Cloning,expression, and characterization of a human 4'-phosphopantetheinyl transferase with broad substrate specificity

A single candidate 4'-phosphopantetheine transferase, identified by BLAST searches of the human genome sequence data base, has been cloned, expressed, and characterized. The human enzyme, which is expressed mainly in the cytosolic compartment in a wide range of tissues, is a 329-residue, monomeric protein. The enzyme is capable of transferring the 4'-phosphopantetheine moiety of coenzyme A to a conserved serine residue in both the acyl carrier protein domain of the human cytosolic multifunctional fatty acid synthase and the acyl carrier protein associated independently with human mitochondria. The human 4'-phosphopantetheine transferase is also capable of phosphopantetheinylation of peptidyl carrier and acyl carrier proteins from prokaryotes. The same human protein also has recently been implicated in phosphopantetheinylation of the alpha-aminoadipate semialdehyde dehydrogenase involved in lysine catabolism (Praphanphoj, V., Sacksteder, K. A., Gould, S. J., Thomas, G. H., and Geraghty, M. T. (2001) Mol. Genet. Metab. 72, 336-342). Thus, in contrast to yeast, which utilizes separate 4'-phosphopantetheine transferases to service each of three different carrier protein substrates, humans appear to utilize a single, broad specificity enzyme for all posttranslational 4'-phosphopantetheinylation reactions.     

4'-phosphopantetheine and coenzyme A biosynthesis in plants

Coenzyme A is required for many synthetic and degradative reactions in intermediary metabolism and is the principal acyl carrier in prokaryotic and eukaryotic cells. Coenzyme A is synthesized in five steps from pantothenate, and recently the CoaA biosynthetic genes in bacteria and human have all been identified and characterized. Coenzyme A biosynthesis in plants is not fully understood, and to date only the AtHAL3a (AtCoaC) gene of Arabidopsis thaliana has been cloned and identified as 4'-phosphopantothenoylcysteine (PPC) decarboxylase (Kupke, T., Hernández-Acosta, P., Steinbacher, S., and Culiá?ez-Macià, F. A. (2001) J. Biol. Chem. 276, 19190-19196). Here, we demonstrate the cloning of the four missing genes, purification of the enzymes, and identification of their functions. In contrast to bacterial PPC synthetases, the plant synthetase is not CTP-but ATP-dependent. The complete biosynthetic pathway from pantothenate to coenzyme A was reconstituted in vitro by adding the enzymes pantothenate kinase (AtCoaA), 4'-phosphopantothenoylcysteine synthetase (AtCoaB), 4'-phosphopantothenoylcysteine decarboxylase (AtCoaC), 4'-phosphopantetheine adenylyltransferase (AtCoaD), and dephospho-coenzyme A kinase (AtCoaE) to a mixture containing pantothenate, cysteine, ATP, dithiothreitol, and Mg2+.     

Mammalian fatty acid synthetase is a structurally and functionally symmetrical dimer

We have explored a comprehensive experimental approach to determine whether the two condensing-enzyme active centers of the mammalian fatty acid synthetase are simultaneously functional. Our strategy involved utilization of trypsinized fatty acid synthetase, which is a nicked homodimer composed of two pairs of 125 + 95-kDa polypeptides. These core polypeptides lack the chain-terminating thioesterase domains but retain all other functional domains of the native enzyme and can assemble long-chain acyl moieties at a rate equal to that of the native enzyme. The 4'-phosphopantetheine content of these enzyme preparations, estimated from the amount of beta-alanine present, from the amount of taurine formed by performic acid oxidation and from the amount of carboxymethylcysteamine formed by alkylation with iodo[2-14C]acetate, was typically 0.86 mol/mol 95-kDa polypeptide. The stoichiometry of long-chain acyl-enzyme synthesis, measured with radiolabeled precursors, indicated that 0.84 mol acyl-chains were assembled/mol 95-kDa polypeptide. When the small amount of apoenzyme present is taken into account, this stoichiometry translates to 1.94 acyl chains per holoenzyme dimer. The 125-kDa polypeptide of one subunit could be cross-linked to the 95-kDa polypeptide of the other subunit by 1,3-dibromo-2-propanone yielding a single molecular species of 220 kDa. Cross-linking was accompanied by a loss of condensing-enzyme activity. This result is consistent with a structurally symmetrical model for the animal fatty acid synthetase [J.K. Stoops and S.J. Wakil (1981) J. Biol. Chem. 256, 5128-5133] in which the juxtaposed 4'-phosphopantetheine and cysteine thiols of opposing subunits that form the two potential catalytic centers for condensing activity are readily susceptible to cross-linking. Both half-maximal cross-linking and 50% inhibition of activity were observed with 1 mol 1,3-dibromo-2-propanone bound/mol enzyme. After assembly of long-chain acyl moieties on the 4'-phosphopantetheine residues, no vacant condensing-enzyme active sites were demonstrable either by cross-linking with 1,3-dibromo-2-propanone or by formation of carboxymethylcysteamine on treatment with iodoacetate. These results are consistent with a structurally and functionally symmetrical model for the mammalian fatty acid synthetase in which the two condensation sites are simultaneously active.     

Characterization of recombinant thioesterase and acyl carrier protein domains of chicken fatty acid synthase expressed in Escherichia coli

Fatty acid synthase of animal tissue is a multifunctional enzyme comprised of two identical subunits, each containing seven partial activities and a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein). We have recently isolated cDNA clones of chicken fatty acid synthase coding for the dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase domains (Chirala, S.S., Kasturi, R., Pazirandeh, M., Stolow, D.T., Huang, W.Y., and Wakil, S.J. (1989) J. Biol. Chem. 264, 3750-3757). To gain insight into the structure and function of the various domains, the portion of the cDNA coding for the acyl carrier protein and thioesterase domains was expressed in Escherichia coli by using an expression vector that utilizes the phage lambda PL promoter. The recombinant protein was efficiently expressed and purified to near homogeneity using anion-exchange and hydroxyapatite chromatography. As expected from the coding capacity of the cDNA expressed, the protein has a molecular weight of 43,000 and reacts with antithioesterase antibodies. The recombinant thioesterase was found to be enzymatically active and has the same substrate specificity and kinetic properties as the native enzyme of the multifunctional synthase. Treatment of the recombinant protein with alpha-chymotrypsin results in the cleavage of the acyl carrier protein and thioesterase domain junction sequence at exactly the same site as with native fatty acid synthase. The amino acid composition of the purified recombinant protein revealed the presence of 0.6 mol of beta-alanine/mol of protein, indicating partial pantothenylation of the recombinant acyl carrier protein domain. These results indicate that the expressed protein has a conformation similar to the native enzyme and that its folding into functionally active domains is independent of the remaining domains of the multifunctional synthase subunit. These conclusions are consistent with the proposal that the multifunctional synthase gene has evolved from fusion of component genes.     

Stoichiometry of substrate binding to rat liver fatty acid synthetase.

Two rat liver fatty acid synthetase preparations, containing 1.6 and 2.0 mol of 4'-phosphopantetheine/mol of synthetase, showed specific activity of 2006 and 2140 nmol of NADPH oxidized/min per mg of protein respectively. The two synthetase preparations could be loaded with either 3.3-4.4 mol of [1-14] acetate or 2.9-3.7 mol of [2-14C]malonate, by incubation with either [1-14C] acetyl-CoA or [2-14C]malonyl-CoA. The 4'-phosphopantetheine site could be more than 90% saturated and the serine site about 80% saturated with malonate derived from malonyl-CoA. However, with acetyl-CoA as substrate, binding at both the 4'-phosphopantetheine and cysteine thiol sites did not reach saturation. We interpret these results to indicate that, whereas the equilibrium constant for transfer of substrates between the serine loading site and the 4'-phosphopantetheine site is close to unity, that for transfer of acetyl moieties between the 4'-phosphopantetheine and cysteine sites favours formation of the 4'-phosphopantetheine thioester. Thus, despite the apparent sub-stoichiometric binding of acetate, the results are consistent with a functionally symmetrical model for the fatty acid synthetase which permits simultaneous substrate binding at two separate active centres.     

Alteration of aminoacyl-tRNA synthetase activities by phosphorylation with casein kinase I

The phosphorylation of a highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes by the cyclic nucleotide-independent protein kinase, casein kinase I, has been examined, and the effects of phosphorylation on the synthetase activities were determined. The synthetase complex, purified as described (Kellermann, O., Tonetti, H., Brevet, A., Mirande, M., Pailliez, J.-P., and Waller, J.-P. (1982) J. Biol. Chem. 257, 11041-11048), contains seven aminoacyl-tRNA synthetases and four unidentified proteins and is free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP results in the phosphorylation of four synthetases, namely, glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I alters binding of the aminoacyl-tRNA synthetase complex to tRNA-Sepharose. The phosphorylated synthetase complex elutes from tRNA-Sepharose at 190 mM NaCl, while the nonphosphorylated complex elutes at 275 mM NaCl. Phosphorylation by casein kinase I results in a significant inhibition of aminoacylation by the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases; the activities of the nonphosphorylated synthetases remain unchanged. These data indicate that phosphorylation of aminoacyl-tRNA synthetases in the high molecular weight complex alters the activities of these enzymes. One of the unidentified proteins present in the complex (Mr 37,000) is also highly phosphorylated by casein kinase I. From a comparison of the properties and phosphopeptide pattern of this protein with that of casein kinase I, it appears that the Mr 37,000 protein in the synthetase complex is an inactive form of casein kinase I. This observation provides further evidence for a physiological role for casein kinase I in regulating synthetase activities.     

Phosphorylation of threonyl- and seryl-tRNA synthetase by cAMP-dependent protein kinase. A possible role in the regulation of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) synthesis

Threonyl-tRNA synthetase has been shown to be phosphorylated in reticulocytes (Dang, C. V., Tan, E. M., and Traugh, J. A., (1988) FASEB J. 2, 2376-2379). Upon incubation of reticulocytes with 8-bromo-cAMP, phosphorylation of threonyl-tRNA synthetase is stimulated approximately 2-fold, an increase similar to that observed with ribosomal protein S6. To analyze the effects of phosphorylation on activity, threonyl-tRNA synthetase has been purified to apparent homogeneity from rabbit reticulocytes utilizing a four-step purification procedure with the simultaneous purification of seryl-tRNA synthetase. Both synthetases are phosphorylated in vitro by the cAMP-dependent protein kinase. Prior to phosphorylation, the two synthetases produce significant amounts of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) in the presence of the cognate amino acid and ATP, with activities comparable to that of lysyl-tRNA synthetase. Phosphorylation has no effect on aminoacylation, but an increase in Ap4A synthesis of up to 6-fold is observed with threonyl-tRNA synthetase and 2-fold with seryl-tRNA synthetase. Thus, cAMP-mediated phosphorylation of specific aminoacyl-tRNA synthetases appears to be a potential mode of regulation of Ap4A synthesis in mammals.     

Protein kinase C phosphorylates glutamyl-tRNA synthetase in rabbit reticulocytes stimulated by tumor promoting phorbol esters

A high Mr synthetase core complex isolated from higher eukaryotes contains aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. Previously, five of the synthetases were shown to be phosphorylated in reticulocytes, and the glutaminyl- and aspartyl-tRNA synthetases were shown to be selectively phosphorylated in response to 8-bromo cAMP (Pendergast, A. M., Venema, R. C., and Traugh, J. A. (1987) J. Biol. Chem. 262, 5939-5942). Exposure of reticulocytes to phorbol 12-myristate 13-acetate stimulates the selective phosphorylation of one synthetase in the complex, glutamyl-tRNA synthetase. Only the glutamyl-tRNA synthetase is modified to a significant extent when the purified complex is phosphorylated in vitro by protein kinase C; up to 0.7 mol of phosphate is incorporated per mol of synthetase. Two-dimensional phosphopeptide mapping shows a single tryptic phosphopeptide, which is identical for the enzyme modified in vitro by protein kinase C or in phorbol 12-myristate 13-acetate-stimulated cells. Phosphorylation in vivo is reproducibly accompanied by a 38 +/- 10% reduction in aminoacylation activity of partially purified glutamyl-tRNA synthetase assayed in vitro. Phosphorylation in vitro has no detectable effect on aminoacylation. This difference may be due to the absence of a required effector molecule which alters activity by interaction with the phosphorylated synthetase. Glutamyl-tRNA synthetase is one of a growing number of translational components, including initiation factors, which are coordinately modified by protein kinase C in response to phorbol 12-myristate 13-acetate.     

The crystal structure of a novel bacterial adenylyltransferase reveals half of sites reactivity

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in bacteria that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis, by transferring an adenylyl group from ATP to 4'-phosphopantetheine, yielding dephospho-CoA (dPCoA). Each phosphopantetheine adenylyltransferase (PPAT) subunit displays a dinucleotide-binding fold that is structurally similar to that in class I aminoacyl-tRNA synthetases. Superposition of bound adenylyl moieties from dPCoA in PPAT and ATP in aminoacyl-tRNA synthetases suggests nucleophilic attack by the 4'-phosphopantetheine on the alpha-phosphate of ATP. The proposed catalytic mechanism implicates transition state stabilization by PPAT without involving functional groups of the enzyme in a chemical sense in the reaction. The crystal structure of the enzyme from Escherichia coli in complex with dPCoA shows that binding at one site causes a vice-like movement of active site residues lining the active site surface. The mode of enzyme product formation is highly concerted, with only one trimer of the PPAT hexamer showing evidence of dPCoA binding. The homologous active site attachment of ATP and the structural distribution of predicted sequence-binding motifs in PPAT classify the enzyme as belonging to the nucleotidyltransferase superfamily.     

Regulation of fatty acid synthetase activity. The 4'-phosphopantetheine hydrolase of rat liver.

The 4'-phosphopantetheine hydrolase of rat liver, partially purified by ammonium sulfate precipitation, catalyzes the hydrolysis of the prosthetic group 4'-phosphopantetheine from the holo-fatty acid synthetase. The two products of the action of this enzyme, 4'-phosphopantetheine and apo-fatty acid synthetase, were isolated by DEAE-cellulose chromatography and by chromatography on a Sepharose epsilon-aminocaproyl pantetheine column, respectively. The resultant apo-fatty acid synthetase was quantitated by immunoprecipitation and it was also converted to the holoprotein with a crude preparation of rat liver 4'-phosphopantetheine transferase. Quantitative determination of the hydrolase reaction product, 4'-phosphopantetheine, by amino acid analysis and microbiological assays confirmed the presence of 1 mol of this compound/mol of holo-fatty acid synthetase.     

An acyl-carrier-protein-thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea. High-level production, purification and characterisation in Escherichia coli

The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176-178]. Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.     

Crystal structure of the plant PPC decarboxylase AtHAL3a complexed with an ene-thiol reaction intermediate

The Arabidopsis thaliana protein AtHAL3a decarboxylates 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine, a step in coenzyme A biosynthesis. Surprisingly, this decarboxylation reaction is carried out as an FMN-dependent redox reaction. In the first half-reaction, the side-chain of the cysteine residue of 4'-phosphopantothenoylcysteine is oxidised and the thioaldehyde intermediate decarboxylates spontaneously to the 4'-phosphopantothenoyl-aminoethenethiol intermediate. In the second half-reaction this compound is reduced to 4'-phosphopantetheine and the FMNH(2) cofactor is re-oxidised. The active site mutant C175S is unable to perform this reductive half-reaction. Here, we present the crystal structure of the AtHAL3a mutant C175S in complex with the reaction intermediate pantothenoyl-aminoethenethiol and FMNH(2). The geometry of binding suggests that reduction of the C(alpha)=C(beta) double bond of the intermediate can be performed by direct hydride-transfer from N5 of FMNH(2) to C(beta) of the aminoethenethiol-moiety supported by a protonation of C(alpha) by Cys175. The binding mode of the substrate is very similar to that previously observed for a pentapeptide to the homologous enzyme EpiD that introduces the aminoethenethiol-moiety as final reaction product at the C terminus of peptidyl-cysteine residues. This finding further supports our view that these homologous enzymes form a protein family of homo-oligomeric flavin-containing cysteine decarboxylases, which we have termed HFCD family.     

The adenine nucleotide-binding site on yeast 3-phosphoglycerate kinase. Affinity labeling of Lys-131 by pyridoxal 5'-diphospho-5'-adenosine

The adenine nucleotide analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) is a potent and highly specific inactivator of yeast 3-phosphoglycerate kinase. Supportive evidence includes the finding that 1) during a 10-min incubation, half-maximal inactivation is given by 10 microM PLP-AMP, 2) covalent incorporation of 1.2 mol of PLP-AMP/mol of enzyme is sufficient to give complete inactivation, and 3) MgATP gives near complete protection against modification and inactivation by PLP-AMP. Following reaction with PLP-AMP and reduction with NaBH4 to form a stable adduct, the enzyme was digested with endoproteinase Lys-C and peptides were separated by reversed-phase high-performance liquid chromatography. The single major labeled peptide was purified and sequenced, and the modified residue was identified as Lys-131. The crystal structure of enzyme in the open conformation shows Lys-131 to reside within a loop of flexible random coil positioned at the outer edge of the central binding cleft, approximately 2 nm from the surface of the cleft that comprises part of the MgATP-binding site (Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kingsman, A. J., and Kingsman, S. M. (1982) EMBO J. 1, 1635-1640). We conclude that the structural element containing Lys-131 undergoes substantial movement during the ligand-induced conformational change known to occur during formation of the ternary complex, resulting in the positioning of a basic residue near a negatively charged substrate. Since similar affinity-labeling results have been presented for hexokinase (Tamura, J. K., LaDine, J. R., and Cross, R. L. (1988) J. Biol. Chem. 263, 7907-7912), we further suggest that movement of positive charge into the central cleft may be a common step in the tight binding of nucleotides by bilobal kinases.     

Phosphopantothenoylcysteine synthetase from Escherichia coli. Identification and characterization of the last unidentified coenzyme A biosynthetic enzyme in bacteria

Phosphopantothenoylcysteine synthase catalyzes the formation of (R)-4'-phospho-N-pantothenoylcysteine from 4'-phosphopantothenate and l-cysteine: this enzyme, involved in the biosynthesis of coenzyme A (CoA), has not previously been identified. Recently it was shown that the NH(2)-terminal domain of the Dfp protein from bacteria catalyzes the next step in CoA biosynthesis, the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to form 4'-phosphopantetheine (Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M., and Steinbacher, S. (2000) J. Biol. Chem. 275, 31838-31846). We have partially purified phosphopantothenoylcysteine decarboxylase from Escherichia coli and demonstrated that the protein encoded by the dfp gene, here renamed coaBC, also has phosphopantothenoylcysteine synthetase activity, using CTP rather than ATP as the activating nucleoside 5'-triphosphate. This discovery completes the identification of all the enzymes involved in the biosynthesis of coenzyme A in bacteria.     

Purification and properties of the fatty acids synthetase complex from Neurospora crassa, and the nature of the fas-mutation.

A procedure is described for the purification of the fatty acid synthetase complex (FAS) from Neurospora crassa. The enzyme complex has a molecular weight of 2.3 times 10(6), contains 6 mol of 4'-phosphopantetheine per mol, and on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives a single band, or a closely spaced doublet, which comigrates with standard myosin (molecular weight, 2 times 10(5)). Since the slightly retarded component in the doublet accounts for all protein-bound 4'-phosphopantetheine, the complex appears to be made up of 11 to 12 equally sized subunits, 6 of which carry the acyl carrier protein function. In this unusual arrangement, notably the lack of the low-molecular-weight acyl carrier protein component seen in other FAS systems, as well as in its enzymatic properties, the Neurospora FAS complex is quite similar to the yeast enzyme. The FAS complex of a saturated fatty acid-requiring mutant, previously disignated cel-, contains less than 2% of the 4'-phosphopantetheine prosthetic groups found in the wild-type complex. The leaky phenotype of this mutant, here designated fas-, is accounted for by a residual fatty acid synthesizing activity in its FAS complex, which is several-fold higher than expected from its residual content of 4'-phosphopanthetheine.     

Molecular characterization of the 4'-phosphopantothenoylcysteine synthetase domain of bacterial dfp flavoproteins

In bacteria, coenzyme A is synthesized in five steps from pantothenate. The flavoprotein Dfp catalyzes the synthesis of the coenzyme A precursor 4'-phosphopantetheine in the presence of 4'-phosphopantothenate, cysteine, CTP, and Mg(2+) (Strauss, E., Kinsland, C., Ge, Y., McLafferty, F. W., and Begley, T. P. (2001) J. Biol. Chem. 276, 13513-13516). It has been shown that the NH(2)-terminal domain of Dfp has 4'-phosphopantothenoylcysteine decarboxylase activity (Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M., and Steinbacher, S. (2000) J. Biol. Chem. 275, 31838-31846). Here I demonstrate that the COOH-terminal CoaB domain of Dfp catalyzes the synthesis of 4'-phosphopantothenoylcysteine. The exchange of conserved amino acid residues within the CoaB domain revealed that the synthesis of 4'-phosphopantothenoylcysteine occurs in two half-reactions. Using the mutant protein His-CoaB N210D the putative acyl-cytidylate intermediate of 4'-phosphopantothenate was detectable. The same intermediate was detectable for the wild-type CoaB enzyme if cysteine was omitted in the reaction mixture. Exchange of the conserved Lys(289) residue, which is part of the strictly conserved (289)KXKK(292) motif of the CoaB domain, resulted in complete loss of activity with neither the acyl-cytidylate intermediate nor 4'-phosphopantothenoylcysteine being detectable. Gel filtration experiments indicated that CoaB forms dimers. Residues that are important for dimerization are conserved in CoaB proteins from eubacteria, Archaea, and eukaryotes.     

Glutamyl-tRNA synthetases of Bacillus subtilis 168T and of Bacillus stearothermophilus. Cloning and sequencing of the gltX genes and comparison with other aminoacyl-tRNA synthetases

The glutamyl-tRNA synthetase (GluRS) of Bacillus subtilis 168T aminoacylates with glutamate its homologous tRNA(Glu) and tRNA(Gln) in vivo and Escherichia coli tRNA(1Gln) in vitro (Lapointe, J., Duplain, L., and Proulx, M. (1986) J. Bacteriol. 165, 88-93). The gltX gene encoding this enzyme was cloned and sequenced. It encodes a protein of 483 amino acids with a Mr of 55,671. Alignment of the amino acid sequences of four bacterial GluRSs (from B. subtilis, Bacillus stearothermophilus, E. coli, and Rhizobium meliloti) gives 20% identity and reveals the presence of several short highly conserved motifs in the first two thirds of these proteins. Conserved motifs are found at corresponding positions in several other aminoacyl-tRNA synthetases. The only sequence similarity between the GluRSs of these Bacillus species and the E. coli glutaminyl-tRNA synthetase (GlnRS), which has no counterpart in the E. coli GluRS, is in a segment of 30 amino acids in the last third of these synthetases. In the three-dimensional structure of the E. coli tRNA(Gln).GlnRS.ATP complex, this conserved peptide is near the anticodon of tRNA(Gln) (Rould, M. A., Perona, J. J., S?ll, D., and Steitz, T. A. (1989) Science 246, 1135-1142), suggesting that this region is involved in the specific interactions between these enzymes and the anticodon regions of their tRNA substrates.     

Mechanism of action of methanol oxidase, reconstitution of methanol oxidase with 5-deazaflavin, and inactivation of methanol oxidase by cyclopropanol

Methanol oxidase isolated from Hansenula polymorpha contains two distinct flavin cofactors in approximately equal amounts. One has been identified as authentic FAD and the other as a modified form of FAD differing only in the ribityl portion of the ribityldiphosphoadenosine side chain. The significance of this finding is as yet unknown. Previous studies have shown that cyclopropanol irreversibly inactivates methanol oxidase [Mincey, T., Tayrien, G., Mildvan, A. S., & Abeles, R. H. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7099-7101]. We have now established that inactivation is accompanied by covalent modification of the flavin cofactor. The stoichiometry of this reaction is 1 mol of cyclopropanol/mol of active flavin. The structure of the covalent adduct was determined by NMR, IR, and UV spectral studies to be an N5,C4a-cyclic 4a,5-dihydroflavin. Reduction of the covalent adduct with NaBH4 at pH 9.0 before removal from the enzyme converted it to the 1-(ribityldiphosphoadenosine)-substituted 4-(3-hydroxypropyl)-2,3-dioxoquinoxaline. Cyclopropyl ring cleavage accompanies inactivation, and covalent bond formation occurs between a methylene carbon of cyclopropanol and N5 of flavin. Methanol oxidase was also reconstituted with 5-deazaflavin adenine dinucleotide (dFAD). Reconstituted enzyme did not catalyze the oxidation of alcohols to the corresponding aldehydes, nor did reduced reconstituted enzyme catalyze the reverse reaction. Incubation of reconstituted enzyme with cyclopropanol resulted in an absorbance decrease at 399 nm, but no irreversible covalent modification of the deazaflavin cofactor. A reversible addition complex between cyclopropanol and dFAD is formed. The structure of that complex was not definitively established, but it is likely that it is formed through the addition of cyclopropoxide to C5 of dFAD. The failure of dFAD-reconstituted methanol oxidase to catalyze the oxidation of substrate, as well as the lack of reaction with cyclopropanol, supports a radical mechanism for alcohol oxidation and cyclopropanol inactivation. Methanol oxidase catalyzes the oxidation of cyclopropylcarbinol to the corresponding aldehyde. No ring-opened products were detected. The failure to form ring-opened products has been used as an argument against radical processes [MacInnes, I., Nonhebel, D. C., Orsculik, S. T., & Suckling, C. J. (1982) J. Chem. Soc., Chem. Commun., 121-122]. We present arguments against this interpretation.     

Endotoxin-binding proteins modulate the susceptibility of bacterial endotoxin to deacylation by acyloxyacyl hydrolase

Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs or LOSs, endotoxin) within intact cells and inflammatory fluids. In cell lysates or as purified enzyme, in contrast, detergent is required for AOAH to act on LPS or LOS (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449 and Katz, S. S., Weinrauch, Y., Munford, R. S., Elsbach, P., and Weiss, J. (1999) J. Biol. Chem. 274, 36579-36584). We speculated that the sequential interactions of endotoxin (E) with endotoxin-binding proteins (lipopolysaccharide-binding protein (LBP), CD14, and MD-2) might produce changes in endotoxin presentation that would allow AOAH greater access to its substrate, lipid A. To test this hypothesis, we measured the activity of purified AOAH against isolated, metabolically labeled meningococcal LOS and Escherichia coli LPS that were presented either as aggregates (LOSagg or LPSagg)+/-LBP or as monomeric protein (sCD14 or MD-2)-endotoxin complexes. Up to 100-fold differences in the efficiency of endotoxin deacylation by AOAH were observed, with the following rank order of susceptibility to AOAH: E:sCD14>or=endotoxin aggregates (Eagg):LBP (molar ratio of E/LBP 100:1)>Eagg, Eagg:LBP (E/LBP approximately 1, mol/mol), or E:MD-2. AOAH treatment of LOS-sCD14 produced partially deacylated LOS still complexed with sCD14. The underacylated LOS complexed to sCD14 transferred to MD-2 and thus formed a complex capable of preventing TLR4 activation. These findings strongly suggest that LBP- and CD14-dependent extraction and transfer of endotoxin monomers are accompanied by increased exposure of fatty acyl chains within lipid A and that the acyl chains are then sequestered when LOS binds MD-2. The susceptibility of the monomeric endotoxin-CD14 complex to AOAH may help constrain endotoxin-induced TLR4 activation when endotoxin and membrane CD14 are present in excess of MD-2/TLR-4.     

Low-resolution structure of the complex of human blood platelet factor 4 with heparin determined by small-angle neutron scattering

Small-angle neutron scattering was used to confirm that human platelet factor 4 was a compact tetrameric globular protein of radius of gyration 1.74 nm and indistinguishable from a sphere. The same technique, when applied to the 1:1 mol/mol complex of platelet factor and heparin of Mr 14000, revealed that the radius of gyration of the particle varied, depending on the relative proportion of 2H2O to H2O in the solvent. Analysis of this variation by the method of Ibel and Stuhrmann (Ibel, K. and Stuhrmann, H.B. (1975) J. Mol. Biol. 93, 255-266) revealed that in the complex the material of greatest neutron-scattering length (the highly sulphated polysaccharide heparin) was furthest from the centre of the particle. This confirms the postulate of Luscombe and Holbrook (Luscombe, M. and Holbrook, J.J. (1983) in Glycoconjugates (Chester, A.M., Heineg?rd, D., Lundblad, A. and Svensson, S., eds.), pp. 818-819, Secretariat, Lund) that the exact 1:1 mole ratio of heparin (Mr greater than 10 000) to platelet factor in this stable complex arises from the heparin winding around the outside of a globular protein core.