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The article presents information on various research papers which will be published in the forthcoming issue of the "European Journal of Biochemistry". Some of the papers discusses are "Enzymatic Synthesis of Neolactotetraosylceramide by the N-Acctyllactosamine Synthase of Human Serum". "Rate of Transplantation and Kinetics of Processing of Newly Synthesized Molecules of Two Major Outer-Membrane Proteins, the OmpA and OmpF Proteins of Escherichia Coli," by the researchers I. Crowlesmith and K. Gamon.

Peptide studies on two acidic proteins, A1 and A2, from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2-protein is Ser-Ile-Thr-Lys, while that of A1-protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1- or A2-protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]

The DNA-binding protein HU from Escherichia coil is a heterodimer constituted of two poly- peptide chains termed HU-1 and HU-2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino-terminal and carboxy-terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116–120]. The amino acid sequences of proteins HU-1 and HU-2 are identical to those of proteins NS-1 and NS-2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395–398]. The amino acid sequences of proteins HU-1 and HU-2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy-terminal part of the molecule. No sequence homology could be established between the proteins HU-1 and HU-2 and any one of the five histones from different eukaryotes. [ABSTRACT FROM AUTHOR]

Reduced and carboxymethylated thioredoxin H from Saccharomuces cerevisiae was digested with trypsin and cleaved with cyanogen bromide in Separate experiments. From the amino-acid sequences of isolated peptides after chymotryptic or peptic hydrolysis an alignment of a 17-residue sequence around the redox-active disulfide of thioredoxin II was possible as follows: -Phe-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys-Met-Ile-Ala-Pro-Met-Ile-Glu-Lys- Comparison of this structure with the corresponding amino-acid sequence of the thioredoxin from Escherichia coli B showed identical residues in 10 consecutive positions which included the tryptophan residue, the disulfide ring and the five residues following this on the COOH- terminal side. The COOH-terminal sequence of yeast thioredoxin II was: -Glu-Ala-Ile-Ala-Ser-Asn-Val and the NH2-terminal residue was valine. The sequence results indicated a considerable homology in primary structure between yeast and E. coli thioredoxins and suggested the existence of a common ancestral gene for thioredoxins. The sequence of a peptide from the active site of yeast thioredoxin I showed that the structure around the disulfide bridge was: -Tyr-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys-. This indicated that thioredoxin I and II from yeast have different primary structures. [ABSTRACT FROM AUTHOR]

Two isogenic derivatives of Escherichia coli K12: strains EA 1 (Met-, Biot-, RCstr and EA 2 (Mei-, Biot-, RCrel) when grown in the presence of ethionine in place of methionine, synthesize ribosomes whose RNA is submethylated and whose proteins contain ethionine. Net ribosome synthesis in the presence of ethionine is greater in the RCrel strain than in the RCstr strain. The precedinig paper describes results obtained with the latter strain. Here we present further studies carried out with the isogenic RCrel strain EA 2 As in the case of the RCstr strain (previous paper) ribosomes synthesized by the RCrel strain grown in the presence of ethionine display defective association properties. A large fraction of these "ethionine" ribosomes is found as free subunits in crude extracts containing 10 mM Mg+ prepared from strain EA 2 grown in an ethionine-containing medium. These free "ethionine" subunits have sedimentation coefficients of 28 S and 45 S approximately while the "ethionine" subunits found associated as 70 S particles in crude bacterial extracts sediment at 30 S and 50 S respectively. Both classes of "ethionine" subunits (free, and associated) can be methylated in vitro in the presence of S-adenosyl methionine with or without the addition of an external source of methylases (100000 x g supernatant of a homologous crude bacterial extract) The latter observation shows that the "ethionine" subunits contain bound methylases. These enzymes do not seem to be normal ribosomal proteins nor to be bound specifically to "ethionine" ribosomes since (a) they are removed by washing the "ethionine" ribosomes with 1 M NH4Cl, and (b) they are found bound to normal methionine ribosomes from which they are also removed by washing with 1 M NH4Cl. [ABSTRACT FROM AUTHOR]

In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem. 269 , 5564–5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position -10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon. [ABSTRACT FROM AUTHOR]

This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of non-covalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 μM. The enzyme binds FAD by a simple second-order process with Kd 0.67 μM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme. [ABSTRACT FROM AUTHOR]

The primary structure of protein S8 from the 30-S ribosomal subunit of Escherichia coil was determined mainly by automatic Edman degradation using a modified Beckman protein sequenator and the solid-phase sequenator of Laursen. The complete sequence, containing 109 amino acids, was derived by analysing peptides from tryptic, chymotryptic, thermolysin, staphylococcal protease and cyanogen bromide digestion of the protein. The amino acid composition was found to be (aspartic acid)6, (asparagine)3, (threonine)5, (serine)5, (glutamic acid)7, (glutamine)6, (proline)5, (glycine)6, (alanine)11, (valine)9, (methionine)4, (isoleucine)7, (leucine)9, (tyrosine)3, (phenylalanine)3, (lysine)11, (arginine)8, (cysteine)1. S8 is a basic protein and binds to the 16-S RNA; knowledge of its sequence is necessary for a detailed study of its interaction with the ribosomal RNA. [ABSTRACT FROM AUTHOR]

By treatment of Bacillus subtilis MO-101-P spoA- met thr- su- with ethyl methanesulfonate, a strong suppressor strain of nonsense mutations, B. subtilis MO-101-P spoA- [met-]+ thr- su+44, was isolated. This strain does not suppress phage Φ29 mutant susB47, selected on a B. subtilis strain containing the su+3 suppressor isolated by Georgopoulos. A revertant from this mutant, susB610, was isolated, being suppressed by both the su+3 and su+44 suppressor strains. The efficiency of suppression by strain su+44 is about 50%. The experiments shown in this paper suggest that strain su+44 contains an amber and strain su+3 an ochre suppressor. [ABSTRACT FROM AUTHOR]

Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenylalanine-resistant strains. The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria. The α and β subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl-tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant a subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation. [ABSTRACT FROM AUTHOR]

Penicillin-binding protein (pbp) 1b, the main DD-transpeptidase/transglycosylase of Escherichia coli, is normally present in the cell in three molecular forms α, β and γ, differenciated by their mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The three molecular forms are enzymatically active in vitro and their relative amounts are kept fairly constant in most labelling experiments with radioactive β-lactam antibiotics. In this paper, we have analyzed the expression of ponB (mrcB), the structural gone for pbp 1b, and the relation among the three forms of pbp 1b in ponB strains lysogenyzed by λ540 (ponB+) recombinant bacteriophages. Our data indicate that ponB is transcribed anti-clockwise on the E. coli chromosome and suggest that pbp 1bx is the first membrane-bound form of pbp 1b able to bind labelled β-lactams, and is the precursor of pbp 1bβ which is, in turn, the precursor of pbp 1bγ. [ABSTRACT FROM AUTHOR]

The accuracy of Qβ translation by Escherichia coli extracts in polymix and a conventional Tris/Mg2+ system has been followed. Misinsertions of histidine and of tryptophan into the phage coat protein were less frequent in polymix than in Tris/Mg2+, as were errors leading to u change in the coat protein pI. Even the lowest Qβ error rates, however, were still an order of magnitude greater than those for poly(U) or poly(U-G) translation. Comparing Qβ translational errors made in vitro to those found in whole cells, histidine misinsertions were almost twice as frequent, errors leading to a coat protein charge change six times more frequent and tryptophan misinsertions at least 15 times more frequent in vitro. The relation of these findings to measurements of translational accuracy and to factors affecting fidelity is discussed. [ABSTRACT FROM AUTHOR]

Partially purified enzymatic fractions from extracts of Escherichia coli B/r catalyse transfer of the isotope label from [adenine-2,8-3H]NAD+ to some bacterial proteins, as well as to hen egg-white lysozyme. The radio-active group in the modified lysozyme was identified as mono(ADP-ribose). Several bacterial proteins were labelled in vivo with 32P; the presence of the label in the form of an ADP-ribosyl group was shown in one of them. [ABSTRACT FROM AUTHOR]

The complete primary structure of elongation factor Tu from Escherichia coli has been elucidated. The protein, which is a mixture of two gene products, consists of a single polypeptide chain of 393 residues. After tryptic digestion of S-carboxymethylated protein, 50 tryptic peptides were isolated covering the complete protein chain. Their alignment was established with overlapping peptides obtained by chemical cleavage with cyanogen bromide and subsequent enzymic subdigestion with Staphylococcus aureus protease, chymotrypsin, elastase and thermolysin. Peptides were sequenced by manual dansyl-Edman and direct Edman degradation procedures. The N-terminal amino acid of EF-Tu is serine and is N-acetylated. The lysine residue at position 56, in the polypeptide chain is partly methylated. The C-terminal residue is a mixture of serine and glycine, and this was the only heterogeneity found in the EF-Tu preparation used in this study. [ABSTRACT FROM AUTHOR]

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-S RNA in the absence of ribosomal protein. These data yield important information on the topography and organization of the 5′ third of the 16-S RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA. [ABSTRACT FROM AUTHOR]

The NADPH-dependent enzymic reduction of disulfide bonds in human choriogonadotropin and its two subunits, α and β, was examined with thioredoxin and thioredoxin reductase from Escherichia coli. With 12 μM thioredoxin and 0.1 μM thioredoxin reductase at pH 7 all disulfide bonds in the α subunit could be reduced in 15 min. The reduction of disulfide bonds was recorded by a simple spectrophotometric assay at 340 nm, which allowed quantitation of the reduction rate and the number of disulfide bonds reduced. Partial reduction of the α subunit with thioredoxin followed by S-carboxymethylation with iodo[2-3H]acetic acid and analysis of tryptic peptides indicated that all S-S bonds in the α subunit were surface oriented and equally reactive. The usefulness of thioredoxin reduction of disulfide bonds as a chemical probe of protein structure was shown by the much slower reaction of disulfide bonds in the intact hormone as compared to its two biologically inactive subunits. [ABSTRACT FROM AUTHOR]

The cyanogens bromide fragments of protein I, a major protein of the Escherichia coli outer cell envelope membrane, have been isolated and characterized. There appear to be two methionineserine or methionine-threonine sequences causing incomplete cleavage but complete conversion of methionine to homoserine. Largerly due to the existence of these overlapping fragments the order of 5 of the 6 fragments present could be deduced. None of the fragments exhibits any remarkable low degree of polarity, and the tryptic fingerprint of the largest fragment (comprising about 60%,of protein I) also does not show any conspicuous large fraction of lipophilic peptides. It is concluded that the domain of protein I that may be buried in the lipid phase of the outer membrane in all likelihood is not very large, and there is, in fact, no definite proof yet that protein I is a membrane protein sensu stricto. [ABSTRACT FROM AUTHOR]

After digestion of protein S4 with trypsin, all 32 tryptic peptides were isolated. Their amino acid compositions were analyzed and the sequence of the amino acids within the tryptic peptides was determined by means of a solid-phase peptide sequenator and by exopeptidases. Alignment of the tryptic peptides was established by analyzing and partially sequencing peptides isolated after digestion of the $4 protein with chymotrypsin, thermolysin and a glutamic-acid-specific protease. Further information about the alignment of peptides came from treatment of S4 with CNBr and with a lysine-modifying reagent. Protein S4 consists of 203 amino acids (Asp8, Asn8, Thr6, Ser10, Glu16, Gln10, Pros, Gly15, Ala18, Va116, Cys1, Met3, Ile8, Leu20, Tyr8, Phe4, His3, Lys20, Arg22 and Trp1) and has a molecular weight of 22550. The basic amino acids are clustered in five regions. Many short repetitions mainly with charged amino acids occur. A prediction is made for regions with or α-helices and with β-sheets. [ABSTRACT FROM AUTHOR]

Two physically and genetically distinct forms of ribosephosphate isomerase have been identified in Escherichia coli K12. The constitutive ribosephosphate isomerase A has a Km for ribose 5-phosphate (4.4 ± 0.5 mM) six times greater than that of the inducible ribosephosphate isomerase B (0.83 ± 0.13 mM). Treatment of the enzymes with 1.25 mM iodoacetate resulted in 100% loss of activity for ribosephosphate isomerase B, whereas ribosephosphate isomerase A was unaffected. Various cellular metabolites were tested and found to be without significant effect on either enzyme. The two enzymes could be separated by filtration on Sephadex G75 superfine and their apparent molecular weights were 45000 for ribosephosphate isomerase A and 32000–34000 for ribosephosphate isomerase B. Under certain conditions the two enzymes showed different patterns of heat inactivation but the results with ribosephosphate isomerase A varied in an unusual way with the protein concentration. Ribosephosphate isomerase B was formed inducibly in a mutant lacking ribosephosphate isomerase A but there was no evidence for the production of ribosephosphate isomerase B in wild-type cells. The formation of ribosephosphate isomerase B was not a consequence of the ribosephosphate isomerase A mutation, since strains could be constructed which formed both enzymes constitutively in the anticipated amounts. The ribosephosphate isomerase formed by a secondary mutant obtained from a ribosephosphate-isomerase-A-negative strain was identified as ribosephosphate isomerase B on the basis of its Km, elution profile from Sephadex G75, inhibition by iodoacetate, and heat inactivation. The ribosephosphate isomerases of another Escherichia coli K12 strain, X289, were investigated, since their properties were reported to be different from many of these described here for ribosephosphate isomerases A and B. In our hands strain X289 contained two ribosephosphate isomerases apparently identical to ribosephosphate isomerases A and B. The evidence to date suggests that ribosephosphate isomerase A catalyses the formation of ribose 5-phosphate from ribulose 5-phosphate and also participates in the reverse reaction during ribose and adenosine catabolism. The normal physiological role of the inducible ribosephosphate isomerase B is still uncertain. [ABSTRACT FROM AUTHOR]

Both rRNA and protein components of Escherichia coli ribosomes have been specifically labelled without any important loss of activity for polyphenylalanine synthesis in the cell-free system. Three adenylic residues per 70-S ribosome were labelled by an anthracenyl derivative and the ribosomal proteins by dansylation of four amino acid residues per 70-S particle. The association between the two complementary subparticles, the formation of the binary and ternary complexes in the presence of mRNA and tRNA were investigated using the concomitant measuring of fluoroscence intensity and polarisation of the two kinds of labels. The formation of these complexes involves the rRNA and the ribosomal protein components of the 30-S and 50-S subparticles directly and/or indirectly. The mobility of the messenger bound to the 70-S ribosome in the presence of tRNA is indicated by use of a polyadenylic acid labelled with the same marker ss that for rRNA. Reversible conformational changes are observed during acidic-alkaline titrations and melting curves of fluorescent ribosomal particles. The irreversible denaturation occurs at the same pH and melting temperature for the rRNA and the ribosomal components: The results are interpreted in terms of highly specific interactions between the rRNA and the proteins, slight modification in the strength and/or nature of these interactions leading to a conformational change of the whole ribosomal particle. [ABSTRACT FROM AUTHOR]

We have studied the ribonucleosidediphosphate reductase of a deoxyuridine-requiring mutant LD195 of Escherichia coli K. This enzyme was purified 50 fold from the mutant and a control strain. In all steps of the purification the mutant strain yielded normal activity of the Bi subunit of the enzyme but approximately 10 % of the wild-type activity of the B2 subunit The activity is similarly reduced for all three substrates tested CDP, UDP, and GDP. The mutation appears to affect the physical structure of the B2 subunit, since addition of high concentrations of sodium acetate can restore enzymatic activity to the partially purified B2 subunit from the mutant strain. In contrast, the same concentrations strongly inhibit the wild- type B2 subunit. Although the mutant has a requirement of deoxyuridine at 42 °C but not at 30 °C (as shown in an accompanying paper), mutant protein B2 is not more thermo-labile than wild-type protein B2 This requirement therefore probably reflects an increased demand of deoxyribonucleotides at higher temperatures. Hydroxyurea inhibits mutant and wild-type ribonucleosidediphosphate reductase to the same extent in vitro. In spite of this, hydroxyurea inhibits growth of the mutant strain at a 20 fold lower concentration than that required for comparable inhibition of growth of the parent. This is explained by the greatly decreased protein B2 activity of the mutant. [ABSTRACT FROM AUTHOR]

Through the use of the analytical active-enzyme-centrifugation method we have been able to measure the sedimentation coefficients of enzymes which have not yet been obtained in highly purified preparations: aspartate-semialdehyde dehydrogenase from Escherichia coli and from yeast and lactate dehydrogenase from rabbit muscle. We have also been able to determine unambiguously the polymerization state of the fully active unit, of β-galactosidase from E. coli, alcohol dehydrogenase from yeast as well as the polymerization state of the fully active unit of two enzymes which undergo an association-dissociation concentration-dependent reaction, glucose-6-phosphate dehydrogenase from yeast and glutamate dehydrogenase from beef liver (for this last enzyme the active-enzyme-centrifugation method has been applied with NAD, NADH, NADP and NADPH as coenzymes and also for its alanine dehydrogenase activity). [ABSTRACT FROM AUTHOR]

The complex protein carrying the two threonine sensitive activities aspartokinase I and homoserine dehydrogenase I is composed of six subunits of a molecular weight 60,000. Furthermore, quantitative determination of the N-terminal amino acids indicates the presence of six methionine residues per mole of native enzyme (mol wt, 360,000). Equilibrium sedimentation studies of N-ethylmaleimide treated protein shows that its six disulfide bridges, revealed by chemical analysis, are intra-chain. Fingerprints of tryptic digests of the protein fail to reveal any difference between the subunits. [ABSTRACT FROM AUTHOR]

The SUMO proteins are a class of small ubiquitin-like modifiers. SUMO is attached to a specific lysine side chain on the target protein via an isopeptide bond with its C-terminal glycine. There are at least four SUMO proteins in humans, which are involved in protein trafficking and targeting. A truncated human SUMO-2 protein that contains residues 9–93 was expressed inEscherichia coliand crystallized in two different unit cells, with dimensions of a = b = 75.25 Å, c = 29.17 Å and a = b = 74.96 Å, c = 33.23 Å, both belonging to the rhombohedral space groupR3. They diffracted X-rays to 1.6 Å and 1.2 Å resolution, respectively. The structures were determined by molecular replacement using the yeast SMT3 protein as a search model. Subsequent refinements yieldedR/ Rfree values of 0.169/0.190 and 0.119/0.185, at 1.6 Å and 1.2 Å, respectively. The peptide folding of SUMO-2 consists of a half-openβ-barrel and two flankingα-helices with secondary structural elements arranged asββαββαβ in the sequence, identical to those of ubiquitin, SMT3 and SUMO-1. Comparison of SUMO-2 with SUMO-1 showed a surface region near the C terminus with significantly different charge distributions. This may explain their distinct intracellular locations. In addition, crystal-packing analysis suggests a possible trimeric assembly of the SUMO-2 protein, of which the biological significance remains to be determined. [ABSTRACT FROM AUTHOR]

Cyclophilins facilitate the peptidyl-prolyl isomerization of a trans-isomer to a cis-isomer in the refolding process of unfolded proteins to recover the natural folding state with cis-proline conformation. To date, only short peptideswith a cis-form proline have been observed in complexes of human and Escherichia coli proteins of cyclophilin A, which is present in cytoplasm. The crystal structures analyzed in this study show two complexes in which peptides having a transform proline, i.e. succinyl-Ala-trans-Pro-Ala-p-nitroanilide and acetyl-Ala-Ala-trans-Pro-Ala-amidomethylcoumarin, are bound on a K163T mutant of Escherichia coli cyclophilin B, the preprotein of which has a signal sequence. Comparison with cis-form peptides bound to cyclophilin A reveals that in any case the proline ring is inserted into the hydrophobic pocket and a hydrogen bond between CO of Pro andNeta2 of Arg is formed to fix the peptide. On the other hand, in the cis-isomer, the formation of two hydrogen bonds of NH and CO of Ala preceding Pro with the protein fixes the peptide, whereas in the trans-isomer formation of a hydrogen bond between CO preceding Ala-Pro and His47 Nepsiv2 via a mediating water molecule allows the large distortion in the orientation of Ala of Ala-Pro. Although loss of double bond character of the amide bond of Ala-Pro is essential to the isomerization pathway occurring by rotating around its bond, these peptides have forms impossible to undergo proton transfer from the guanidyl group of Arg to the prolyl N atom, which induces loss of double bond character. Guanylate cyclase-activating proteins (GCAPs) are neuronal calcium sensors that activate membrane bound guanylate cyclases (EC 4.6.1.2.) of vertebrate photoreceptor cells when cytoplasmic Ca2+ decreases during illumination. GCAPs contain four EF-hand Ca2+-binding motifs, but the first EF-hand is nonfunctional. It was concluded that for GCAP-2, the loss of Ca2+-binding ability of EF-hand 1 resulted in a region that is crucial for targeting guanylate cyclase [Ermilov, A.N.,Olshevskaya, E.V.& Dizhoor,A.M. (2001) J. Biol. Chem. 276, 48143-48148]. In this study we tested the consequences of mutations in EF-hand 1 of GCAP-1 with respect to Ca2+ binding, Ca2+-induced conformational changes and target activation. When the nonfunctional first EF-hand in GCAP-1 is replaced by a functional EF-hand the chimeric mutant CaM-GCAP-1 bound four Ca2+ and showed similar Ca2+-dependent changes in tryptophan fluorescence as the wild-type. CaM- GCAP-1 neither activated nor interacted with guanylate cyclase. Size exclusion chromatography revealed that the mutant tended to form inactive dimers instead of active monomers like the wild-type. Critical amino acids in EFhand 1 ofGCAP-1 are cysteine at position 29 and proline at position 30, as changing these to glycine was sufficient to cause loss of target activation without a loss of Ca2+- induced conformational changes. The latter mutation also promoted dimerization of the protein. Our results show that EF-hand 1 in wild-typeGCAP-1 is critical for providing the correct conformation for target activation. [ABSTRACT FROM AUTHOR]

Although isopentenyl diphosphate–dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH. [ABSTRACT FROM AUTHOR]

Signal sequences often contain α-helix-destabilizing amino acids within the hydrophobic core. In the precursor of the Escherichia coli outer-membrane protein PhoE, the glycine residue at position -10 (Gly-10 ) is thought to be responsible for the break in the α-helix. Previously, we showed that substitution of Gly-10 by α-helix-promoting residues (Ala, Cys or Leu) reduced the proton-motive force dependency of the translocation of the precursor, but the actual role of the helix breaker remained obscure. Here, we considered the possibility that extension of the α-helical structure in the signal sequence resulting from the Gly-10 substitutions affects the targeting pathway of the precursor. Indeed, the mutations resulted in reduced dependency on SecB for targeting in vivo . In vitro cross-linking experiments revealed that the G-10L and G-10C mutant PhoE precursors had a dramatically increased affinity for P48, one of the constituents of the signal-recognition particle (SRP). Furthermore, in vitro cross-linking experiments revealed that the G-10L mutant protein is routed to the SecYEG translocon via the SRP pathway, the targeting pathway that is exploited by integral inner-membrane proteins. Together, these data indicate that the helix breaker in cleavable signal sequences prevents recognition by SRP and is thereby, together with the hydrophobicity of the signal sequence, a determinant of the targeting pathway. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria are enveloped by a system of two membranes, and they use specialized multicomponent, energy-driven pumps to transport molecules directly across this double-layered partition from the cell interior to the extra-cellular environment. One component of these pumps is embedded in the outer-membrane, and the paradigm for its structure and function is the TolC protein from Escherichia coli. A common component of a wide variety of efflux pumps, TolC and its homologues are involved in the export of chemically diverse molecules ranging from large protein toxins, such as α-hemolysin, to small toxic compounds, such as antibiotics. TolC family members thus play important roles in conferring pathogenic bacteria with both virulence and multidrug resistance. These pumps assemble reversibly in a transient process that brings together TolC or its homologue, an inner-membrane-associated periplasmic component, an integral inner-membrane translocase and the substrate itself. TolC can associate in this fashion with a variety of different partners to participate in the transport of diverse substrates. We review here the structure and function of TolC and the other components of the efflux/transport pump. [ABSTRACT FROM AUTHOR]

GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase and d-proline reductase, respectively, that are processed post-translationally to form a catalytic active pyruvoyl group. The cleavage occurred on the N-terminal side of a cysteine residue, which is thus the precursor of a pyruvoyl moiety. Both proproteins could be over-expressed in Escherichia coli and conditions were developed for in vitro processing. GrdE could be expressed as full-size protein, whereas PrdA had to be truncated N-terminally to achieve successful over-expression. Both proproteins were cleaved at the in vivo observed cleavage site after addition of 200 mm NaBH4 in Tris buffer (pH 7.6) at room temperature as analysed by SDS/PAGE and MS. Cleavage of GrdE was observed with a half-time of ≈ 30 min. Cys242, as the precursor of the pyruvoyl group in GrdE, was changed to alanine, serine, or threonine by site-directed mutagenesis. The Cys242→Ser and Cys242→Thr mutant proteins were also cleaved under similar conditions with extended half-times. However, the Cys242→Ala mutant protein was not cleaved indicating a pivotal role of the thiol group of cysteine or hydroxyl group of serine and threonine during the processing of pyruvoyl group-dependent reductases. [ABSTRACT FROM AUTHOR]

The putative catalytic domain of an open reading frame from Plasmodium falciparum with similarity to the ispF gene of Escherichia coli specifying 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase was expressed in a recombinant E. coli strain. The recombinant protein was purified to homogeneity and was found to catalyze the formation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate from 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate at a rate of 4.3 µmol·mg-1·min-1. At lower rates, the recombinant protein catalyzes the formation of 2-phospho-2C-methyl-d-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate and the formation of 2C-methyl-d-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-d-erythritol. Divalent metal ions such as magnesium or manganese are required for catalytic activity. The enzyme has a pH optimum at pH 7.0. Recombinant expression of the full-length open reading frame afforded insoluble protein that could not be folded in vitro. The enzyme is a potential target for antimalarial drugs directed at the nonmevalonate pathway of isoprenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Examines the characterization of MB-1 protein. Description of the protein; Role of Escherichia coli in the purification of MB-1; Use of circular dichroism and fluorospectroscopy.

We previously used fhuF as a sensitive reporter gene of the iron status of Escherichia coli. In this report, the fhuF gene was identified as open reading frame f262b at 99.2 min on the genome sequence map of E. coli K-12. The FhuF protein was labeled with a His-tag and then purified to electrophoretic homogeneity. Based on sulfur determinations and Mössbauer and EPR spectroscopy, FhuF was identified as a [2Fe-2S] protein. The g values (gx = 1.886, gy = 1.961, gz = 1.994) and some of the Mössbauer parameters of FhuF obtained [oxidized protein as isolated : ΔEQ,4.2K = 0.474 mm s-1; Fe3+ (reduced protein) : ΔEQ = 0.978 mm s-1] are not typical of common [2Fe-2S] proteins and indicate that FhuF has unusual structural properties. The primary sequence of FhuF does not show any sequence similiarities to known [2Fe-2S] proteins. By site-directed mutagenesis, each of the six cysteines of FhuF was replaced by serine. EPR of the six reduced mutant proteins revealed that the terminal cysteine residues 244, 245, 256, and 259 form the [2Fe-2S]Cys4 cluster. Mutants having the Cys-to-Ser replacement at positions 244, 245, 256, or 259 did not complement a fhuF mutant. The motif Cys-Cys-Xaa10-Cys-Xaa2-Cys in FhuF differs considerably from the motif Cys-Xaa2-Cys-Xaa9-15-Cys-Xaa2-Cys found in other [2Fe-2S] proteins. The unusual Cys-Cys terminal group of the cluster may explain the atypical EPR and Mössbauer spectroscopic properties of the FhuF protein; possibly the tetrahedral symmetry at the ferric ion site is distorted. The phenotype of fhuF mutants and the structural features of the FhuF protein suggest that FhuF is involved in the reduction of ferric iron in cytoplasmic ferrioxamine B. [ABSTRACT FROM AUTHOR]

The gene strQ was identified as the last gene of a putative transcription unit, strB1FGHPQ, located in the gene cluster for the production of 5′-hydroxy-streptomycin (OH-Sm) in Streptomyces glaucescens GLA.0. [In contrast, the corresponding operon in the str/sts-gene cluster of the Sm-producer Streptomyces griseus, strB1FGHIK, differs in the two distal genes; Mansouri, K. & Piepersberg, W. (1991) Mol. Gen. Genet. 228, 459-469.] The deduced StrQ protein exhibited similarities to members of the enzyme family of hexose-1-phosphate nucleotidylyltransferases (NDP-hexose synthases or pyrophosphorylases), with the strongest similarity to the subfamily of α-D-glucose-1-phosphate cytidylyltransferases (CDP-D-glucose synthases). The StrQ protein was heterologously expressed in Escherichia coli. The purified protein revealed an enzyme activity of that of a CDP-D-glucose synthase and a substrate specificity restricted to CTP and α-D-glucose 1-phosphate. The Km and Vmax values determined for CTP are 44 μM and 920 μM and for α-D-glucose 1-phosphate 195 μM and 1.06 mM, respectively. The CDP-D-glucose synthase activity was also detected in cells of S. glaucescens under the conditions of antibiotic production, but was absent from cells of the streptomycin producer S. griseus N2-3-11. Also, the genomes of several strains of S. griseus did not seem to possess strQ-related genes. In contrast, hybridisation experiments indicated that genes homologous to strQ were probably present in various other actinomycetes producing aminoglycosides. A possible function of the StrQ protein in the OH-Sm biosynthetic pathway of GLA.0 is discussed. [ABSTRACT FROM AUTHOR]

Ornithine carbamoyltransferase (OTCase) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is a 400 ± 20-kDa polymer of a 35-kDa subunit, in keeping with the corresponding gene sequence [Roovers, M., Hethke, C., Legrain, C., Thomm, M. & Glansdorff, N. (1997) Isolation of the gene encoding Pyrococcus furiosus ornithine cabamoyltransferase and study of its expression profile in vivo and in vitro, Eur. J. Biochem. 247, 1038-1045]. In contrast with the dodecameric catabolic OTCase of Pseudomonas aeruginosa, P. furiosus OTCase exhibits no substrate cooperativity. In keeping with other data discussed in the text, this suggests that the enzyme serves an anabolic function. Half-life estimates for the purified enzyme ranged over 21-65 min at 100°C according to the experimental conditions and reached several hours in the presence of ornithine and phosphate. The stability was not markedly influenced by the protein concentration. Whereas comparative examination of OTCase sequences did not point to any outstanding feature possibly related to thermophily, modelling the enzyme on the X-ray structure of R aeruginosa OTCase (constituted by four trimers assembled in a tetrahedral manner) suggests that the molecule is stabilized, at least in part, by a set of hydrophobic interactions at the interfaces between the trimers. The comparison between P. aeruginosa and P. furiosus OTCases suggests that two different properties, allostery and thermostability, have been engineered starting from a similar quaternary structure of high internal symmetry. Recombinant P. furiosus OTCase synthesised by Escherichia coli proved less stable than the native enzyme. In Saccharomyces cerevisiae, however, an enzyme apparently identical to the native one could be obtained. [ABSTRACT FROM AUTHOR]

Recombinant, normal human medium-chain acyl-CoA dehydrogenase (MCADH) and the common, human disease-causing K304E mutant ([Glu304]MCADH) protein were expressed in Escherichia coli using an optimized system, and the enzymes were purified to apparent homogeneity. The crucial factor leading to the production of active [Glu304]MCADH protein is the expression in E. coli cells at reduced temperature (28°C). Expression in the same system at 37°C results in very low amounts of active mutant protein. Several catalytic and physicochemical parameters of these two proteins have been determined and were compared to those of purified pig kidney MCADH. Although [Glu304]MCADH has approximately the same rate of substrate reduction with dodecanoyl-CoA and the same Vmax as human MCADH with the best substrate for the latter, octanoyl-CoA, the Km in the mutant MCADH is fourfold higher, which generates a correspondingly lower catalytic efficiency. Importantly, Vmax obtained using the natural acceptor, electron transfer flavoprotein, is only a third that for human MCADH. The Vmax/Km versus chain-length profile of the mutant shows a maximum with dodecanoyl-CoA which differs markedly from that of human MCADH, which has maximal efficiency with octanoyl-CoA. The substrate specificity of the mutant is broader with a less pronounced activity peak resembling long-chain acyl-CoA dehydrogenase. The purified mutant enzyme exhibits a reduced, thermal stability compared to human wild-type MCADH. The major difference between the two proteins expressed in E. coli is the more pronounced lability of the K304E mutant in crude extracts, which suggests a higher susceptibility to attack by endogenous proteases. Differences between tetrameric [Glu304]MCADH which survives the first step(s) of purifiction and corresponding MCADH are minor. The overall differences in properties of [Glu304]MCADH together with its impaired folding and tetramer assembly may contribute to the generation of the abnormalities observed in patients homozygous for the K304E mutation. [ABSTRACT FROM AUTHOR]

Autoantibodies to the islet-cell 65-kDa variant of glutamate decarboxylase (GAD65) are found in most insulin-dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease. As IDDM-preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti-GAD immunochemical tests. These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs. Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues. Thus, the conformational integrity of GAD65 is essential for an appropriate anti-GAD assay. Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems. Since the large-scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative. A number of DNA constructs intended to export the enzyme to the pedplasmic space or to improve its cytoplasmic solubility were designed and tested. Our results provide a solution to the two main problems associated with the expression of GAD65 in E. coli: misfolding, leading to the formation of inclusion bodies; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65. We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N-terminal fusion protein with thioredoxin. [ABSTRACT FROM AUTHOR]

A 6-kb DNA fragment from an extreme thermophile, Thermus thermophilus, carrying the genes for cytochrome oxidase ba3 subunit I (cbaA) and the ribosomal protein S15 (rpsO) was cloned into Escherichia coli. The gene rpsO was sequenced. The deduced amino acid sequence exhibits 59% identity to the corresponding protein from E. coli. Expression of rpsO in E. coli requires the use of a fully repressed inducible promoter because S15 from T. thermophilus is toxic for E. coli cells, When purified without denaturation from either overproducing E. coli strain or from T. thermophilus ribosomes, the S15 protein is stable and binds a cloned T. thermophilus 16S rRNA fragment (nucleotides 559-753), with low identical dissociation constants (2.5 nM), thus demonstrating that the thermophilic protein folds correctly in a mesophilic bacterium. The rRNA fragment bound corresponds in position and structure to the 16S rRNA fragment of E. coli. A similar high affinity was also found for the binding of S 15 from T. thermophilus or E. coli to the corresponding E. coli 16S rRNA fragment, whereas a slightly lower affinity was observed in binding experiments between E. coli S15 and T. thermophilus 16S rRNA fragment. These results suggest that S15 from T. thermophilus recognizes similar determinants in both rRNA fragments. Competition experiments support this conclusion. [ABSTRACT FROM AUTHOR]

The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N-terminal hexa-histidine tag The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absentee of calcium. In contrast to S. hyicuss lipase, not only strontium but also barium can replace calcium with full retention of activity of S, aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases. [ABSTRACT FROM AUTHOR]

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to the translocation of protons across a membrane. The NAD(H)-binding domain of transhydrogenase (domain I protein) from Rhodospirillum rubrum and from Escherchia coli were overexpressed and purified. Nucleoude binding to the domain I proteins was determined by equilibrium dialysis. NADH and its analogue, acetylpyridine adenine dinucleotide (reduced form), bound with relatively high affinity (Kd = 32 μM and 120 μM). respectively, for the R. rubrum protein). The binding affinity was similar at pH 8.0 and pH 9.0 in zwitterionic buffers and at pH 7.5 in sodium phosphate buffer, NAD+hound with lower affinity (Kd = 300 μM). NADPH bound only very weakly (Kd1> 1 mM). Using a centrifugation procedure, Yamaguchi and Hatefi [Yamaguchi, M & Hatefi, Y. (1993) J. Biol. Chem, 268, 1781-17877 ] found that mitochondrial transhydrogenase, and a proteolytically derrived domain I fragment from that exnzyme, bound one NADH per dimer. They suggested that this result implied half-of-the-sites reactivity for the interaction between the nucleotide ligand and the protein. However our studies on both the E. coli and the R. rubrum recombinant transhydrogenase domain I protein using equilibrum dialysis show that the binding stoichiometry for both NADH and the reduced form of acetylpyridine adenine dinucleotide (AcPdADH) is two nucleotides per dimer: no interaction between the monochondrial transhydrogenases are discussed. [ABSTRACT FROM AUTHOR]

The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-150; Paulsen, H., Rumler, U. & Rudiger, W. (1990) Planta (Heidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII. [ABSTRACT FROM AUTHOR]

The gene for the translation termination factor RF-3 in Escherichia coli has been cloned and sequenced. Only small amounts of the protein have been purified until now, not sufficient for detailed investigation of the structure and function of this factor. For such studies, we have developed an overexpression system and a purification procedure suitable for large quantities of RF-3. The gene prfC was cloned into the osmo-inducible plasmid pOSEX3 and subsequently transformed into the E. coli strain MKH13. The expression of prfC in this plasmid, which is under the control of the osmotic pressure in the growth medium, leads to a level of RF-3 more than 100-times higher than that in wild-type cells. Using a new two step FPLC protein purification procedure consisting of ion-exchange chromatography on Q-Sepharose FF and S-Sepharose HP, we obtain 220 mg pure Rf-3 from 10 g overproducing cells, corresponding to 55 mg RF-3/I medium. The identity of the purified protein was confirmed by matrix-assisted laser desorption/ionisation mass spectrometry of tryptolytic fragments and by N-terminal amino acid sequencing. The activity of the purified factor was tested in vitro by measuring the stimulation of RF-2 dependent formymethionine release from a ribosomal termination complex and the binding capacity of GTP and GDP. All assays showed that the purified RF-3 was highly active with a specific activity of approximately 2000 units/mg. [ABSTRACT FROM AUTHOR]

To identify the ssDNA-binding domain of Escherichia coli RecA protein, we examined the ssDNA-binding capabilities of synthetic peptides, the sequences of which were derived from the C- and N-termini and from sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure. Synthetic peptides derived from amino acid residues 185–219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter-binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E. coti RecA in this region did not. The binding of RecA to ssDNA examined using a gel-shill assay was inhibited by a synthetic peptide derived from this ssDNA-binding region, but not by synthetic peptides derived from amino acid residues 301–329 of the C-terminus or from N-terminal residues 6–39. A peptide corresponding to amino acid positions 152–169 of the RecA molecule and spanning loop L1 and its flanking regions did not bind ssDNA at peptide concentrations up to 250 μM. We have also defined a synthetic 20-amino-acid peptide that comprises amino acid residues 193–212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region of RecA. Finally, two maltose-binding protein-RecA fusion proteins were made, one containing amino acid residues 185–224 of RecA and the other the last 51 C-terminal residues of RecA (amino acid residues 303–353). In contrast to the C-terminus-derived fusion protein, the fusion protein containing the putative DNA-binding site demonstrated significant binding to single-stranded oligonucleotides in both filter-binding and gel-shift assays. These findings suggest that a portion of the region extending from amino acid residues 193–212 is either part of or the whole ssDNA-binding domain of the RecA protein. [ABSTRACT FROM AUTHOR]

The active form of protein R2, the small subunit of ribonucleotide reductase, contains a diferric center and a free radical localized at Tyr122. Hydroxyurea scavenges this radical but leaves the iron center intact. the resulting metR2 protein is inactive. The introduction of a radical into metR2 is dependent on the reduction of the iron center. In Escherichia coli, this is achieved by an enzyme system consisting of a NAD(P)H:flavin oxidoreductase and a poorly defined protein fraction, fraction b. Assuming that the iron center is deeply buried within the protein, electron transfer is suggested to occur over long distances. Site-directed mutagenesis allowed us to identify two invariant residues. Tyr356 at the C-terminal part of the protein and Tyr122 located 0.5 nm away from the closest iron atom, as mediators of this electron transfer. We also found that deazaflavins were excellent catalysts in the photoreduction of the iron center of meR2 and generation of the tyrosyl radical, providing the simplest and most efficient model for the physiological flavin reductase/fraction b activating system. The properties of the model reaction are described. [ABSTRACT FROM AUTHOR]

We have utilized a T7 polymerase/promoter system for the high-level incorporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high-level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high-level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray structural analysis, supplementing the traditional trial-and-error preparation of heavy-atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high-level incorporation of amino acid analogs can provide single-atom mutations for the detailed study of the structure and function of proteins. [ABSTRACT FROM AUTHOR]

Focuses on [sup 1]H, [sup 13]C, [sup 15[N-NRM resonance assignments of oxidized thioredoxin h from the eukaryotic green alga Chlamydomonas reinhardtii using novel methods based on two-dimensional triple-resonance NRM spectroscopy and computer-assisted backbone assignment. Overexpression of cystolic thioredoxin h in E. coli; Secondary structure characteristics of thioredoxin h.

A genomic library from Lactobacillus delbrueckii subsp, bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and tiros unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD+-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD+-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp, bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched β-carbon. [ABSTRACT FROM AUTHOR]

The TlpA protein of Bradyrhizobium japonicum was previously identified genetically as a membrane-anchored, periplasmic thioredoxin-like protein. Here we describe the heterologous expression in Escherichia coli, subsequent purification and biochemical characterization of TlpA. A soluble form of TlpA, which lacks its N-terminal membrane anchor, was overexpressed in E. coli and purified by a two-step procedure. Pure TlpA was shown to be a monomer in solution and was active in reducing the disulfides of insulin and in reactivating reduced, denatured RnaseA. Evidence is presented that two non-active-site cysteine residues form an intramolecular disulfide bond, a feature that is not normally found in other prokaryotic thioredoxins. [ABSTRACT FROM AUTHOR]

The mob mutants in Escherichia coil are pleiotropically defective in all molybdoenzyme activities. They synthesise molybdopterin, the unique core of the molybdenum cofactor, but are unable to attach the GMP moiety to molybdopterin to form molybdopterin guanine dinucleotide, the functional molybdenum cofactor in Escherichia coli. A partially purified preparation termed protein FA (protein factor d'association), is able to restore molybdoenzyme activities to broken cell preparations of mob mutants. A fragment of DNA capable of complementing mob mutants has been isolated from an E. coli genomic library. Strains carrying this DNA in a multicopy plasmid, express 30-fold more protein FA activity than the wild-type bacterium. Protein FA has been purified to homogeneity by a combination of ion-exchange, affinity and gel-filtration chromatography. Protein FA consists of a single polypeptide of molecular mass 22 kDa and is monomeric in solution. N-terminal amino acid sequencing confirmed that protein FA is a product of the first gene at the mob locus. The purified protein FA was required in stoichiometric rather than catalytic amounts in the process that leads to the activation of the precursor of the molybdoenzyme nitrate reductase, which is consistent with the requirement of a further component in the activation. [ABSTRACT FROM AUTHOR]

The enzyme myristoyl-CoA:protein N-myristoyltransferase is responsible for the attachment of a myristoyl group to the N-terminal glycine of a number of cell, viral and fungal proteins. In order to overcome the difficulties of purification of this enzyme from tissue sources, we have produced an N-terminally polyhistidine-tagged version of the enzyme and expressed this in Escherichia coli. The resulting enzyme has a molecular mass of 53 kDa arid is fully active showing the expected specificity for myristic acid and causing the N-terminal inyristoylation of both synthetic peptide and protein substrates in vitro. The enzyme exhibits a broad pH optimum peaking at a pH of 8.0 and has a Km for myristoyl-CoA of 7.6 μM. The two synthetic peptide substrates based on the N-terminal sequence of the catalytic subunit of protein kinase A (GNAAAARR) and of p60src (GSS- KSKPKDPSQRRRY) have different kinetic parameters with Km, values of 115.2 μM and 44.2 μM and Vmax values of 95 and 120 nmol - min-1 - mg-2, respectively. The expressed enzyme is partially inhibited (50%) by iodoacetamide at 5 mM and fully inhibited by diethylpyrocarbonate at 10mM. This latter inhibition can be prevented by including histidine in the incubation of the enzyme and inhibitor. Antisera raised to synthetic peptides based on sequences derived from the N- and C- terminus of the human enzyme reacted with the expressed protein on Western blots, but only the N-terminal sequence reacted with the native protein suggesting that the C-terminus may he riot he accessible. The enzyme can catalyse the removal of a myristoyl group from myristoylated peptides but does so only in the presence of added coenzyme A. [ABSTRACT FROM AUTHOR]

A novel protein derivative has been found during process development of biosynthetic human growth hormone; it has been characterized as human growth hormone with a Cys182-Cys189 trisulphide bridge. We have not been able to find a previous report in the literature about this kind of derivative. The characterization was obtained partly on the full-length derivative and partly on a tryptic fragment of the derivative. The full-length derivative was characterized by reduction with 1.4-dithiothreitol followed by electrospray mass spectrometry, treatment with cysteine and measurement of hydrogen sulphide liberation upon cysteine treatment. The tryptic fragment from peptide mapping was characterized by amino acid analysis, amino acid sequencing and mass spectrometry. All data indicated an extra sulphur atom in the Cys182-Cys189 cystine bridge. [ABSTRACT FROM AUTHOR]