Crystal Structure and Functional Analysis of the Glutaminyl Cyclase from Xanthomonas campestris

Huang, Wei-Lin; Wang, Yu-Ruei; Ko, Tzu‐Ping; Chia, C.Y.; Huang, Kai‐Fa; Wang, Andrew H.J.

doi:10.1016/j.jmb.2010.06.012

Cited by 22 publications

(25 citation statements)

References 53 publications

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“…To test the feasibility of intracellular autonomous pGlu formation, we first constructed an expression vector in Escherichia coli system attempting to produce the bQC(E45Q)-TEVP(S219V)-rsTEV-MCPx-6His fusion proteins, where bQC(E45Q) represents a gain-of-function mutant of the glutaminyl cyclase from the plant pathogenic bacterium Xanthomonas campestris [17], TEVP(S219V) is a high-stability mutant of TEV protease [39], and MCPx is the monocyte chemoattractant protein 1 or 2 [25]. However, after testing a variety of competent cells and induction conditions, we found that large fractions of the IPTG-induced fusion proteins were present in the insoluble aggregates, and the obtained MCPx-6His products were very few.…”

Section: Resultsmentioning

confidence: 99%

“…The resulting DNA fragment was inserted into the vector pRSF-1b (Novagen) via the Nco I and Sal I sites. Second, the DNA encoding enhanced green fluorescence protein (EGFP) was amplified from the plasmid pMBP-rsTEV-EGFP as reported previously [27], and the DNA encoding bQC(E45Q) was amplified from the expression vector of Xanthomonas campestris QC [17]. The two resulting DNA fragments were simultaneously inserted into the vector pET-32 Ek-LIC via the LIC Duet Minimal Adaptor (Novagen).…”

Section: Methodsmentioning

confidence: 99%

“…Type I QCs display a five-bladed β-propeller fold and are mainly identified in plants, several pathogenic bacteria, and human parasites [15]–[17], while type II QCs adopt an α/β topology and are abundant in the neuroendocrine tissues and peripheral blood lymphocytes of mammals [11], [12], [18]–[20]. In addition, on the basis of different subcellular localizations, two isoforms of mammalian QCs have been reported, i.e., secreted and Golgi-resident QCs, which are encoded by genes located at different chromosomes [20]–[22].…”

Section: Introductionmentioning

confidence: 99%

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Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

et al. 2014

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Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

et al. 2014

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“…This domain can consist of 4 to 10 blades in a large diversity of proteins that mediate different functions such as substrate/protein binding [29], transferase [30], lyase [31], signaling domain [32] and others [33, 34]. POP presents a seven-bladed β-propeller domain lacking the canonical closure [35] and hydrogen or disulfide bonds between the first and the seventh blade.…”

Section: Overall Structure and Catalytic Featuresmentioning

confidence: 99%

Parasite Prolyl Oligopeptidases and the Challenge of Designing Chemotherapeuticals for Chagas Disease, Leishmaniasis and African Trypanosomiasis

Bastos

Motta²,

Grellier³

et al. 2013

CMC

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The trypanosomatids Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp. cause Chagas disease, leishmaniasis and human African trypanosomiasis, respectively. It is estimated that over 10 million people worldwide suffer from these neglected diseases, posing enormous social and economic problems in endemic areas. There are no vaccines to prevent these infections and chemotherapies are not adequate. This picture indicates that new chemotherapeutic agents must be developed to treat these illnesses. For this purpose, understanding the biology of the pathogenic trypanosomatid-host cell interface is fundamental for molecular and functional characterization of virulence factors that may be used as targets for the development of inhibitors to be used for effective chemotherapy. In this context, it is well known that proteases have crucial functions for both metabolism and infectivity of pathogens and are thus potential drug targets. In this regard, prolyl oligopeptidase and oligopeptidase B, both members of the S9 serine protease family, have been shown to play important roles in the interactions of pathogenic protozoa with their mammalian hosts and may thus be considered targets for drug design. This review aims to discuss structural and functional properties of these intriguing enzymes and their potential as targets for the development of drugs against Chagas disease, leishmaniasis and African trypanosomiasis.

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“…This domain can consist of 4 to 10 blades in a large diversity of proteins that mediate different functions such as substrate/protein binding [29], transferase [30], lyase [31], signaling domain [32] and others [33,34]. POP presents a seven-bladed -propeller domain lacking the canonical closure [35] and hydrogen or disulfide bonds between the first and the seventh blade.…”

Section: Prolyl Oligopeptidase (Ec 342126)mentioning

confidence: 99%

Parasite Prolyl Oligopeptidases and the Challenge of Designing Chemotherapeuticals for Chagas Disease, Leishmaniasis and African Trypanosomiasis

Bastos¹,

Motta²,

Grellier³

et al. 2013

CMC

View full text Add to dashboard Cite

Abstract:The trypanosomatids Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp. cause Chagas disease, leishmaniasis and human African trypanosomiasis, respectively. It is estimated that over 10 million people worldwide suffer from these neglected diseases, posing enormous social and economic problems in endemic areas. There are no vaccines to prevent these infections and chemotherapies are not adequate. This picture indicates that new chemotherapeutic agents must be developed to treat these illnesses. For this purpose, understanding the biology of the pathogenic trypanosomatid-host cell interface is fundamental for molecular and functional characterization of virulence factors that may be used as targets for the development of inhibitors to be used for effective chemotherapy. In this context, it is well known that proteases have crucial functions for both metabolism and infectivity of pathogens and are thus potential drug targets. In this regard, prolyl oligopeptidase and oligopeptidase B, both members of the S9 serine protease family, have been shown to play important roles in the interactions of pathogenic protozoa with their mammalian hosts and may thus be considered targets for drug design. This review aims to discuss structural and functional properties of these intriguing enzymes and their potential as targets for the development of drugs against Chagas disease, leishmaniasis and African trypanosomiasis.

show abstract

Crystal Structure and Functional Analysis of the Glutaminyl Cyclase from Xanthomonas campestris

Cited by 22 publications

References 53 publications

Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

Parasite Prolyl Oligopeptidases and the Challenge of Designing Chemotherapeuticals for Chagas Disease, Leishmaniasis and African Trypanosomiasis

Parasite Prolyl Oligopeptidases and the Challenge of Designing Chemotherapeuticals for Chagas Disease, Leishmaniasis and African Trypanosomiasis

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