scholarly journals Hetero-oligomeric CPN60 resembles highly symmetric group I chaperonin structure revealed by Cryo-EM

2018 ◽  
Author(s):  
Qian Zhao ◽  
Xiang Zhang ◽  
Frederik Sommer ◽  
Na Ta ◽  
Ning Wang ◽  
...  
Keyword(s):  
Symmetric Group ◽  
Chlamydomonas Reinhardtii ◽  
Surface Properties ◽  
Growth Condition ◽  
Model System ◽  
Uneven Distribution ◽  
E Coli ◽  
Group I ◽  
Key Enzyme

AbstractThe chloroplast chaperonin system is indispensable for the biogenesis of Rubisco, the key enzyme in photosynthesis. Using Chlamydomonas reinhardtii as the model system, we revealed that chloroplast chaperonin is consisted of CPN60α, CPN60β1, and CPN60β2, and co-chaperonin is composed of three subunits CPN20, CPN11 and CPN23 in vivo. CPN20 homo-oligomers and all possible other chloroplast co-chaperonin hetero-oligomers are functional, but only CPN11/20/23-CPN60αβ1β2 pair can fully replace GroES/GroEL in E. coli at stringent growth condition. Endogenous CPN60 was purified and its stoichiometry was determined to be 6:2:6 for CPN60α:CPN60β1:CPN60β2. The cryo-EM structures of endogenous CPN60αβ1β2/ADP and CPN60αβ1β2/co-chaperonin/ADP were solved at resolutions of 4.06 Å and 3.82Å, respectively. In both hetero-oligomeric complexes the chaperonin subunits within each ring are highly symmetric. The chloroplast co-chaperonin CPN11/20/23 formed seven GroES-like domains through hetero-oligomerization which symmetrically interact with CPN60αβ1β2. Our structures also reveal an uneven distribution of roof-like structures in the dome-shaped CPN11/20/23 and potentially diversified surface properties in the folding cavity of CPN60αβ1β2 that might enable the chloroplast chaperonin system to assist in the folding of specific substrates.

scholarly journals Boosting toxic protein expression: transient in vivo inactivation of engineered bacterial alkaline phosphatase

2020 ◽  
Author(s):  
Natalia Krawczun ◽  
Marta Bielawa ◽  
Kasjan Szemiako ◽  
Beata Lubkowska ◽  
Ireneusz Sobolewski ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Sulfhydryl Group ◽  
Antibody Detection ◽  
Bacterial Alkaline Phosphatase ◽  
E Coli ◽  
Metal Affinity ◽  
Novel Approach ◽  
Key Enzyme

Abstract Background:The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts’ metabolism. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host’s viability. Bacterial alkaline phosphatase (BAP) from Escherichia coli ( E. coli ) is a key enzyme used in protein/antibody detection and molecular cloning. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors’ background. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. coli periplasm. Then, the leader is clipped off and dimers are formed upon oxidation.Results: We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal his6-tag, cloned and expressed in E. coli in a P BAD promoter expression vector. The expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. coli cytoplasm, preventing formation of the active enzyme. A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn 2+ , Mg 2+ and phosphate. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3’-protruding).Conclusions: The developed method appears well suited for the industrial production of ultrapure BAP. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.

scholarly journals Boosting toxic protein expressionbiosynthesis: transient in vivo inactivation of engineered bacterial alkaline phosphatase

2020 ◽  
Author(s):  
Natalia Krawczun ◽  
Marta Bielawa ◽  
Kasjan Szemiako ◽  
Beata Lubkowska ◽  
Ireneusz Sobolewski ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Sulfhydryl Group ◽  
Antibody Detection ◽  
Bacterial Alkaline Phosphatase ◽  
E Coli ◽  
Metal Affinity ◽  
Novel Approach ◽  
Key Enzyme

Abstract Background The biotechnology production of enzymes is often troubled by the toxicity of the recombinant products of cloned and expressed genes, which interferes with the recombinant hosts’ metabolism. Various approaches have been taken to overcome these limitations, exemplified by tight control of recombinant genes or secretion of recombinant proteins. An industrial approach to protein production demands maximum possible yields of biosynthesized proteins, balanced with the recombinant host’s viability. Bacterial alkaline phosphatase (BAP) from Escherichia coli (E. coli) is a key enzyme used in protein/antibody detection and molecular cloning. As it removes terminal phosphate from DNA, RNA and deoxyribonucleoside triphosphates, it is used to lower self-ligated vectors’ background. The precursor enzyme contains a signal peptide at the N-terminus and is secreted to the E. coli periplasm. Then, the leader is clipped off and dimers are formed upon oxidation.Results We present a novel approach to phoA gene cloning, engineering, expression, purification and reactivation of the transiently inactivated enzyme. The recombinant bap gene was modified by replacing a secretion leader coding section with a N-terminal his6-tag, cloned and expressed in E. coli in a PBAD promoter expression vector. The gene expression was robust, resulting in accumulation of His6-BAP in the cytoplasm, exceeding 50% of total cellular proteins. The His6-BAP protein was harmless to the cells, as its natural toxicity was inhibited by the reducing environment within the E. coli cytoplasm, preventing formation of the active enzyme. A simple protocol based on precipitation and immobilized metal affinity chromatography (IMAC) purification yielded homogeneous protein, which was reactivated by dialysis into a redox buffer containing reduced and oxidized sulfhydryl group compounds, as well as the protein structure stabilizing cofactors Zn2+, Mg2+ and phosphate. The reconstituted His6-BAP exhibited high activity and was used to develop an efficient protocol for all types of DNA termini, including problematic ones (blunt, 3’-protruding).Conclusions The developed method appears well suited for the industrial production of ultrapure BAP. Further, the method of transient inactivation of secreted toxic enzymes by conducting their biosynthesis in an inactive state in the cytoplasm, followed by in vitro reactivation, can be generally applied to other problematic proteins.

scholarly journals The state of oligomerization of Rubisco controls the rate of synthesis of the Rubisco large subunit in Chlamydomonas reinhardtii

2021 ◽  
Author(s):  
Wojciech Wietrzynski ◽  
Eleonora Traverso ◽  
Francis-André Wollman ◽  
Katia Wostrikoff
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Large Subunit ◽  
Assembly Process ◽  
Bisphosphate Carboxylase ◽  
Photosynthetic Organisms ◽  
The Core ◽  
Key Enzyme ◽  
Assembly Intermediate ◽  
Life On Earth

Abstract Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is present in all photosynthetic organisms and is a key enzyme for photosynthesis-driven life on Earth. Its most prominent form is a hetero-oligomer in which small subunits (SSU) stabilize the core of the enzyme built from large subunits (LSU), yielding, after a chaperone-assisted multistep assembly process, an LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii and a combination of site-directed mutants to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU are associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis, in which LSU translation is controlled by its ability to assemble with the SSU, via the mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model whereby the last assembly intermediate, an LSU8-RAF1 complex, provides the platform for SSU binding to form the Rubisco enzyme, and when SSU is not available, converts to a key regulatory form that exerts negative feed-back on the initiation of LSU translation.

scholarly journals Architecture and self-assembly of the Clostridium sporogenes/botulinum spore surface illustrate a general protective strategy across spore formers

2020 ◽  
Author(s):  
Thamarai K. Janganan ◽  
Nic Mullin ◽  
Ainhoa Dafis-Sagarmendi ◽  
Jason Brunt ◽  
Svetomir B. Tzokov ◽  
...  
Keyword(s):  
Self Assembly ◽  
Clostridium Sporogenes ◽  
E Coli ◽  
Cysteine Rich Protein ◽  
Reducing Conditions ◽  
Group I ◽  
Protective Strategy ◽  
Additional Protein ◽  
Protective Structures

AbstractSpores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives have similar spore architectures incorporating protective proteinaceous envelopes. We reveal in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other Clostridial relatives, forms a hexagonally symmetric molecular filter. A cysteine-rich protein, CsxA, when expressed in E. coli, self-assembles into a highly thermally stable structure identical to native exosporium. Like exosporium, CsxA arrays require harsh reducing conditions for disassembly. We conclude that in vivo, CsxA self-organises into a highly resilient, disulphide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar in Bacillus spores, despite lack of protein homology. In both cases, intracellular disulphide formation is favoured by the high lattice symmetry. We propose that cysteine-rich proteins identified in distantly related spore formers may adopt a similar strategy for intracellular assembly of robust protective structures.

scholarly journals The state of oligomerization of Rubisco controls the rate of LSU translation in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Wojciech Wietrzynski ◽  
Eleonora Traverso ◽  
Francis-André Wollman ◽  
Katia Wostrikoff
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Large Subunit ◽  
Small Subunit ◽  
Bisphosphate Carboxylase ◽  
Photosynthetic Organisms ◽  
Initiation Of Translation ◽  
Key Enzyme ◽  
Assembly Intermediate ◽  
Life On Earth

AbstractRibulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is a key enzyme for photosynthesis-driven life on Earth. While present in all photosynthetic organisms, its most prominent form is a hetero-oligomer in which a Small Subunit (SSU) stabilizes the core of the enzyme built from Large Subunits (LSU), yielding, after a chaperone-assisted multistep assembly, a LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii, and a combination of site-directed mutants, to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU is associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis which places translation of its large subunit under the control of its ability to assemble with the small subunit, by a mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model where the last assembly intermediate, an octameric LSU8-RAF1 complex which delivers LSU to SSU to form the Rubisco enzyme, converts to a key regulator form able to exert a negative feed-back on the initiation of translation of LSU, when SSU is not available.

scholarly journals The Catalytic Role of RuBisCO for in situ CO2 Recycling in Escherichia coli

2020 ◽  
Vol 8 ◽  
Author(s):  
Ju-Jiun Pang ◽  
Jong-Shik Shin ◽  
Si-Yu Li
Keyword(s):  
Escherichia Coli ◽  
Rubisco Activase ◽  
Bisphosphate Carboxylase ◽  
Metabolite Production ◽  
E Coli ◽  
Theoretical Yield ◽  
Key Enzyme ◽  
Sugar Phosphates

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme responsible for biological CO2 assimilation. RuBisCO can be heterologously expressed in Escherichia coli so that glucose and CO2 are co-metabolized to achieve high mixotrophic metabolite production, where the theoretical yield of mixotrophic metabolite production is 2.4 mol(ethanol+acetate+pyruvate)/molglucose. However, RuBisCO is known for its low kcat and for forming inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates, yet the inhibited form of RuBisCO can be reversed by RuBisCO activase (Rca). In this study, RuBisCO forms I and II were cloned and expressed in Escherichia coli for in situ CO2 recycling, where CO2 produced during glucose fermentation was recycled and co-metabolized with the glucose. In addition, forms I and II RuBisCO activases were co-expressed with RuBisCO in E. coli to determine their in vivo effects on in situ CO2 recycling. Form I RuBisCO activase (Rca1) was co-expressed with form I RuBisCO and form II RuBisCO activase (Rca2) was co-expressed with form II RuBisCO. The results showed that both form I and form II RuBisCO exhibit comparable activities in E. coli and generated similar levels of in situ CO2 recycling. A significant increase in the total metabolite yield from 1.5 ± 0.1 to 2.2 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose occurred when Rca2 was co-expressed with form II RuBisCO. Meanwhile, the total metabolite yield increased from 1.7 ± 0.1 to 2.0 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose when Rca1 was co-expressed with form I RuBisCO. This data suggests that both forms I and II RuBisCO are subject to in vivo RuBP inhibition yet can be relieved by the co-expression of Rca. Interestingly, it is suggested that the in vivo RuBP inhibition of form II RuBisCO can be more easily reversed compared to form I. When the catalytic power of RuBisCO is maintained by Rca, the high activity of phosphoribulokinase (Prk) plays an important role in directing glucose to the RuBisCO-based engineered pathway and fermentation yields of 2.1–2.3 mol(ethanol+acetate+pyruvate)/molglucose can be obtained. This study is the first to demonstrate that in vivo RuBP inhibition of RuBisCO can be a bottleneck for in situ CO2 recycling in E. coli.

Deoxyribonucleotide Synthesis in an Escherichia coli Mutant (H 1491) which Lacks Ribonucleotide Reductase Subunit B2

1989 ◽  
Vol 44 (7-8) ◽  
pp. 715-718 ◽  
Author(s):  
Jens Harder ◽  
Hartmut Follmann ◽  
Klaus Hantke
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Strictly Anaerobic ◽  
Anaerobic Conditions ◽  
Sensitive Mutant ◽  
E Coli ◽  
Key Enzyme ◽  
Minimal Media

An iron-sensitive mutant of E. coli with a Mudl phage insertion in the nrdB gene lacks subunit B2 of the key enzyme of DNA synthesis, ribonucleotide reductase. Nevertheless, these cells are capable of growing in minimal media under anaerobic conditions, indicating a second enzyme or pathway for deoxyribonucleotide synthesis. We here show that ribonucleotide reduction cannot be unambiguously measured in bacterial extracts whereas phosphorylase- catalyzed deoxyribosyl transfer does occur; however these salvage reactions could not function in vivo in the absence of deoxyribosides. It is suggested that the cells possess a specific, anaerobic ribonucleotide reductase which escapes detection under aerobic standard conditions, similar to the situation found in strictly anaerobic methanogens.

scholarly journals In vivo susceptibility of ESBL producing Escherichia coli to ceftriaxone in children with acute pyelonephritis

2012 ◽  
Vol 140 (5-6) ◽  
pp. 321-325 ◽  
Author(s):  
Amira Peco-Antic ◽  
Dusan Paripovic ◽  
Svetlana Buljugic ◽  
Brankica Spasojevic-Dimitrijeva ◽  
Mirjana Cvetkovic ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acute Pyelonephritis ◽  
Empiric Therapy ◽  
Confirmatory Test ◽  
Diffusion Method ◽  
E Coli ◽  
Group I ◽  
Days Of Therapy ◽  
Group Ii

Introduction. The choice of empiric therapy of acute pyelonephritis (APN) in children should be based on the knowledge of Escherichia coli (E. coli) as the most common uropathogen and its antibiotic sensitivities considering that nowadays ESBL-producing [ESBL (+)] E. coli is on the rise worldwide. Objective. To examine in vivo susceptibility of ESBL (+) E. coli to ceftriaxone (CTX), and to evaluate the options for empiric therapy for APN in children. Methods. Retrospective study of CTX empiric therapy of APN in children treated at the University Children?s Hospital in Belgrade from January 2005 to December 2009. ESBL phenotypic confirmatory test with ceftazidime, CTX and cefotaxime was performed for all urine isolates by disc diffusion method on Mueller-Hinton agar plates. In vivo sensitivity of CTX documented by clinical response to empiric CTX therapy was compared between two groups of children: group I with ESBL (+) E. coli and group II with ESBL (-) E. coli APN. Results. Group I with ESBL (+) APN consisted of 94 patients and group II of 120 patients with ESBL (-) APN, respectively. All patients received CTX as empiric therapy at a mean dose of 66.9 mg during 7.2?2.6 days of therapy. Clinical effect of CTX was similar in patients with ESBL (+) compared to those with ESBL (-) APN. Conclusions. In vitro resistance of ESBL E. coli to CTX determined by standard methods is not sufficiently predictive for its in vivo sensitivity. Therefore CTX may be used as empiric therapy for acute pyelonephritis in children.

scholarly journals Ribosome surface properties may impose limits on the nature of the cytoplasmic proteome

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Paul E Schavemaker ◽  
Wojciech M Śmigiel ◽  
Bert Poolman
Keyword(s):  
Surface Properties ◽  
Diffusion Coefficients ◽  
Molecular Motion ◽  
Fluorescent Proteins ◽  
Translational Diffusion ◽  
Net Charge ◽  
E Coli ◽  
Surface Modified

Much of the molecular motion in the cytoplasm is diffusive, which possibly limits the tempo of processes. We studied the dependence of protein mobility on protein surface properties and ionic strength. We used surface-modified fluorescent proteins (FPs) and determined their translational diffusion coefficients (D) in the cytoplasm of Escherichia coli, Lactococcus lactis and Haloferax volcanii. We find that in E. coli D depends on the net charge and its distribution over the protein, with positive proteins diffusing up to 100-fold slower than negative ones. This effect is weaker in L. lactis and Hfx. volcanii due to electrostatic screening. The decrease in mobility is probably caused by interaction of positive FPs with ribosomes as shown in in vivo diffusion measurements and confirmed in vitro with purified ribosomes. Ribosome surface properties may thus limit the composition of the cytoplasmic proteome. This finding lays bare a paradox in the functioning of prokaryotic (endo)symbionts.

Studies on the Synthesis of Phosphoglycerides in Escherichia coli

1972 ◽  
Vol 50 (1) ◽  
pp. 16-19 ◽  
Author(s):  
Georgina Benns ◽  
P. Proulx
Keyword(s):  
Escherichia Coli ◽  
Isotope Ratio ◽  
Uneven Distribution ◽  
Dihydroxyacetone Phosphate ◽  
Glycerol Moiety ◽  
E Coli ◽  
Prior Oxidation

The incorporation of various labelled precursors into E. coli lipids was studied. In growing cells, incorporation of glycerol, doubly labelled with 14C and 3H, occurs without a change in isotope ratio. In cell homogenates incorporation of sn-glycero-3-phosphate-U-14C was greater in the polyglycerophosphatide fraction than in phosphatidylethanolamine and higher in the unacylated than in the acylated glycerol moiety of phosphatidylglycerol. This uneven distribution of label in phosphatidylglycerol was not affected by addition of unlabelled dihydroxyacetone phosphate and occurred to the same extent when glucose-3,4-14C was the precursor. Our results obtained in vivo indicate that glycerophosphate is acylated without prior oxidation. Furthermore, labelling patterns obtained in vitro suggest an extensive dilution by endogenous diacyl glycerol precursors not formed by acylation of dihydroxyacetone phosphate.

Sign in / Sign up

Export Citation Format

Share Document