Many-molecule encapsulation by an icosahedral shell

We computationally study how an icosahedral shell assembles around hundreds of molecules. Such a process occurs during the formation of the carboxysome, a bacterial microcompartment that assembles around many copies of the enzymes ribulose 1,5-bisphosphate carboxylase/ oxygenase and carbonic anhydrase to facilitate carbon fixation in cyanobacteria. Our simulations identify two classes of assembly pathways leading to encapsulation of many-molecule cargoes. In one, shell assembly proceeds concomitantly with cargo condensation. In the other, the cargo first forms a dense globule; then, shell proteins assemble around and bud from the condensed cargo complex. Although the model is simplified, the simulations predict intermediates and closure mechanisms not accessible in experiments, and show how assembly can be tuned between these two pathways by modulating protein interactions. In addition to elucidating assembly pathways and critical control parameters for microcompartment assembly, our results may guide the reengineering of viruses as nanoreactors that self-assemble around their reactants.

Download Full-text

A Multiprotein Bicarbonate Dehydration Complex Essential to Carboxysome Function in Cyanobacteria

Journal of Bacteriology ◽

10.1128/jb.01283-07 ◽

2007 ◽

Vol 190 (3) ◽

pp. 936-945 ◽

Cited By ~ 82

Author(s):

Swan S.-W. Cot ◽

Anthony K.-C. So ◽

George S. Espie

Keyword(s):

Carbonic Anhydrase ◽

Protein Interactions ◽

Specific Protein ◽

Yeast Two Hybrid ◽

Biochemical Activity ◽

Catalytic Core ◽

Bisphosphate Carboxylase ◽

Molecular Approaches ◽

Two Hybrid ◽

Protein Shell

ABSTRACT Carboxysomes are proteinaceous biochemical compartments that constitute the enzymatic “back end” of the cyanobacterial CO2-concentrating mechanism. These protein-bound organelles catalyze HCO3 − dehydration and photosynthetic CO2 fixation. In Synechocystis sp. strain PCC6803 these reactions involve the β-class carbonic anhydrase (CA), CcaA, and Form 1B ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The surrounding shell is thought to be composed of proteins encoded by the ccmKLMN operon, although little is known about how structural and catalytic proteins integrate to form a functional carboxysome. Using biochemical activity assays and molecular approaches we have identified a catalytic, multiprotein HCO3 − dehydration complex (BDC) associated with the protein shell of Synechocystis carboxysomes. The complex was minimally composed of a CcmM73 trimer, CcaA dimer, and CcmN. Larger native complexes also contained RbcL, RbcS, and two or three immunologically identified smaller forms of CcmM (62, 52, and 36 kDa). Yeast two-hybrid analyses indicated that the BDC was associated with the carboxysome shell through CcmM73-specific protein interactions with CcmK and CcmL. Protein interactions between CcmM73 and CcaA, CcmM73 and CcmN, or CcmM73 and itself required the N-terminal γ-CA-like domain of CcmM73. The specificity of the CcmM73-CcaA interaction provided both a mechanism to integrate CcaA into the fabric of the carboxysome shell and a means to recruit this enzyme to the BDC during carboxysome biogenesis. Functionally, CcaA was the catalytic core of the BDC. CcmM73 bound H14CO3 − but was unable to catalyze HCO3 − dehydration, suggesting that it may potentially regulate BDC activity.

Download Full-text

A Novel Evolutionary Lineage of Carbonic Anhydrase (ε Class) Is a Component of the Carboxysome Shell

Journal of Bacteriology ◽

10.1128/jb.186.3.623-630.2004 ◽

2004 ◽

Vol 186 (3) ◽

pp. 623-630 ◽

Cited By ~ 190

Author(s):

Anthony K.-C. So ◽

George S. Espie ◽

Eric B. Williams ◽

Jessup M. Shively ◽

Sabine Heinhorst ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Active Sites ◽

Carbon Fixation ◽

Total Carbon ◽

Bisphosphate Carboxylase ◽

Evolutionary Lineage ◽

Shell Protein ◽

Essential Components ◽

Halothiobacillus Neapolitanus ◽

Chemolithoautotrophic Bacteria

ABSTRACT A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO2 fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO2-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (ε-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO2 necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.

Download Full-text

In Salmonella enterica, Ethanolamine Utilization Is Repressed by 1,2-Propanediol To Prevent Detrimental Mixing of Components of Two Different Bacterial Microcompartments

Journal of Bacteriology ◽

10.1128/jb.00215-15 ◽

2015 ◽

Vol 197 (14) ◽

pp. 2412-2421 ◽

Cited By ~ 18

Author(s):

Ryan Sturms ◽

Nicholas A. Streauslin ◽

Shouqiang Cheng ◽

Thomas A. Bobik

Keyword(s):

Protein Interactions ◽

Regulatory Protein ◽

Gene Clusters ◽

Content Type ◽

Bacterial Microcompartments ◽

Shell Protein ◽

Bacterial Microcompartment ◽

Genes Encoding ◽

Shell Proteins ◽

High Conservation

ABSTRACTBacterial microcompartments (MCPs) are a diverse family of protein-based organelles composed of metabolic enzymes encapsulated within a protein shell. The function of bacterial MCPs is to optimize metabolic pathways by confining toxic and/or volatile metabolic intermediates. About 20% of bacteria produce MCPs, and there are at least seven different types. Different MCPs vary in their encapsulated enzymes, but all have outer shells composed of highly conserved proteins containing bacterial microcompartment domains. Many organisms have genes encoding more than one type of MCP, but given the high homology among shell proteins, it is uncertain whether multiple MCPs can be functionally expressed in the same cell at the same time. In these studies, we examine the regulation of the 1,2-propanediol (1,2-PD) utilization (Pdu) and ethanolamine utilization (Eut) MCPs inSalmonella. Studies showed that 1,2-PD (shown to induce the Pdu MCP) represses transcription of the Eut MCP and that the PocR regulatory protein is required. The results indicate that repression of the Eut MCP by 1,2-PD is needed to prevent detrimental mixing of shell proteins from the Eut and Pdu MCPs. Coexpression of both MCPs impaired the function of the Pdu MCP and resulted in the formation of hybrid MCPs composed of Eut and Pdu MCP components. We also show that plasmid-based expression of individual shell proteins from the Eut MCP or the β-carboxysome impaired the function of Pdu MCP. Thus, the high conservation among bacterial microcompartment (BMC) domain shell proteins is problematic for coexpression of the Eut and Pdu MCPs and perhaps other MCPs as well.IMPORTANCEBacterial MCPs are encoded by nearly 20% of bacterial genomes, and almost 40% of those genomes contain multiple MCP gene clusters. In this study, we examine how the regulation of two different MCP systems (Eut and Pdu) is integrated inSalmonella. Our findings indicate that 1,2-PD (shown to induce the Pdu MCP) represses the Eut MCP to prevent detrimental mixing of Eut and Pdu shell proteins. These findings suggest that numerous organisms which produce more than one type of MCP likely need some mechanism to prevent aberrant shell protein interactions.

Download Full-text

Self-assembling shell proteins PduA and PduJ have essential and redundant roles in bacterial microcompartment assembly

10.1101/2020.07.06.187062 ◽

2020 ◽

Author(s):

Nolan W. Kennedy ◽

Svetlana P. Ikonomova ◽

Marilyn Slininger Lee ◽

Henry W. Raeder ◽

Danielle Tullman-Ercek

Keyword(s):

Self Assembly ◽

Carbon Fixation ◽

Enteric Bacteria ◽

Bacterial Microcompartments ◽

Bacterial Microcompartment ◽

Self Assembling ◽

Assembly Mechanism ◽

High Propensity ◽

Serovar Typhimurium ◽

Shell Proteins

AbstractProtein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.

Download Full-text

CARBONIC ANHYDRASE AND CARBON FIXATION IN COCCOLITHOPHORIDS

Journal of Phycology ◽

10.1111/j.1529-8817.1982.tb03205.x ◽

1982 ◽

Vol 18 (3) ◽

pp. 423-426 ◽

Cited By ~ 27

Author(s):

C. Steven Sikes ◽

A. P. Wheeler

Keyword(s):

Carbonic Anhydrase ◽

Carbon Fixation

Download Full-text

Distinctive Responses of Ribulose-1,5-Bisphosphate Carboxylase and Carbonic Anhydrase in Wheat Leaves to Nitrogen Nutrition and their Possible Relationships to CO2-Transfer Resistance

PLANT PHYSIOLOGY ◽

10.1104/pp.100.4.1737 ◽

1992 ◽

Vol 100 (4) ◽

pp. 1737-1743 ◽

Cited By ~ 135

Author(s):

Amane Makino ◽

Hiroshi Sakashita ◽

Jun Hidema ◽

Tadahiko Mae ◽

Kunihiko Ojima ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Nitrogen Nutrition ◽

Transfer Resistance ◽

Bisphosphate Carboxylase ◽

Wheat Leaves

Download Full-text

Design and in vitro realization of carbon-conserving photorespiration

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1812605115 ◽

2018 ◽

Vol 115 (49) ◽

pp. E11455-E11464 ◽

Cited By ~ 33

Author(s):

Devin L. Trudeau ◽

Christian Edlich-Muth ◽

Jan Zarzycki ◽

Marieke Scheffen ◽

Moshe Goldsmith ◽

...

Keyword(s):

Carbon Fixation ◽

Calvin Cycle ◽

Computational Design ◽

Carbon Loss ◽

Fixation Rate ◽

Bisphosphate Carboxylase ◽

Stoichiometric Model ◽

Synthetic Routes ◽

Coa Reductase

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO2. Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO2. Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO2 release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss. We further developed a kinetic–stoichiometric model that indicates that the identified synthetic shunts could potentially enhance carbon fixation rate across the physiological range of irradiation and CO2, even if most of their enzymes operate at a tenth of Rubisco’s maximal carboxylation activity. Glycolate reduction to glycolaldehyde is essential for several of the synthetic shunts but is not known to occur naturally. We, therefore, used computational design and directed evolution to establish this activity in two sequential reactions. An acetyl-CoA synthetase was engineered for higher stability and glycolyl-CoA synthesis. A propionyl-CoA reductase was engineered for higher selectivity for glycolyl-CoA and for use of NADPH over NAD+, thereby favoring reduction over oxidation. The engineered glycolate reduction module was then combined with downstream condensation and assimilation of glycolaldehyde to ribulose 1,5-bisphosphate, thus providing proof of principle for a carbon-conserving photorespiration pathway.

Download Full-text

Protein Folding by Domain V of Escherichia coli 23S rRNA: Specificity of RNA-Protein Interactions

Journal of Bacteriology ◽

10.1128/jb.01800-07 ◽

2008 ◽

Vol 190 (9) ◽

pp. 3344-3352 ◽

Cited By ~ 36

Author(s):

Dibyendu Samanta ◽

Debashis Mukhopadhyay ◽

Saheli Chowdhury ◽

Jaydip Ghosh ◽

Saumen Pal ◽

...

Keyword(s):

Escherichia Coli ◽

Protein Folding ◽

Carbonic Anhydrase ◽

Protein Interactions ◽

23S Rrna ◽

Unfolded Proteins ◽

Bovine Carbonic Anhydrase ◽

Egg White Lysozyme ◽

General Protein ◽

Domain V

ABSTRACT The peptidyl transferase center, present in domain V of 23S rRNA of eubacteria and large rRNA of plants and animals, can act as a general protein folding modulator. Here we show that a few specific nucleotides in Escherichia coli domain V RNA bind to unfolded proteins and, as shown previously, bring the trapped proteins to a folding-competent state before releasing them. These nucleotides are the same for the proteins studied so far: bovine carbonic anhydrase, lactate dehydrogenase, malate dehydrogenase, and chicken egg white lysozyme. The amino acids that interact with these nucleotides are also found to be specific in the two cases tested: bovine carbonic anhydrase and lysozyme. They are either neutral or positively charged and are present in random coils on the surface of the crystal structure of both the proteins. In fact, two of these amino acid-nucleotide pairs are identical in the two cases. How these features might help the process of protein folding is discussed.

Download Full-text