In vitro refolding of carboxypeptidase T precursor from Thermoactinomyces vulgaris obtained in Escherichia coli as cytoplasmic inclusion bodies

Trachuk, Lesya A.; Letarov, Andrei V.; Kudelina, Irina A.; Yusupova, M. P.; Chestukhina, G. G.

doi:10.1016/j.pep.2004.10.020

Cited by 14 publications

(4 citation statements)

References 20 publications

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“…Various metalloproteases have been expressed in E. coli, in the form of inclusion bodies (Trachuk et al, 2005;Oneda and Inouye, 1999), and thus active enzymes have to be recovered from inclusion bodies by denaturation and refolding processes. Therefore, an extracellular production system in which secreted protein can be directly applied in activity assays is highly preferable.…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning and sequence analysis of a novel zinc-metalloprotease gene from the Salinivibrio sp. strain AF-2004 and its extracellular expression in E. coli

Karbalaei‐Heidari

Ziaee

Amoozegar

et al. 2008

Gene

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Section: Discussionmentioning

confidence: 99%

Molecular cloning and sequence analysis of a novel zinc-metalloprotease gene from the Salinivibrio sp. strain AF-2004 and its extracellular expression in E. coli

Karbalaei‐Heidari

Ziaee

Amoozegar

et al. 2008

Gene

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“…Native CPT was isolated from inclusion bodies as described in [27]. The inclusion bodies were separated by centrifugation, washed with 0.05% CHAPS (w/v), 2 M NaCl and water, and dissolved in 8 M urea up to the final concentration of 5 mg/mL.…”

Section: Methodsmentioning

confidence: 99%

The nature of the ligand’s side chain interacting with the S1'-subsite of metallocarboxypeptidase T (from Thermoactinomyces vulgaris) determines the geometry of the tetrahedral transition complex

et al. 2019

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The carboxypeptidase T (CPT) from Thermoactinomyces vulgaris has an active site structure and 3D organization similar to pancreatic carboxypeptidases A and B (CPA and CPB), but differs in broader substrate specificity. The crystal structures of CPT complexes with the transition state analogs N-sulfamoyl-L-leucine and N-sulfamoyl-L-glutamate (SLeu and SGlu) were determined and compared with previously determined structures of CPT complexes with N-sulfamoyl-L-arginine and N-sulfamoyl-L-phenylalanine (SArg and SPhe). The conformations of residues Tyr255 and Glu270, the distances between these residues and the corresponding ligand groups, and the Zn-S gap between the zinc ion and the sulfur atom in the ligand's sulfamoyl group that simulates a distance between the zinc ion and the tetrahedral sp 3 -hybridized carbon atom of the converted peptide bond, vary depending on the nature of the side chain in the substrate's C-terminus. The increasing affinity of CPT with the transition state analogs in the order SGlu, SArg, SPhe, SLeu correlates well with a decreasing Zn-S gap in these complexes and the increasing efficiency of CPT-catalyzed hydrolysis of the corresponding tripeptide substrates (ZAAL > ZAAF > ZAAR > ZAAE). Thus, the side chain of the ligand that interacts with the primary specificity pocket of CPT, determines the geometry of the transition complex, the relative orientation of the bond to be cleaved by the catalytic groups of the active site and the catalytic properties of the enzyme. In the case of PLOS ONE | https://doi.

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“…Native CpT was isolated from inclusion bodies as described previously . The inclusion bodies were separated by centrifugation, washed with 0.05% CHAPS (w/v), 2 m NaCl and water, and dissolved in 8 m urea up to a final concentration of 5 g·L −1 .…”

Section: Methodsmentioning

confidence: 99%

Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris

et al. 2015

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The crystal structures of carboxypeptidase T (CpT) complexes with phenylalanine and arginine substrate analogs -benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid -were determined by the molecular replacement method at resolutions of 1.57 A and 1.62 A to clarify the broad substrate specificity profile of the enzyme. The conservative Leu211 and Leu254 residues (also present in both carboxypeptidase A and carboxypeptidase B) were shown to be structural determinants for recognition of hydrophobic substrates, whereas Asp263 was for recognition of positively charged substrates. Mutations of these determinants modify the substrate profile: the CpT variant Leu211Gln acquires carboxypeptidase B-like properties, and the CpT variant Asp263Asn the carboxypeptidase A-like selectivity. The Pro248-Asp258 loop interacting with Leu254 and Tyr255 was shown to be responsible for recognition of the substrate's C-terminal residue. Substrate binding at the S1 0 subsite leads to the ligand-dependent shift of this loop, and Leu254 side chain movement induces the conformation rearrangement of the Glu277 residue crucial for catalysis. This is a novel insight into the substrate selectivity of metallocarboxypeptidases that demonstrates the importance of interactions between the S1 0 subsite and the catalytic center.

show abstract

In vitro refolding of carboxypeptidase T precursor from Thermoactinomyces vulgaris obtained in Escherichia coli as cytoplasmic inclusion bodies

Cited by 14 publications

References 20 publications

Molecular cloning and sequence analysis of a novel zinc-metalloprotease gene from the Salinivibrio sp. strain AF-2004 and its extracellular expression in E. coli

Molecular cloning and sequence analysis of a novel zinc-metalloprotease gene from the Salinivibrio sp. strain AF-2004 and its extracellular expression in E. coli

The nature of the ligand’s side chain interacting with the S1'-subsite of metallocarboxypeptidase T (from Thermoactinomyces vulgaris) determines the geometry of the tetrahedral transition complex

Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris

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