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F Bossa

Publications and source records attributed to F Bossa.

At least 37 records · Page 2Linked to original sources
PubMed1998

The structure of serine hydroxymethyltransferase as modeled by homology and validated by site-directed mutagenesis.

We describe a model for the three-dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of the alpha-family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of site-directed mutants. New active site features are also proposed for further experimental testing.

Amino Acid Sequence↗
PubMed1998

Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase.

A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked by 12 bp at the 5' end and 287 bp at the 3' end. The full-length cDNA was then cloned into a pET22b vector as a NdeI-EcoRI insert. HMS174(DE3) cells were transformed with this plasmid and, after induction with isopropyl beta-D-thiogalactopyranoside, expressed a catalytically active serine hydroxymethyltransferase. The enzyme was purified and shown to be the expressed rabbit enzyme lacking the first methionine residue. Spectral characteristics of the bound pyridoxal phosphate and kinetic constants for the natural substrates L-serine and tetrahydrofolate were essentially identical to the values obtained previously for the rabbit cytosolic enzyme. The pattern of bands shown by the pure recombinant enzyme on an isoelectric focusing gel containing 6 M urea showed a major band and a minor band representing about 15-20% of the protein. Upon incubation of the recombinant enzyme at pH 7.3 and 37 degreesC, three new bands were observed on isoelectric focusing with the concomitant formation of isoaspartyl residues, as determined by reactivity with protein isoaspartyl methyltransferase. These results are consistent with deamidation of Asn residues to isoaspartyl during the in vitro incubation. The enzyme purified from rabbit liver has previously been shown to contain isoaspartyl residues.

Animals↗
PubMed1998

Characterization and analysis of D-amino acids.

The need to screen a large number of natural extracts, with the aim of detecting D-amino acids or isolating and characterizing peptides containing them, has stimulated the development of novel and improved procedures for the analysis of amino acid enantiomeric mixtures, with special attention paid to automation. Different methods for the analysis of D-amino acids are described and discussed.

Amino Acids↗
PubMed1997

Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis.

The cytosolic and mitochondrial alcohol dehydrogenases from Kluyveromyces lactis (KlADHs) were purified and characterised. Both the N-terminally blocked cytosolic isozymes, KlADH I and KlADH II, were strictly NAD-dependent and exhibited catalytic properties similar to those previously reported for other yeast ADHs. Conversely, the mitochondrial isozymes, KlADH III and KlADH IV, displayed Ala and Asn, respectively, as N-termini and were able to oxidise at an increased rate primary alcohols with aliphatic chains longer than ethanol, such as propanol, butanol, pentanol and hexanol. Interestingly, the mitochondrial KlADHs, at variance with cytosolic isozymes and the majority of ADHs from other sources, were capable of accepting as a cofactor, and in some case almost equally well, either NAD or NADP. Since Asp-223 of horse liver ADH, thought to be responsible for the selection of NAD as coenzyme, is strictly conserved in all the KlADH isozymes, this amino-acid residue should not be considered critical for the coenzyme discrimination with respect to the other residues lining the coenzyme binding pocket of the mitochondrial isozymes. The relatively low specificity of the mitochondrial KlADHs both toward the alcohols and the cofactor could be explained on the basis of an enhanced flexibility of the corresponding catalytic pockets. An involvement of the mitochondrial KlADH isozymes in the physiological reoxidation of the cytosolic NADPH was also hypothesized. Moreover, both cytosolic and KlADH IV isozymes have an additional cysteine, not involved in zinc binding, that could be responsible for the increased activity in the presence of 2-mercaptoethanol.

Alcohol Dehydrogenase↗
PubMed1997

Stability of aspartate aminotransferase from Sulfolobus solfataricus.

Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is an extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C. The structural stability of this protein was investigated by coupling isothermally and thermally induced denaturation studies to molecular modeling. Gel filtration analysis indicated that SsAspAT unfolds with an N2 reversible 2D mechanism. In the molecular model, a cluster of hydrophobic residues was shown at the interface between the subunits of SsAspAT and suggested this cluster as a structural feature stabilizing the enzyme quaternary structure. At 25 degrees C, SsAspAT is less resistant to guanidinium chloride-induced denaturation than the cytosolic aspartate aminotransferase from pig heart (cpAspAT), which was chosen as a mesophilic counterpart in the thermodynamic analysis since it shares with SsAspAT the two-state unfolding mechanism. Therefore, in the case of aspartate aminotransferases, thermal stability does not correlate with the stability against chemical denaturants. Isothermal denaturation curves at 25 degrees C and melting profiles recorded in the presence of guanidinium chloride showed that the delta G degrees (H2O) at 25 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; the parameter delta n, related to the number of binding sites for the denaturant differentially exposed in unfolded and folded states, is higher for SsAspAT than for cpAspAT; and delta Cp is lower for the thermophilic enzyme than for the mesophilic one by 8 kJ/K.mol. These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT. In agreement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network.

Amino Acid Sequence↗
PubMed1997

Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.

Archaeal Proteins↗
PubMed1996

Amino acid sequence of chicken Cu, Zn-containing superoxide dismutase and identification of glutathionyl adducts at exposed cysteine residues.

The copper, zinc-containing superoxide dismutase electromorphs from chicken erythrocytes have been isolated, their complete amino acid sequence determined and the identity of the protein moieties established. All electromorphs are constituted by a polypeptide chain made of 153 amino acid residues, corresponding to a molecular mass of 15,598 Da. Accurate molecular mass determination by electrospray mass spectrometry of the separated electromorphs unequivocally proved that, in the chicken superoxide dismutase, either one or two cysteine residues/subunit are involved in a mixed disulfide with glutathione. The same post-translational modification has been proven to occur in human superoxide dismutase. A different rate of S-thiolation by endogenous glutathione was also demonstrated to be responsible for charge heterogeneity in cells. Effect of this modification on the catalytic and molecular properties of superoxide dismutases, and possible mechanisms for the S-thiolation process, were also investigated and discussed.

Amino Acid Sequence↗
PubMed1996

Structure of fuscopeptins, phytotoxic metabolites of Pseudomonas fuscovaginae.

The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined. The combined use of FAB mass spectroscopy NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to Z-Dhb-D-Pro-L-Leu-D-Ala-D-Ala-D-Ala-D-Ala-D-Val-Gly-D-Ala-D-Val-D-Ala-D- Val-Z-Dhb-Da-Thr-L-Ala-L-Dab-D-Dab-L-Phe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue. The N-terminus is in turn acylated by 3-hydroxyoctanoate in fuscopeptin A and 3-hydroxydecanoate in fuscopeptin B. Some preliminary data on the biological activity of fuscopeptins are also reported.

Amino Acid Sequence↗
PubMed1996

Site-directed mutagenesis techniques in the study of Escherichia coli serine hydroxymethyltransferase.

The 3340-bp fragment containing the Escherichia coli glyA gene coding for serine hydroxymethyltransferase was reduced in size by PCR, and the 1600-bp fragment obtained was cloned into the vector pBR322 in both orientations (5'-3', and 3'-5'). This DNA manipulation allowed us to perform site-directed mutagenesis by PCR on the glyA gene. To overcome the problem of the presence of wild-type protein in the various mutant enzyme preparations, the E. coli strain GS245 used to express recombinant serine hydroxymethyltransferase was made recA deficient through generalized transduction mediated by phage P1. The new strain was used for the production of a mutant form of the enzyme, in which the pyridoxal 5'-phosphate binding lysine was substituted by a glutamine. The preparation of this mutant form was completely devoid of wild-type enzyme contamination and measurements of its catalytic activity in the transamination reactions of L- and D-alanine confirmed the suggestion that the active site lysine is not the base that removes the alpha-proton from the substrate.

Alanine↗
PubMed1996

Isolation, overexpression, and biochemical characterization of the two isoforms of glutamic acid decarboxylase from Escherichia coli.

Escherichia coli glutamic acid decarboxylase is a pyridoxal phosphate-dependent enzyme that catalyzes the alpha-decarboxylation of glutamate to yield 4-amino-butyrate and CO2. The E. coli chromosome contains two genes encoding for this enzyme, gadA and gadB, which map at distinct loci. Their protein products differ in only five amino acid residues, four of which are located in the N-terminal region (Smith et al., 1992, J. Bacteriol. 174, 5820-5826). Herein, we report the sequences of the two gad genes, including their regulatory regions. Both genes were separately cloned into the vector pQE60, for overexpression under the control of the lac promoter. In this way, we have succeded in separately expression large quantities of each pure isoform. The two isoforms were characterized biochemically and all evidence, including that from analysis of the complex pre-steady-state kinetic behavior of the enzymes, indicates that the functional properties of the two isoenzymes are identical.

Base Sequence↗
PubMed1995

Solution conformation of the Pseudomonas syringae pv. syringae phytotoxic lipodepsipeptide syringopeptin 25-A. Two-dimensional NMR, distance geometry and molecular dynamics.

Syringopeptin 25-A is a phytotoxic amphiphilic lipodepsipeptide containing 25 amino acid residues, produced by some isolates of the plant pathogenic bacterium Pseudomonas syringae pv. syringae. Previous papers have reported its covalent structure and some of its biological properties. Attention has now been directed to define its conformation in solution, a structural feature regarded as important for understanding its possible role in the bacterial colonization of host plants, and its toxic action on the plant cell. Here we report the stereochemistry of its amino acid components, the complete interpretation of the two-dimensional NMR spectra and NOE data, and finally the structure obtained by computer simulations applying distance geometry and molecular dynamics procedures. The conformation of syringopeptin 25-A in aqueous solution includes three different structural regions interrupted by rigid 2,3-dehydro-2-aminobutyric acid residues: a loop from residue 2 to 6, a helicoidal zone from 8 to 15, and the lactone ring from 18 to 25. The three-dimensional structure of the lactone moiety is very similar to that of two previously studied bioactive lipodepsinonapeptides. Preliminary circular dichroism evidence of conformational variations in solution of trifluoroethanol, which stimulates a membrane-like environment, are also reported.

Amino Acid Sequence↗
PubMed1995

Primary structure and tissue distribution of human 4-aminobutyrate aminotransferase.

cDNA encoding human 4-aminobutyrate aminotransferase (aminobutyrate:2-oxoglutarate aminotransferase) was prepared by polymerase chain reaction using mRNA from human neuroblastoma cells as the template and oligonucleotides synthesized on the basis of the information obtained from direct protein sequencing. The cDNA-deduced sequence enabled peptides, sequenced by automated Edman degradation, to be aligned for confirmation of the complete primary structure. The results are compared with the recently published sequence of the rat enzyme deduced entirely from DNA sequencing [Medina-Kauwe, L. K., Tillakaratne, N. J. K., Wu, J.-Y. & Tobin, A. J. (1994) J. Neurochem. 62, 1267-1275]. Although the sequences are almost identical for most of their length, they differ in a segment of 36 residues. Almost complete identity of the two sequences is established if it is assumed that a frame-shift error was introduced into the reported rat cDNA sequence. The human cDNA was used to probe for the presence of 4-aminobutyrate aminotransferase mRNA in human tissues and a significant transcript was found in heart, placenta and in tissues usually associated with the expression of this enzyme.

4-Aminobutyrate Transaminase↗
PubMed1994

Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins.

The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon-generated mutant of a Pseudomonas syringae wild-type strain proposed for the biological control of Dutch elm disease, have been determined. While two pseudomycins are identical to the known syringopeptins 25-A and 25-B, pseudomycins A, B, C, C' are new lipodepsinonapeptides. For all of these the peptide moiety corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH) -L-Thr (4-Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser. This is in turn N-acylated by 3,4-dihydroxytetradecanoate in pseudomycin A, by 3-hydroxytetradecanoate in pseudomycin B, by 3,4-dihydroxyhexadecanoate in pseudomycin C, and by 3-hydroxyhexadecanoate in pseudomycin C'. Some preliminary data on the biological activity of pseudomycin A are reported.

Amino Acid Sequence↗
PubMed1994

The function of arginine 363 as the substrate carboxyl-binding site in Escherichia coli serine hydroxymethyltransferase.

Both the highly conserved Arg363 and Arg372 residues of Escherichia coli serine hydroxymethyltransferase were changed to alanine and lysine residues. Each of the four mutant proteins were purified to homogeneity and characterized with respect to spectral properties of the enzyme-bound pyridoxal phosphate and kinetic properties with substrates and substrate analogs. The R372A and R372 K mutant enzymes exhibited spectra and kinetic properties close to those of the wild-type enzyme. The R363 K mutant enzyme exhibited only 0.03% of the catalytic activity of the wild-type enzyme and a 15-fold reduction in affinity for glycine and serine. The R363A mutant enzyme did not bind serine and glycine and showed no activity with serine as the substrate. Both R363 K and R363A enzymes bound amino acid esters at the active site and catalyzed the retro-aldol cleavage of serine ethyl ester and serinamide. The catalytic activity of the R363 K and R363A enzymes with the serine ethyl ester were about 0.006% and 0.1% of wild-type enzyme activity with serine, respectively. The R363A mutant enzyme catalyzed the half transamination of D-alanine methyl ester and L-alanine methyl ester at rates similar to the rates of transamination of D-alanine and L-alanine by the wild-type enzyme. The results are interpreted to show that R363 is the binding site of the amino acid substrate carboxyl group and that forming an ion pair between R363 and the substrate carboxyl group is an important feature in catalysis by serine hydroxymethyltransferase. Evidence is also provided that R363 may play a role in the substrate-induced open to closed conformational change of the active site.

Amino Acid Sequence↗
PubMed1994

Antimicrobial peptides from skin secretions of Rana esculenta. Molecular cloning of cDNAs encoding esculentin and brevinins and isolation of new active peptides.

Three cytolytic peptides, termed brevinin-1E, brevinin-2E, and esculentin, were isolated from skin secretions of the European frog Rana esculenta (Simmaco, M., Mignogna, G., Barra, D., and Bossa, F. (1993) FEBS Lett. 324, 159-161). Nucleotide sequence analysis of cDNAs coding for the corresponding precursors revealed that in all of them a single copy of the sequence of the mature peptide is present preceded by a dibasic cleavage site and followed by a stop codon. The signal peptides of these precursors show a clear homology to the corresponding region of the precursor of dermorphin, a neuropeptide occurring in the skin of amphibians of the subfamily Phyllomedusinae. Ten new peptides, ranging in size from 24 to 46 residues, all possessing an intramolecular disulfide bridge located at the carboxyl-terminal end, were isolated from skin secretions of R. esculenta. These peptides can be grouped into four subfamilies on the basis of their distinctive structural and/or functional properties. All of these new peptides have antimicrobial and/or hemolytic activities typical for the respective subfamily. In addition, we demonstrate that esculentin-1 also inhibits the growth of Pseudomonas aeruginosa, Candida albicans, and Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1994

Similarity between pyridoxal/pyridoxamine phosphate-dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes.

A multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate-dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate-dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.

Amino Acid Sequence↗
PubMed1994

The amino acid sequence of glutamate dehydrogenase from Pyrococcus furiosus, a hyperthermophilic archaebacterium.

The complete amino acid sequence of glutamate dehydrogenase from the archaebacterium Pyrococcus furiosus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage with cyanogen bromide, Asp-N endoproteinase, trypsin, or pepsin. The enzyme subunit is composed of 420 amino acid residues yielding a molecular mass of 47,122 D. In the recently determined primary structure of glutamate dehydrogenase from another thermophilic archaebacterium, Sulfolobus solfataricus, the presence of some methylated lysines was detected and the possible role of this posttranslational modification in enhancing the thermostability of the enzyme was discussed (Maras, B., Consalvi, V., Chiaraluce, R., Politi, L., De Rosa, M., Bossa, F., Scandurra, R., and Barra, D. (1992), Eur. J. Biochem. 203, 81-87). In the primary structure reported here, such posttranslational modification has not been found, indicating that the role of lysine methylation should be revisited. Comparison of the sequence of glutamate dehydrogenase from Pyrococcus furiosus with that of S. solfataricus shows a 43.7% similarity, thus indicating a common evolutionary pathway.

Amino Acid Sequence↗