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1.
Cell Mol Life Sci ; 61(12): 1455-74, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15197470

ABSTRACT

N-terminal methionine excision (NME) is the major proteolytic pathway responsible for the diversity of N-terminal amino acids in proteins. Dedicated NME components have been identified in all organisms, in all compartments in which protein synthesis occurs: cytoplasm, plastids and mitochondria. Recent studies have revealed that NME is regulated at various levels and plays an important role in controlling protein turnover. NME is essential in Eubacteria and lower eukaryotes and is the target of many natural and synthetic inhibitors. Such inhibitors have considerable potential for use in the treatment of various human diseases, from cancer to bacterial and parasitic infections.


Subject(s)
Amidohydrolases/chemistry , Methionine/chemistry , Proteins/chemistry , Aminopeptidases/chemistry , Animals , Cations , Cell Nucleus/metabolism , Cytoplasm/metabolism , Humans , Metalloproteases/metabolism , Mitochondria/metabolism , Models, Chemical , Phylogeny , Plastids/metabolism , Protein Structure, Tertiary , Proteome/chemistry
2.
Trends Plant Sci ; 6(12): 566-72, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11738381

ABSTRACT

Most mature proteins do not retain their initial N-terminal amino acid (methionine in the cytosol and N-formyl methionine in the organelles). Recent studies have shown that dedicated machinery is involved in this process in plants. In addition to cytosolic and organelle-targeted methionine aminopeptidases, organellar peptide deformylases have been identified. Here, we attempt to answer questions about the mechanism, specificity and significance of N-terminal methionine cleavage in plant organelles. It seems to be universal because orthologues of plant deformylases are found in most living organisms.


Subject(s)
Amidohydrolases , Aminopeptidases/metabolism , Methionine/metabolism , Organelles/enzymology , Plant Proteins/metabolism , Protein Processing, Post-Translational , Chloroplasts/metabolism , Cytosol/metabolism , Evolution, Molecular
3.
J Mol Biol ; 314(4): 695-708, 2001 Dec 07.
Article in English | MEDLINE | ID: mdl-11733990

ABSTRACT

Peptide deformylases (PDFs) are essential enzymes of the N-terminal protein processing pathway of eubacteria. The recent discovery of two types of PDFs in higher plants, PDF1A and PDF1B, and the detection of PDF1A in humans, have raised questions concerning the importance of deformylation in eukaryotes. Here, we have characterized fully in vitro and compared the properties of the two classes of eukaryotic PDFs, PDF1A and PDF1B, using the PDFs from Arabidopsis thaliana and Lycopersicon esculentum. We have shown that the PDFs of a given class (1A or 1B) all display similar features, independently of their origin. We also observed similar specificity of all plant PDFs for natural substrate peptides, but identified a number of biochemical differences between the two classes (1A or 1B). The main difference lies at the level of the bound cofactor, iron for PDF1B-like bacterial PDFs, and zinc for PDF1A. The nature of the metal cation has important consequences concerning the relative sensitivity to oxygen of the two plant PDFs. Investigation of the specificity of these enzymes with unusual substrates revealed additional differences between the two types of PDFs, enabling us to identify specific inhibitors with a lower affinity against PDF1As. However, the two plant PDFs were inhibited equally strongly in vitro by actinonin, an antibiotic that specifically acts on bacterial PDFs. Uptake of actinonin by A. thaliana seedlings was used to investigate the function of PDFs in the plant. Because it induces an albino phenotype, we conclude that deformylation is likely to play an essential role in the chloroplast.


Subject(s)
Amidohydrolases , Aminopeptidases/chemistry , Aminopeptidases/classification , Arabidopsis/enzymology , Eukaryotic Cells/enzymology , Solanum lycopersicum/enzymology , Aminopeptidases/antagonists & inhibitors , Aminopeptidases/isolation & purification , Arabidopsis/drug effects , Arabidopsis/metabolism , Binding Sites , Catalytic Domain , Chloroplasts/drug effects , Chloroplasts/enzymology , Chloroplasts/metabolism , Coenzymes/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Stability , Eukaryotic Cells/drug effects , Eukaryotic Cells/metabolism , Half-Life , Hydroxamic Acids/pharmacology , Iron/metabolism , Oxygen/metabolism , Plant Proteins/antagonists & inhibitors , Plant Proteins/chemistry , Plant Proteins/classification , Plant Proteins/isolation & purification , Spectrophotometry, Atomic , Substrate Specificity , Zinc/metabolism
4.
Expert Opin Ther Targets ; 5(1): 41-57, 2001 Feb.
Article in English | MEDLINE | ID: mdl-15992167

ABSTRACT

Peptide deformylases (PDFs) constitute a growing family of hydrolytic enzymes previously believed to be unique to Eubacteria. Recent data from our laboratory have demonstrated that PDF orthologues are present in many eukaryotes, including several parasites. In this report we aim to explain why PDF could be considered to be a potent target for human and veterinary antiparasitic treatments.

5.
EMBO J ; 19(21): 5916-29, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11060042

ABSTRACT

The N-terminal protein processing pathway is an essential mechanism found in all organisms. However, it is widely believed that deformylase, a key enzyme involved in this process in bacteria, does not exist in eukaryotes, thus making it a target for antibacterial agents such as actinonin. In an attempt to define this process in higher eukaryotes we have used Arabidopsis thaliana as a model organism. Two deformylase cDNAs, the first identified in any eukaryotic system, and six distinct methionine aminopeptidase cDNAs were cloned. The corresponding proteins were characterized in vivo and in vitro. Methionine aminopeptidases were found in the cytoplasm and in the organelles, while deformylases were localized in the organelles only. Our work shows that higher plants have a much more complex machinery for methionine removal than previously suspected. We were also able to identify deformylase homologues from several animals and clone the corresponding cDNA from human cells. Our data provide the first evidence that lower and higher eukaryotes, as well as bacteria, share a similar N-terminal protein processing machinery, indicating universality of this system.


Subject(s)
Amidohydrolases , Aminopeptidases/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Aminopeptidases/genetics , Animals , Arabidopsis/genetics , Arabidopsis/metabolism , Cell Compartmentation , Cloning, Molecular , DNA, Complementary/genetics , DNA, Plant/genetics , Drosophila/genetics , Drosophila/metabolism , Escherichia coli/genetics , Eukaryotic Cells , Gene Expression , Genes, Plant , Humans , Methionyl Aminopeptidases , Molecular Sequence Data , Plant Proteins/metabolism , Sequence Homology, Amino Acid
6.
Mol Microbiol ; 36(6): 1197-205, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10931273

ABSTRACT

Peptide deformylase was discovered 30 years ago, but as a result of its unusually unstable activity it was not fully characterized until very recently. The aim of this paper is to review the many recent data concerning this enzyme and to try to assess its potential as a target for future antimicrobial drugs.


Subject(s)
Amidohydrolases , Aminopeptidases/physiology , Anti-Infective Agents , Enzyme Inhibitors , Aminopeptidases/antagonists & inhibitors , Aminopeptidases/chemistry , Animals , Anti-Infective Agents/pharmacology , Enzyme Inhibitors/pharmacology , Humans
7.
Parasitol Today ; 16(4): 165-8, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10725904

ABSTRACT

Peptide deformylase is found only in Eubacteria, making it a logical target for discovering new antibacterial agents. Although this protein is absent from animal or fungal cells, evidence supports its existence in eukaryotic protists, including the causative agents of malaria, sleeping sickness, Chagas disease and leishmaniosis. Here, Thierry Meinnel discusses the idea that deformylase inhibitors could be used as very broad-spectrum antibiotics against bacterial infections, as well as parasitic diseases.


Subject(s)
Amidohydrolases , Aminopeptidases/antagonists & inhibitors , Aminopeptidases/metabolism , Antiprotozoal Agents/pharmacology , Eukaryota/drug effects , Eukaryota/enzymology , Amino Acid Sequence , Aminopeptidases/chemistry , Aminopeptidases/genetics , Animals , Antiprotozoal Agents/therapeutic use , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Humans , Molecular Sequence Data , Protozoan Infections/drug therapy
9.
J Mol Biol ; 290(4): 825-37, 1999 Jul 23.
Article in English | MEDLINE | ID: mdl-10398584

ABSTRACT

Translation initiation factor IF3, one of three factors specifically required for translation initiation in Escherichia coli, inhibits initiation on any codon other than the three canonical initiation codons, AUG, GUG, or UUG. This discrimination against initiation on non-canonical codons could be due to either direct recognition of the two last bases of the codon and their cognate bases on the anticodon or to some ability to "feel" codon-anticodon complementarity. To investigate the importance of codon-anticodon complementarity in the discriminatory role of IF3, we constructed a derivative of tRNALeuthat has all the known characteristics of an initiator tRNA except the CAU anticodon. This tRNA is efficiently formylated by methionyl-tRNAfMettransformylase and charged by leucyl-tRNA synthetase irrespective of the sequence of its anticodon. These initiator tRNALeuderivatives (called tRNALI) allow initiation at all the non-canonical codons tested, provided that the complementarity between the codon and the anticodon of the initiator tRNALeuis respected. More remarkably, the discrimination by IF3, normally observed with non-canonical codons, is neutralised if a tRNALIcarrying a complementary anticodon is used for initiation. This suggests that IF3 somehow recognises codon-anticodon complementarity, at least at the second and third position of the codon, rather than some specific bases in either the codon or the anticodon.


Subject(s)
Codon, Initiator , Escherichia coli/genetics , Peptide Chain Initiation, Translational , Peptide Initiation Factors/genetics , Anticodon , Base Sequence , Eukaryotic Initiation Factor-3 , Gene Expression Regulation, Bacterial , Genotype , Kinetics , Models, Genetic , Molecular Sequence Data , RNA, Transfer, Leu/genetics , RNA, Transfer, Met/genetics
10.
J Mol Biol ; 289(5): 1445-57, 1999 Jun 25.
Article in English | MEDLINE | ID: mdl-10373378

ABSTRACT

The substrate specificity of Escherichia coli peptide deformylase was investigated by measuring the efficiency of the enzyme to cleave formyl- peptides of the general formula Fo-Xaa-Yaa-NH2, where Xaa represents a set of 27 natural and unusual amino acids and Yaa corresponds to a set of 19 natural amino acids. Substrates with bulky hydrophobic side-chains at the P1' position were the most efficiently cleaved, with catalytic efficiencies greater by two to five orders of magnitude than those associated with polar or charged amino acid side-chains. Among hydrophobic side-chains, linear alkyl groups were preferred at the P1' position, as compared to aryl-alkyl side-chains. Interestingly, in the linear alkyl substituent series, with the exception of norleucine, deformylase exhibits a preference for the substrate containing Met in the P1' position. Next, the influence in catalysis of the second side-chain was studied after synthesis of 20 compounds of the formula Fo-Nle-Yaa-NH2. Their deformylation rates varied within a range of only one order of magnitude. A 3D model of the interaction of PDF with an inhibitor was then constructed and revealed indeed the occurrence of a deep and hydrophobic S1' pocket as well as the absence of a true S2' pocket. These analyses pointed out a set of possible interactions between deformylase and its substrates, which could be the ground driving substrate specificity. The validity of this enzyme:substrate docking was further probed with the help of a set of site-directed variants of the enzyme. From this, the importance of residues at the bottom of the S1' pocket (Ile128 and Leu125) as well as the hydrogen bond network that the main chain of the substrate makes with the enzyme were revealed. Based on the numerous homologies that deformylase displays with thermolysin and metzincins, a mechanism of enzyme:substrate recognition and hydrolysis could finally be proposed. Specific features of PDF with respect to other members of the enzymes with motif HEXXH are discussed.


Subject(s)
Amidohydrolases , Aminopeptidases/metabolism , Metalloendopeptidases/metabolism , Peptides/metabolism , Thermolysin/metabolism , Aminopeptidases/chemistry , Aminopeptidases/genetics , Binding Sites , Dipeptides/metabolism , Escherichia coli/enzymology , Mutagenesis, Site-Directed , Peptide Library , Protein Conformation , Substrate Specificity
11.
Biochemistry ; 38(14): 4287-95, 1999 Apr 06.
Article in English | MEDLINE | ID: mdl-10194346

ABSTRACT

Series of substrates derivatives of peptide deformylase were systematically synthesized and studied for their capacities to undergo hydrolysis. Data analysis indicated the requirement for a hydrophobic first side chain and for at least two main chain carbonyl groups in the substrate. For instance, Fo-Met-OCH3 and Fo-Nle-OCH3 were the minimal substrates of peptide deformylase obtained in this study, while positively charged Fo-Nle-ArgNH2 was the most efficient substrate (kcat/Km = 4.5 x 10(5) M-1.s-1). On the basis of this knowledge, 3-mercapto-2-benzylpropanoylglycine (thiorphan), a known inhibitor of thermolysin, could be predicted and further shown to inhibit the deformylation reaction. The inhibition by this compound was competitive and proved to depend on the hydrophobicity at the P1' position. Spectroscopic evidence that the sulfur group of thiorphan binds next to the active site metal ion on the enzyme could be obtained. Consequently, a small thiopseudopeptide derived from Fo-Nle-OCH3 was designed and synthesized. This compound behaved as a competitive inhibitor of peptide deformylase with KI = 52 +/- 5 microM. Introduction of a positive charge to this thiopeptide via addition of an arginine at P2' improved the inhibition constant up to 2.5 +/- 0.5 microM, a value 4 orders of magnitude smaller than that of the starting inhibitors. Evidence that this inhibitor, imino[(5-methoxy-5-oxo-4-[[2-(sulfanylmethyl)hexanoyl]amino]pentyl )am ino]methanamine, binds inside the active site cavity of peptide deformylase, while keeping intact the 3D fold of the protein, was provided by NMR. A fingerprint of the interaction of the inhibitor with the residues of the enzyme was obtained.


Subject(s)
Amidohydrolases , Aminopeptidases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Oligopeptides/chemical synthesis , Aminopeptidases/metabolism , Binding, Competitive , Captopril/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Hydrolysis , Metalloendopeptidases/chemistry , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Oligopeptides/chemistry , Oligopeptides/metabolism , Substrate Specificity , Thermolysin/chemistry , Zinc/chemistry
12.
J Mol Biol ; 280(3): 515-23, 1998 Jul 17.
Article in English | MEDLINE | ID: mdl-9665853

ABSTRACT

Previous work indicated that peptide deformylase behaves as a metalloenzyme since the Escherichia coli enzyme was shown to copurify with a zinc ion. The present study establishes that nickel:enzyme complexes can also be isolated provided that nickel salts were added in the buffers throughout the purification. Similar results were obtained with the deformylases from Thermus thermophilus and Bacillus stearothermophilus. As a result of nickel binding, the catalytic efficiencies of peptide deformylases increased by two to three orders of magnitude with respect to those of the forms previously characterized. Using the model substrate N-formyl-Met-Ala-Ser, kcat/Km values of 5.4, 1.2 and 25 10(4)M-1s-1 could be obtained for the E. coli, T. thermophilus and B. stearothermophilus enzymes, respectively. This value satisfyingly accounts for the deformylation turnover required in the cell. In vitro characterization of the E. coli enzyme shows that zinc can readily substitute for the bound nickel with the catalytic efficiency decreasing to 80 M-1s-1 in turn. Conversely, the activity of the zinc-containing protein can be significantly improved by addition of nickel to the enzymatic assay.


Subject(s)
Amidohydrolases , Aminopeptidases/metabolism , Bacterial Proteins/metabolism , Cations/metabolism , Nickel/metabolism , Aminopeptidases/isolation & purification , Enzyme Activation , Escherichia coli/enzymology , Manganese/metabolism , Thermus thermophilus/enzymology , Zinc/metabolism
13.
J Mol Biol ; 280(3): 501-13, 1998 Jul 17.
Article in English | MEDLINE | ID: mdl-9665852

ABSTRACT

In the accompanying paper, we report that zinc is unlikely to be the co-factor supporting peptide deformylase activity in vivo. In contrast, nickel binding promotes full enzyme activity. The three-dimensional structure of the resulting nickel-containing peptide deformylase (catalytic domain, residues 1 to 147) was solved by NMR using a 13C-15N-doubly labelled protein sample. A set of 2261 restraints could be collected, with an average of 15.4 per amino acid. The resolution, which shows a good definition for the position of most side-chains, is greatly improved compared to that previously reported for the zinc-containing, inactive form. A comparison of the two stuctures indicates however that both share the same 3D organization. This shows that the nature of the bound metal is the primary determinant of the hydrolytic activity of this enzyme. Site-directed mutagenesis enabled us to determine the conserved residues of PDF involved in the structure of the active site. In particular, a buried arginine appears to be critical for the positioning of Cys90, one of the metal ligands. Furthermore, the 3D structure of peptide deformylase was compared to thermolysin and metzincins. Although the structural folds are very different, they all display a common structural motif involving an alpha-helix and a three-stranded beta-sheet. These conserved structural elements build a common scaffold which includes the active site, suggesting a common hydrolytic mechanism for these proteases. Finally, an invariant glycine shared by both PDF and metzincins enables us to extend the conserved motif from HEXXH to HEXXHXXG.


Subject(s)
Amidohydrolases , Aminopeptidases/chemistry , Nickel/chemistry , Amino Acid Sequence , Aminopeptidases/metabolism , Binding Sites , Magnetic Resonance Spectroscopy , Metalloendopeptidases/chemistry , Models, Molecular , Molecular Sequence Data , Nickel/metabolism , Protein Conformation , Sequence Alignment , Sequence Homology, Amino Acid , Thermolysin/chemistry , Zinc/chemistry
14.
Biochemistry ; 37(17): 6041-9, 1998 Apr 28.
Article in English | MEDLINE | ID: mdl-9558342

ABSTRACT

In this study, a protocol for the purification of fully active Escherichia coli RNase P holoenzyme from a strain overproducing both the C5 protein and the M1 RNA components is described. A total of 0. 8 mg of homogeneous enzyme, with a 1:1 protein/RNA subunit stoichiometry, was recovered from a 1 L bacterial culture. In addition, a convenient and reliable method based on capillary gel electrophoresis was developed to measure initial rates of pre-tRNA maturation by RNase P. Using these tools, the kinetic parameters of cleavage by RNase P of various mutants of pre-tRNAfMet showing maturation defects in vivo [Meinnel and Blanquet (1995) J. Biol. Chem. 270, 15906-15914] were investigated in vitro and the locations of cleavage sites were determined from the length of the various products of the reaction. The nucleotide at position -2 of pre-tRNAfMet is shown to be important only in the selection of the cleavage site, whereas it has no role in the efficiency of the reaction. It is concluded that base G-2 acts as an antideterminant by preventing an alternative cleavage by RNase P. In addition, the presence of G-2 alone is enough to fully compensate for the lack of a G at position +1 of pre-tRNAfMet.


Subject(s)
Endoribonucleases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Guanine/physiology , RNA Precursors/physiology , RNA, Catalytic/metabolism , RNA, Transfer, Met/physiology , Base Composition , Base Sequence , Binding Sites , Catalysis , Coenzymes/chemistry , Coenzymes/isolation & purification , Coenzymes/metabolism , Electrophoresis, Capillary , Endoribonucleases/chemistry , Endoribonucleases/isolation & purification , Escherichia coli/genetics , Hydrolysis , Molecular Sequence Data , RNA Precursors/genetics , RNA Processing, Post-Transcriptional , RNA, Catalytic/chemistry , RNA, Catalytic/isolation & purification , RNA, Transfer, Met/genetics , Ribonuclease P
15.
J Mol Biol ; 267(3): 749-61, 1997 Apr 04.
Article in English | MEDLINE | ID: mdl-9126850

ABSTRACT

Thermus thermophilus peptide deformylase was characterized. Its enzymatic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite few sequence identities. In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly conserved amino acids was noticed, EGCLS, the cysteine of which corresponds to the third zinc ligand. The study of site-directed mutants of the E. coli deformylase shows that the residues of this stretch are crucial for the structure and/or catalytic efficiency of the active enzyme. Both aforementioned sequences were used as markers of the peptide deformylase family in protein sequence databases. Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearothermophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtilus and Synechocystis sp. could be identified. The characterization of the product of the open reading frame from B. stearothermophilus confirmed that it actually corresponded to a peptide deformylase with properties similar to those of the E. coli enzyme. Alignment of the nine peptide deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved. This motif is also located in the active site according to the three-dimensional structure of the E. coli enzyme. Site-directed variants of E. coli peptide deformylase showed the involvement of the corresponding residues for maintaining an active and stable enzyme. Altogether, these data allow us to propose that the three identified conserved motifs of peptide deformylases build up the active site around a metal ion. Finally, an analysis of the location of the other conserved residues, in particular of the hydrophobic ones, was performed using the three-dimensional model of the E. coli enzyme. This enables us to suggest that all bacterial peptide deformylases adopt a constant overall tertiary structure.


Subject(s)
Amidohydrolases , Aminopeptidases/chemistry , Conserved Sequence/genetics , Metalloendopeptidases/chemistry , Zinc/chemistry , Amino Acid Sequence , Aminopeptidases/genetics , Binding Sites , Escherichia coli/enzymology , Geobacillus stearothermophilus/enzymology , Metalloendopeptidases/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Thermus thermophilus/enzymology
17.
J Mol Biol ; 262(3): 375-86, 1996 Sep 27.
Article in English | MEDLINE | ID: mdl-8845003

ABSTRACT

Escherichia coli peptide deformylase, a member of the zinc metalloproteases family, is made up of an active core domain composed of 147 residues and of an additional and dispensable C-terminal tail of 21 residues. The three-dimensional structure of the catalytic core could be studied by NMR. 1H and 15N NMR resonances assignments were obtained by two-dimensional and three-dimensional heteronuclear spectroscopy. The structure could be calculated using a set of 1015 restraints for the 147 residues of the enzyme. The overall structure is composed of a series of antiparallel beta-strands which surround two perpendicular alpha-helices. The C-terminal helix contains the HEXXH motif, which is crucial for activity. This helical arrangement and the way the histidines bind the zinc ion clearly are structurally reminiscent of the other members of the metalloprotease family, such as thermolysin or metzincins. Nevertheless, the overall arrangement of secondary and tertiary structures of peptide deformylase and the positioning of its third zinc ligand (a cysteine) are quite different from those of the other members of the family. These discrepancies, together with several biochemical differences, lead us to propose that peptide deformylase is the first example of a new class of the zinc-metalloproteases family. Studies of the interaction of peptide deformylase with either an inhibitor of the reaction or a product of the catalysed reaction, Met-Ala-Ser, as well as comparisons with the structures of other enzymes of the family, have enabled us to delineate the area corresponding to their binding site. The structural basis of the specificity of recognition of the formyl group is discussed in the context of the protease superfamily.


Subject(s)
Amidohydrolases , Aminopeptidases/chemistry , Metalloendopeptidases/classification , Zinc/metabolism , Amino Acid Sequence , Aminopeptidases/antagonists & inhibitors , Aminopeptidases/metabolism , Binding Sites , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Magnetic Resonance Spectroscopy , Metalloendopeptidases/chemistry , Models, Molecular , Molecular Sequence Data , N-Formylmethionine/chemistry , N-Formylmethionine/metabolism , Peptides/metabolism , Protein Conformation , Substrate Specificity
18.
FEBS Lett ; 385(1-2): 91-5, 1996 Apr 29.
Article in English | MEDLINE | ID: mdl-8641475

ABSTRACT

Upon trypsinolysis, the 18 C-terminal residues of Escherichia coli peptide deformylase were removed but the resulting form exhibited full activity. Moreover, a mutant fms gene encoding the first 145 out of the 168 residues of the enzyme was able to complement a fms(Ts) strain and exhibited full activity. Upon progressive truncation up to residue 139, both activity and stability decreased up to complete inactivation. Mutagenesis of residues of the 138-145 region highlights the importance of Leu-141 and Phe-142. N-Terminal deletions were also carried out. Beyond two residues off, the enzyme showed a dramatic instability. Finally, NMR and thermostability studies of the full-length enzyme and comparison to the 1-147 form strongly suggest that the dispensable residues are disordered in solution.


Subject(s)
Amidohydrolases , Aminopeptidases/chemistry , Aminopeptidases/genetics , Aminopeptidases/isolation & purification , Aminopeptidases/metabolism , Binding Sites , Enzyme Stability , Escherichia coli/enzymology , Genetic Complementation Test , Hot Temperature , Molecular Weight , Mutagenesis, Site-Directed , Peptide Fragments , Sequence Deletion , Structure-Activity Relationship , Trypsin , Zinc/analysis
19.
Nucleic Acids Res ; 24(5): 907-13, 1996 Mar 01.
Article in English | MEDLINE | ID: mdl-8600459

ABSTRACT

Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.


Subject(s)
RNA, Transfer, Met/isolation & purification , RNA, Transfer, Phe/isolation & purification , Chromatography, High Pressure Liquid , Cloning, Molecular , Deuterium , Escherichia coli/genetics , Escherichia coli/metabolism , RNA, Transfer, Met/biosynthesis , RNA, Transfer, Met/genetics , RNA, Transfer, Phe/biosynthesis , RNA, Transfer, Phe/genetics
20.
Biochimie ; 78(7): 543-54, 1996.
Article in English | MEDLINE | ID: mdl-8955898

ABSTRACT

Selection of the proper start codon for the synthesis of a polypeptide by the Escherichia coli translation initiation apparatus involves several macromolecular components. These macromolecules interact in a specific and concerted manner to yield the translation initiation complex. This review focuses on recent data concerning the properties of the initiator tRNA and of enzymes and factors involved in the translation initiation process. The three initiation factors, as well as methionyl-tRNA synthetase and methionyl-tRNA(f)Met formyltransferase are described. In addition, the tRNA recognition properties of EF-Tu and peptidyl-tRNA hydrolase are considered. Finally, peptide deformylase and methionine aminopeptidase, which catalyze the amino terminal maturation of nascent polypeptides, can also be associated to the translation initiation process.


Subject(s)
Amidohydrolases , Escherichia coli/genetics , Protein Biosynthesis , Aminopeptidases/metabolism , Anticodon , Bacterial Proteins/metabolism , Base Sequence , Carboxylic Ester Hydrolases/metabolism , Methionine-tRNA Ligase/metabolism , Methionyl Aminopeptidases , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Elongation Factor Tu/metabolism , Peptide Initiation Factors/metabolism , Prokaryotic Initiation Factor-2 , Prokaryotic Initiation Factor-3 , RNA, Messenger/metabolism , RNA, Transfer, Met/metabolism , Ribosomes/metabolism , Software
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