the fatty acid synthetase was incubated with radiolabelled acetyl-CoA or malonyl-CoA, labelled acyl-enzyme was isolated. The results are consistent with the idea that fatty acid synthesis proceeds by a multisite substituted-enzyme mechanism involving Ping Pong reactions at the following enzyme sites: acetyl transacylase, malonyl transacylase, beta-oxo acyl-enzyme synthetase and fatty acyl transacylase.

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. The acyltransferase reaction progresses through formation of a binary complex between acyl-ACP and HlyC to a reactive acyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. The homologous acyltransferases of the family have a number of conserved amino acid residues that may be catalytically important. Experiments to illuminate the reaction mechanism were done. The formation of an acyl-enzyme intermediate suggested that the reaction likely proceeded through two partial reactions. The reversibility of the first partial reaction was shown by using separately subcloned, purified, and expressed substrates and enzyme. The effects of single site-directed mutations of conserved residues of HlyC on different portions of reaction progress (binary complex formation, acyl-enzyme formation, and enzyme activity, including kinetic parameters) were determined. Mutations of His23, the only residue essential for activity, formed normal binary complexes but were unable to form acyl-HlyC. The same was seen with S20A, a mutant with greatly impaired activity. Mutation of two conserved tyrosines separately to glycines results in greatly impaired binary complex and acyl-HlyC formation, but mutation of those residues to phenylalanines restored behavior to wild-type.

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. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1999) Biochemistry 38, 9541-9548]. The ping pong kinetic mechanism implied by these findings was validated using two different acyl-ACP substrates, thus verifying the independence of the previously demonstrated two partial reactions. Assessments of the stability of the acyl-HlyC intermediate revealed an increased stability at pH 8.6 compared to more acidic pHs. Mutations of a single conserved histidine residue essential for catalysis gave minimal activity when substituted with a tyrosine residue and no activity with a lysine residue. Unlike numerous other His23 mutants, however, the H23K enzyme showed significant acyl-HlyC formation although it was unable to transfer the acyl group from the proposed amide bond intermediate to proHlyA. These findings are compatible with transient formation of acyl-His23 during the course of HlyC catalysis. The effects of several other single site-directed mutations of conserved residues of HlyC on different portions of the reaction progress were examined using a 39 500 kDa fragment of proHlyA which was a more effective substrate than intact proHlyA.