Crystal structure of carboxypeptidase T from <i>Thermoactinomyces vulgaris</i>

Teplyakov, A.; Polyakov, K. M.; Obmolova, G.; Strokopytov, B.V.; Куранова, И. П.; Osterman, Andrei L.; Grishin, N.V.; Smulevitch, Sergei; Zagnitko, Olga; Galperina, Olga; Matz, Michael E.; Stepanov, V. M.

doi:10.1111/j.1432-1033.1992.tb17184.x

Cited by 74 publications

(56 citation statements)

References 31 publications

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“…It occurs in quite unrelated proteins such as the J-Atracotoxin from Hadronyche versuta (26), methanol dehydrogenase from Methylobacterium extorquens (27)(28)(29), the ethanol dehydrogenase from Pseudomonas aeruginosa (30), acetylcholine-binding protein from Lymnaea stagnalis (31), thioesterase I from Bos taurus (32), carboxypeptidase T from Thermoacinomyces vulgaris (33), and antimicrobial peptide hepcidin-25 from Homo sapiens (34) (Fig. 4 A-G).…”

Section: Resultsmentioning

confidence: 99%

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Cemazar

Zahariev

Lopez

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The oxidative folding of the Amaranthus ␣-amylase inhibitor, a 32-residue cystine-knot protein with three disulfide bridges, was studied in vitro in terms of the disulfide content of the intermediate species. A nonnative vicinal disulfide bridge between cysteine residues 17 and 18 was found in three of five fully oxidized intermediates. One of these, the most abundant folding intermediate (MFI), was further analyzed by 1 H NMR spectroscopy and photochemically induced dynamic nuclear polarization, which revealed that it has a compact structure comprising slowly interconverting conformations in which some of the amino acid side chains are ordered. NMR pulsed-field gradient diffusion experiments confirmed that its hydrodynamic radius is indistinguishable from that of the native protein. Molecular modeling suggested that the eight-membered ring of the vicinal disulfide bridge in MFI may be located in a loop region very similar to those found in experimentally determined 3D structures of other proteins. We suggest that the structural constraints imposed on the folding intermediates by the nonnative disulfides, including the vicinal bridge, may play a role in directing the folding process by creating a compact fold and bringing the cysteine residues into close proximity, thus facilitating reshuffling to native disulfide bridges.T he cystine knot, or the knottin fold, is a compact structure consisting of a short triple-stranded antiparallel ␤-sheet reinforced by three disulfide bridges that form a topological knot [see Amaranthus ␣-amylase inhibitor (AAI) structure, Fig. 1A Inset]. It is found both in small peptides and as a domain of larger proteins (1) and in addition, it is a recurrent substructure of larger cysteine-rich motifs such as the well known conotoxin fold (2). Despite their small size, cystine knots can fulfill a large variety of functions ranging from ion-channel blocking to enzyme inhibition, which makes them ideal protein engineering scaffolds for industrial applications (3, 4). Because cystine knots occur in many unrelated species (fungi, plants, snails, and spiders, etc.), it is presumed that this fold has emerged via convergent evolution (2). The compactness and versatility of this fold are best illustrated by the fact that both the shortest peptide inhibitor of ␣-amylases, AAI [32 amino acids (Fig. 1 A)] (5-8) and the shortest lectin molecule known so far (9), are members of this family.Cystine knot peptides are known to form readily in vitro under the conditions of oxidative folding (4). In this work, we describe an analysis of the oxidative folding intermediates of AAI, which shows that the native protein (N) forms via several fully oxidized intermediates with nonnative disulfide bonds, including the vicinal disulfide 17-18. NMR spectroscopy revealed that the main folding intermediate has a structure as compact as the completely folded peptide, comprising a number of slowly interconverting backbone conformations. On the basis of the experimental data, we suggest that the compactness, a prerequisi...

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Section: Resultsmentioning

confidence: 99%

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Cemazar

Zahariev

Lopez

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…Nevertheless, given the numerous trypsin cleavage sites in the molecule, the absence of fragments including the potential Cys 32 is not surprising since such a disulfide bond is known to be energetically unfavorable, although such a bond formation has been reported for a protein, the carboxypeptidase T (PDB code: 1obr) (39), and recently for the hepcidin peptide (PDB code: 1m4e.pdb and 1m4f.pdb), an iron-regulatory hormone which also displays antimicrobial activity (40). In contrast, the mass of two fragments from Pen-3a corresponding to a three fragment-peptide cross-linked by the three disulfide bonds was measured.…”

Section: Discussionmentioning

confidence: 99%

Solution Structure of the Recombinant Penaeidin-3, a Shrimp Antimicrobial Peptide

Yang

Poncet

Garnier

et al. 2003

Journal of Biological Chemistry

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Penaeidins are a family of antimicrobial peptides of 47-63 residues isolated from several species of shrimp. These peptides display a proline-rich domain (N-terminal part) and a cysteine-rich domain (C-terminal part) stabilized by three conserved disulfide bonds whose arrangement has not yet been characterized. The recombinant penaeidin-3a of Litopenaeus vannamei (63 residues) and its [T8A]-Pen-3a analogue were produced in Saccharomyces cerevisiae and showed similar antimicrobial activity. The solution structure of the [T8A]-Pen3a analogue was determined by using two-dimensional 1 H NMR and simulated annealing calculations. The proline-rich domain, spanning residues 1-28 was found to be unconstrained. In contrast, the cysteine-rich domain, spanning residues 29 -58, displays a well defined structure, which consists of an amphipathic helix (41-50) linked to the upstream and the downstream coils by two disulfide bonds (Cys 32 -Cys 47 and Cys 48 -Cys 55 ). These two coils are in turn linked together by the third disulfide bond (Cys 36 -Cys 54 ). Such a disulfide bond packing, which is in agreement with the analysis of trypsin digests by ESI-MS, contributes to the highly hydrophobic core. Side chains of Arg 45 and Arg 50 , which belong to the helix, and side chains of Arg 37 and Arg 53 , which belong to the upstream and the downstream coils, are located in two opposite parts of this globular and compact structure. The environment of these positively charged residues, either by hydrophobic clusters at the surface of the cysteine-rich domain or by sequential hydrophobic residues in the unconstrained proline-rich domain, gives rise to the amphipathic character required for antimicrobial peptides. We hypothesize that the antimicrobial activity of penaeidins can be explained by a cooperative effect between the proline-rich and cysteine-rich features simultaneously present in their sequences.Antimicrobial peptides are major elements of host-defense systems represented in all species from plants to vertebrate and invertebrate animals. Among these antimicrobial molecules, cysteine-rich peptides are the most widespread. They are structurally classified into (i) peptides with a ␤-sheet structure such as the mammalian defensins (1), (ii) peptides with a ␤-hairpin-like fold such as tachyplesins from horseshoe crabs (2), thanatin (3), porcine protegrins (4, 5), androctonin (6), or gomesin (7), and (iii) peptides adopting the cystine-stabilized ␣␤-motif, including invertebrate and plant defensins (8Ϫ10).Only recently, such effectors of innate immunity were isolated from crustaceans, whereas numerous peptides have been characterized from other arthropods, both insects and chelicerates. Three peptides, named penaeidins (Pen), 1 were initially purified in their active form (5.48Ϫ6.62 kDa) from the hemocytes of the shrimp Litopenaeus (Penaeus) vannamei, and they were fully characterized at the amino acid level (Pen-1, Pen-2, Pen3a) and by cDNA cloning from a hemocyte library (pen-2, -3a, -3b, and-3c) (11). Penaeidins are an original p...

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“…A thermostable CP is useful for high-temperature analysis of the C-terminal amino acid sequences of proteins. Recently, several thermostable CPs from the thermophilic bacteria Thermoactinomyces vulgaris (35,36) and Thermus aquaticus (27,28,29) and the thermophilic archaea Sulfolobus solfataricus (12,13,38) and Pyrococcus furiosus (8) have been characterized. Using genome sequencing for P. horikoshii (20, 21), we found two kinds of genes encoding CP-homologous proteins.…”

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confidence: 99%

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

Ishikawa¹,

Ishida²,

Matsui³

et al. 2001

Appl Environ Microbiol

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Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of Sulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.Pyrococcus horikoshii OT3 is one of the thermophilic archaea collected from a volcanic vent in the Okinawa Trough (16,20,21). This archaeon's optimum growth temperature ranges from 90 to 105°C. Most of the proteins from P. horikoshii are expected to be thermostable and active at high temperatures.Carboxypeptidase (CP) (peptidyl-L-amino-acid hydrolase; EC 3.4.12.1), which hydrolyzes the peptide bond at the C termini of peptides and proteins, is widely distributed in many organisms. Mammalian carboxypeptidase A (CPA) and CPB have been studied in detail (1,11,24). A thermostable CP is useful for high-temperature analysis of the C-terminal amino acid sequences of proteins. Recently, several thermostable CPs from the thermophilic bacteria Thermoactinomyces vulgaris (35,36) and Thermus aquaticus (27,28,29) and the thermophilic archaea Sulfolobus solfataricus (12,13,38) and Pyrococcus furiosus (8) have been characterized. Using genome sequencing for P. horikoshii (20, 21), we found two kinds of genes encoding CP-homologous proteins. One protein has 40% identity to the CP of T. aquaticus (28). The other has approximately 45% identity to the CP of S. solfataricus (13) and aminoacylase (N-acyl-L-amino acid amidohydrolase; EC 3.5.1.14) (3) of Bacillus stearothermophilus (33). In the latter protein, some of the amino acids which corresponded to the essential acti...

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Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris

Cited by 74 publications

References 31 publications

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Solution Structure of the Recombinant Penaeidin-3, a Shrimp Antimicrobial Peptide

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

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