Structural and biochemical studies of serine acetyltransferase reveal why the parasite Entamoeba histolytica cannot form a cysteine synthase complex

Cysteine (Cys) plays a major role in growth and survival of the human parasite Entamoeba histolytica. We report here the crystal structure of serine acetyltransferase (SAT) isoform 1, a cysteine biosynthetic pathway enzyme from E. histolytica (EhSAT1) at 1.77 Å, in complex with its substrate serine (Ser) at 1.59 Å and inhibitor Cys at 1.78 Å resolution. EhSAT1 exists as a trimer both in solution as well as in crystal structure, unlike hexamers formed by other known SATs. The difference in oligomeric state is due to the N-terminal region of the EhSAT1, which has very low sequence similarity to known structures, also differs in orientation and charge distribution. The Ser and Cys bind to the same site, confirming that Cys is a competitive inhibitor of Ser. The disordered C-terminal region and the loop near the active site are responsible for solvent-accessible acetyl-CoA binding site and, thus, lose inhibition to acetyl-CoA by the feedback inhibitor Cys. Docking and fluorescence studies show that EhSAT1 C-terminal-mimicking peptides can bind to O-acetyl serine sulfhydrylase (EhOASS), whereas native C-terminal peptide does not show any binding. To test further, C-terminal end of EhSAT1 was mutated and found that it inhibits EhOASS, confirming modified EhSAT1 can bind to EhOASS. The apparent inability of EhSAT1 to form a hexamer and differences in the C-terminal region are likely to be the major reasons for the lack of formation of the large cysteine synthase complex and loss of a complex regulatory mechanism in E. histolytica.     

Active-site residues of a plant membrane-bound fatty acid elongase β-ketoacyl-CoA synthase,FAE1 KCS

The fatty acid elongase-1 β-ketoacyl-CoA synthase, FAE1 KCS, a seed-specific elongase condensing enzyme from Arabidopsis, is involved in the production of eicosenoic (C20:1) and erucic (C22:1) acids. Alignment of the amino acid sequences of FAE1 KCS, KCS1, and five other putative elongase condensing enzymes (KCSs) revealed the presence of six conserved cysteine and four conserved histidine residues. Each of the conserved cysteine and histidine residues was individually converted by site-directed mutagenesis to both alanine and serine, and alanine and lysine respectively. After expression in yeast cells, the mutant enzymes were analyzed for their fatty acid elongase activity. Our results indicated that only cysteine 223 is an essential residue for enzyme activity, presumably for acyl chain transfer. All histidine substitutions resulted in complete loss of elongase activity. The loss of activity of these mutants was not due to their lower expression level since immunoblot analysis confirmed each was expressed to the same extent as the wild type FAE1 KCS.     

Expression and characterization of Flavikunin: A Kunitz‐type serine protease inhibitor identified in the venom gland cDNA library of Bungarus flaviceps

Trancriptomic analysis of the venom gland cDNA library of Bungarus flaviceps revealed Kunitz‐type serine protease inhibitor as one of the major venom protein families with three groups A, B, C. One of the group B isoforms named Flavikunin, which lacked an extra cysteine residue involved in disulfide bond formation in β‐bungarotoxin, was synthesized, cloned, and overexpressed in Escherichia coli. To decipher the structure‐function relationship, the P1 residue of Flavikunin, histidine, was mutated to alanine and arginine. Purified wild‐type and mutant Flavikunins were screened against serine proteases‐thrombin, factor Xa, trypsin, chymotrypsin, plasmin, and elastase. The wild‐type and mutant Flavikunin (H?R) inhibited plasmin with an IC ₅₀ of 0.48 and 0.35 µM, respectively. The in‐silico study showed that P1 residue of wild‐type and mutant (H?R) Flavikunin interacted with S1′ and S1 site of plasmin, respectively. Thus, histidine at the P1 position was found to be involved in plasmin inhibition with mild anticoagulant activity.     

Aromatic boronic acids as probes of the catalytic site of human plasma lecithin-cholesterol acyltransferase

We have recently proposed a catalytic mechanism for human plasma lecithin-cholesterol acyltransferase (EC 2.3.1.43) (J. Biol. Chem. (1986) 261, 7032-7043), implicating single serine and histidine residues in phosphatidylcholine cleavage and two cysteine residues in cholesterol esterification. We now confirm the involvement of serine and histidine in catalysing the phospholipase A2 action of lecithin-cholesterol acyltransferase by demonstrating the inhibition of this activity by phenylboronic acid (Ki = 1.23 mM) and m-aminophenylboronic acid (Ki = 2.32 mM), inhibitors of known serine/histidine hydrolases. The specificity of the interaction of aromatic boronic acids with catalytic serine and histidine residues and the putative formation of a tetrahedral adduct between boron and the lecithin-cholesterol acyltransferase serine hydroxyl group which is similar to the transition-state intermediate formed between phosphatidylcholine and the catalytic serine residue was suggested by: substrate protection against inhibition by phenylboronic acids; a much reduced incorporation of phenylmethane[35S]sulphonyl fluoride into the enzyme in the presence of phenylboronic acid; the lack of interaction of histidine- or serine-modified enzyme with immobilized phenylboronic acid in the presence of glycerol (Ve/Vo = 2.7 and 2.3 respectively) when compared to the native enzyme (Ve/Vo = 5.25). Fatty acyl-lecithin-cholesterol acyltransferase, produced by incubation of the enzyme with a lecithin-apolipoprotein A-I proteoliposome substrate, was not retarded upon the sorbent column (Ve/Vo = 1.5). Modification of the enzyme's two free cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) or potassium ferricyanide reduced (Ve/Vo = 3.5) but did not abolish retardation on the sorbent column, indicating that these modifications resulted in steric hinderance of the interaction of the boron atom with the lecithin-cholesterol acyltransferase serine hydroxyl group. These data suggest that the serine and histidine residues are proximal within the enzyme catalytic site and that both cysteine thiol groups are close to the serine hydroxyl group. The presence of significant amino-acid sequence homologies between lecithin-cholesterol acyltransferase, triacylglycerol lipases and the transacylases of fatty acid synthase is also reported.     

Studies on the structure of lecithin:cholesterol acyltransferase (LACT)--comparisons of the active site region and secondary structure of the human and the porcine enzymes

1. Chemical modification of essential serine, histidine and cysteine residues of porcine LCAT were accompanied by loss of enzymatic activity. 2. Modification of cysteine with DTNB inactivated the enzyme which could not be reactivated by KCN suggesting direct involvement of the cysteine residue(s) in catalysis. 3. About half of the primary structure of the porcine enzyme was determined. 4. Respective regions of the human and porcine LCAT are highly homologous; especially, the amino-terminus and the region surrounding the DFP-labeled serine residues. 5. The observed primary structure differences represent amino acid substitutions that are projected to induce significant changes in secondary structure.     

Staphylococcal lactose phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis on the lacE gene gives evidence that a cysteine residue is responsible for phosphorylation

The lactose-specific phosphocarrier protein enzyme II of the bacterial phosphoenol-pyruvate-dependent phosphotransferase system of Staphylococcus aureus was modified by site-specific mutagenesis on the corresponding lacE gene in order to replace the histidine residues 245, 274 and 510 and the cysteine residue 476 of the amino acid sequence with a serine residue. The wild-type and mutant genes were expressed in Escherichia coli and the gene products were characterized in different in vitro test systems. In vitro phosphorylation studies on mutant derivatives of the lactose-specific enzyme II led to the conclusion that cysteine residue 476 is the active-site for phosphorylation of this enzyme II by a phospho-enzyme III of the same sugar specificity. A cysteine residue phosphorylated intermediate was first postulated for the mannitol-specific enzyme II of E. coli and studies performed independently concerning the lactose-specific enzyme II of Lactobacillus casei are in agreement with the above results.     

Simple modifications of the serpin reactive site loop convert SCCA2 into a cysteine proteinase inhibitor: a critical role for the P3' proline in facilitating RSL cleavage

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the serpin family that are 92% identical in their amino acid sequence. Despite this similarity, they inhibit distinct classes of proteinases. SCCA1 neutralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and human mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-like cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactive site loop (RSL) is important for cysteine proteinase inhibition. The inhibition of catS by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motifs outside and only eight residues within the RSL that were directing catS-specific inhibition. The purpose of this study was to determine which of these residues might account for the marked difference in the ability of SCCA1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules containing different RSL mutations showed that no single amino acid substitution could convert SCCA2 into a more potent cysteine proteinase inhibitor. Rather, different combinations of mutations led to incremental increases in catS inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and SCCA2. Interestingly, the RSL cleavage site differed between wild-type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of catS by both wild-type SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase.     

Role of cysteine residues in 4-oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1

4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K(m)-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl(2); and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.     

A catalytic role for histidine 237 in rat mammary gland thioesterase II

The involvement of a histidyl residue in the catalytic mechanism of thioesterase II, a serine active-site enzyme that catalyzes the chain terminating reaction in de novo fatty acid synthesis, has been inferred from studies with the inhibitor diethyl pyrocarbonate. Its likely location has been predicted by identification of conserved residues in related thioesterases and ultimately confirmed by site-directed mutagenesis. Diethyl pyrocarbonate inactivated the enzyme with a second-order rate constant of 49 M-1 s-1 at pH 6, 10 degrees C. Data analysis indicated that although several residues reacted with the reagent, modification of a single residue was responsible for the inactivation. Removal of a single ethoxycarbonyl moiety by treatment with neutral hydroxylamine completely restored enzyme activity. Prior ethoxycarbonylation of the histidyl residue blocked the ability of the active-site serine to react with phenylmethanesulfonyl fluoride. Comparison of the amino acid sequences of five structurally related proteins indicated that only 1 histidine has been completely conserved. Replacement of this residue in rat thioesterase II (His-237) with arginine and leucine by mutagenesis reduced the catalytic activity by 2-3 orders of magnitude. The activity of the mutant thioesterases, unlike that of the wild-type enzyme, was relatively insensitive to inhibition by diethyl pyrocarbonate and phenylmethylsulfonyl fluoride. These studies provide strong evidence that His-237 is involved directly in catalysis and suggest that its role is to increase the nucleophilic character of the active-site Ser-101 by acting as a proton acceptor thus facilitating acylation of the seryl residue. The mechanism appears to share certain common features with the charge-relay system characteristic of other esterases.     

A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.     

Human plasma lecithin-cholesterol acyltransferase. An elucidation of the catalytic mechanism

Human plasma lecithin-cholesterol acyltransferase (LCAT) transacylates the sn-2 fatty acid of lecithin to cholesterol forming cholesteryl ester and lysolecithin. Measurement of the phospholipase A2 and transacylase activities of the enzyme using proteoliposome substrates and following selective chemical modification of serine, histidine, and cysteine residues of pure homogeneous LCAT indicated the following catalytic mechanism: HS-Cys-E-Ser-OH + lecithin in equilibrium HS-Cys-E-Ser-O-FA + lysolecithin, HS-Cys-E-Ser-O-FA in equilibrium FA-S-Cys-E-Ser-OH, FA-S-Cys-E-Ser-OH + cholesterol-OH in equilibrium HS-Cys-E-Ser-OH + cholesterol-O-FA, where FA denotes fatty acid. Modification of 2 LCAT cysteine residues with 5,5'-dithiobis-(2-nitrobenzoic acid) or treatment with ferricyanide inactivated the transacylase but not the phospholipase A2 activity. Modification of 1 serine residue with phenylmethanesulfonyl fluoride or 1 histidine residue with diethyl pyrocarbonate inhibited cholesteryl ester formation and phospholipase A2 activity. Proteoliposome substrates protected both activities against chemical inactivation. Lecithin alone protected the phospholipase A2 activity against phenylmethanesulfonyl fluoride inactivation but not the transacylase against 5,5'-dithiobis-(2-nitrobenzoic acid) inactivation. Incubation of native LCAT with arachidonyl-CoA or the lecithin-apo-A-I proteoliposome resulted in acylation of three enzyme sites, only one of which was stable to neutral hydroxylamine after denaturation. Fatty acylenzyme oxy- and thioesters were demonstrable in both cases. No transfer of arachidonic acid from iodoacetamide-modified LCAT to cholesterol occurred, indicating that the fatty-acylated serine residue cannot directly esterify cholesterol. Cholesterol arachidonate was formed upon incubation of phenylmethanesulfonyl fluoride-modified LCAT with arachidonyl-CoA.     

Chemical modification of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.

Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var. alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule. Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result. No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge. Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one. Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1. Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min). The inhibition was partially reversible. CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site. Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased. Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.     

The contribution of cysteine residues to antigenicity and extent of processing of herpes simplex virus type 1 glycoprotein D.

Glycoprotein D (gD) is an envelope component of herpes simplex virus types 1 (gD-1) and 2 (gD-2). The gD-1 polypeptide contains seven cysteine residues among its 369 amino acids; six are located on the N-terminal or luminal portion of the glycoprotein, and a seventh is located in the transmembrane region. Previous studies used a panel of monoclonal antibodies (MAbs) to define gD epitopes as continuous or discontinuous. Purified gD, denatured by reduction and alkylation, loses discontinuous epitopes, whereas continuous epitopes are retained. The contribution of disulfide bonds to maintenance of discontinuous epitopes is, therefore, significant. In the present study, our objective was to determine the contribution of individual cysteine residues to folding of gD-1 into its native conformation. Site-directed oligonucleotide mutagenesis was used to create seven mutants, each with a serine residue replacing a cysteine. The mutated genes were cloned into a eucaryotic expression vector and transfected into COS-1 cells, and the proteins were separated by nondenaturing polyacrylamide gel electrophoresis, followed by immunoblotting. Replacement of cysteine 7 (residue 333) had only a minimal effect on the antigenic properties of gD-1. In contrast, replacement of any one of the other six cysteine residues resulted in either a major reduction or a complete loss of binding of those MAbs that recognize discontinuous epitopes, with no effect on the binding of MAbs which recognize continuous epitopes. These mutations also had profound effects on the extent of oligosaccharide processing of gD-1. This was determined by digestion of the expressed proteins with various endoglycosidases, followed by electrophoresis and Western blotting (immunoblotting) to observe any mobility changes. Three mutant gD proteins which did not express discontinuous epitopes contained only high-mannose-type oligosaccharides, suggesting that processing had not proceeded beyond the precursor stage. Two mutant forms of gD exhibited reduced binding of MAbs to discontinuous epitopes. A small proportion of the molecules which accumulated at 48 h posttransfection contained complex oligosaccharides. One mutant exhibited reduced binding of MAbs to discontinuous epitopes, but was present at 48 h posttransfection only in the precursor form. The cysteine 7 mutant was processed to the same extent as wild-type gD. We conclude that the first six cysteine residues are critical to the correct folding, antigenic structure, and processing of gD-1, and we speculate that they form three disulfide-bonded pairs.     

Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low K_m) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low K_m of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys¹⁶⁰ of LAT1 and Cys¹¹⁰ of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl₂ with a K_i of 0.56 ± 0.11 μM. The inhibitory effect of HgCl₂ for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl₂ for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys⁸⁸ and Cys⁴³⁹, altered amino acid transport. The V_max was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys⁴³⁹ residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.     

Assignment of interchain disulfide bonds in platelet-derived growth factor (PDGF) and evidence for agonist activity of monomeric PDGF.

Platelet-derived growth factor (PDGF) is a dimeric factor stabilized by disulfide bonds. Using an approach involving partial reduction of PDGF, we have identified the 2nd and 4th cysteine residues in the PDGF chains as the cysteine residues forming interchain disulfide bonds. Analysis of PDGF mutants in which the 2nd and 4th cysteine residues were mutated to serine residues revealed that the disulfide bonds are arranged in a cross-wise manner, with the 2nd cysteine residue in one chain being linked to the 4th cysteine residue in the other. A PDGF B-chain mutant, in which both the 2nd and 4th cysteine residues were substituted with serine residues, migrated as a monomer in sodium dodecyl sulfate gel electrophoresis and retained receptor binding activity. When analyzed in receptor dimerization and autophosphorylation assays, this mutant showed agonistic activity. Thus, structural information has been obtained that will allow the large scale production of properly folded monomeric PDGF, as well as design of specific PDGF heterodimers.     

Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain

A mutant of papain, where an inter-domain hydrogen bond between the side chain hydroxyl group of a serine residue at position 176 and the side chain carbonyl oxygen of a glutamine residue at position 19 has been removed by site-directed mutagenesis, has been produced and characterized kinetically. The mutation of Ser176 to an alanine has only a small effect on the kinetic parameters, the kcat/Km for hydrolysis of CBZ-Phe-Arg-MCA by the Ser176Ala enzyme being of 8.1 x 10(4) /M/s compared with 1.2 x 10(5) /M/s for papain. Serine 176 is therefore not essential for the catalytic functioning of papain, even though this residue is conserved in all cysteine proteases sequenced. The pH-activity profiles were shown to be narrower in the mutant enzyme by up to 1 pH unit at high ionic strength. This result is interpreted to indicate that replacing Ser176 by an alanine destabilizes the thiolate-imidazolium form of the catalytic site Cys25-His159 residues of papain. Possible explanations for that effect are given and the role of a serine residue at position 176 in papain is discussed.     

Purification and characterization of a mutant ATP phosphoribosyltransferase hypersensitive to histidine feedback inhibition.

A mutant form of ATP phosphoribosyltranferase (EC 2.4.2.17), hisG1708c, which results in abnormally slow growth of Salmonella typhimurium at 20 °C was purified to homogeneity and kinetic and chemical behavior were characterized. Initial velocity steady-state substrate kinetics of wild-type and mutant enzymes at 37 °C were consistent with sequential kinetics and demonstrated that standard assay concentrations of substrates were sufficient to substantially saturate both enzymes. Nearly time-independent inhibition by histidine at 37 °C could be obtained only after incubation in the presence of product and histidine. Studies at 37 °C showed that the mutant enzyme is 24 times more sensitive to histidine than the wild type in a negatively cooperative manner instead of the positively cooperative manner seen for wild type. Pure mutant enzyme exhibits two major electrophoretic species of native enzyme. Although one less cysteine is titratable in native mutant enzyme, the amino acid compositions of mutant and wild-type enzymes are similar. Histidine produces an ultraviolet difference spectrum in mutant enzyme closely resembling that produced in wild type. Binding of histidyl-tRNA to mutant enzyme is substantially inhibited by histidine. It is concluded that the hisG1708c mutation alters some conformational processes coupled to the histidine binding site while not affecting others.     

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. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. HlyC and hemolysin belong to a family of at least 13 toxins produced by Gram-negative bacteria. The homologous acyltransferases of the family show a number of conserved residues that are possible candidates for participation in acyl transfer. Specific chemical reagents and site-directed mutagenesis showed that neither the single conserved cysteine nor the three conserved serine residues were required for enzyme activity. Treatment with the reversible histidine-modifying diethyl pyrocarbonate (DEPC) inhibited acyltransferase activity, and acyltransferase activity was restored following hydroxylamine treatment. The substrate myristoyl-ACP protected HlyC from DEPC inhibition. These findings and spectral absorbance changes suggested that histidine, particularly a histidine proximal to the substrate binding site, was essential for enzyme activity. Site-directed mutageneses of the single conserved histidine residue, His23, to alanine, cysteine, or serine resulted in each instance in complete inactivation of the enzyme.     

Two glutamate residues, Glu 208 alpha and Glu 197 beta, are crucial for phosphorylation and dephosphorylation of the active-site histidine residue in succinyl-CoA synthetase.

Succinyl-CoA synthetase catalyzes the reversible reaction succinyl-CoA + NDP + P(i) <--> succinate + CoA + NTP (N denoting adenosine or guanosine). The enzyme consists of two different subunits, designated alpha and beta. During the reaction, a histidine residue of the alpha-subunit is transiently phosphorylated. This histidine residue interacts with Glu 208 alpha at site I in the structures of phosphorylated and dephosphorylated Escherichia coli SCS. We postulated that Glu 197 beta, a residue in the nucleotide-binding domain, would provide similar stabilization of the histidine residue during the actual phosphorylation/dephosphorylation by nucleotide at site II. In this work, these two glutamate residues have been mutated individually to aspartate or glutamine. Glu 197 beta has been additionally mutated to alanine. The mutant proteins were tested for their ability to be phosphorylated in the forward or reverse direction. The aspartate mutant proteins can be phosphorylated in either direction, while the E208 alpha Q mutant protein can only be phosphorylated by NTP, and the E197 beta Q mutant protein can only be phosphorylated by succinyl-CoA and P(i). These results demonstrate that the length of the side chain at these positions is not critical, but that the charge is. Most significantly, the E197 beta A mutant protein could not be phosphorylated in either direction. Its crystal structure shows large differences from the wild-type enzyme in the conformation of two residues of the alpha-subunit, Cys 123 alpha-Pro 124 alpha. We postulate that in this conformation, the protein cannot productively bind succinyl-CoA for phosphorylation via succinyl-CoA and P(i).