scholarly journals Decoding the absolute stoichiometric composition and structural plasticity of α-carboxysomes

2021 ◽  
Author(s):  
Yaqi Sun ◽  
Victoria M. Harman ◽  
James R. Johnson ◽  
Taiyu Chen ◽  
Gregory F. Dykes ◽  
...  
Keyword(s):  
Rational Design ◽  
Carbon Fixation ◽  
Stoichiometric Composition ◽  
Quantitative Mass Spectrometry ◽  
Shell Protein ◽  
Self Assembling ◽  
The Absolute ◽  
Halothiobacillus Neapolitanus ◽  
Protein Shell ◽  
Insight Into

AbstractCarboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria and some chemoautotrophs. This self-assembling organelle encapsulates the key CO2-fixing enzymes, Rubisco, and carbonic anhydrase using a polyhedral protein shell that is constructed by hundreds of shell protein paralogs. The α-carboxysome from the chemoautotroph Halothiobacillus neapolitanus serves as a model system in fundamental studies and synthetic engineering of carboxysomes. Here we adopt a QconCAT-based quantitative mass spectrometry to determine the absolute stoichiometric composition of native α-carboxysomes from H. neapolitanus. We further performed an in-depth comparison of the protein stoichiometry of native and recombinant α-carboxysomes heterologously generated in Escherichia coli to evaluate the structural variability and remodeling of α-carboxysomes. Our results provide insight into the molecular principles that mediate carboxysome assembly, which may aid in rational design and reprogramming of carboxysomes in new contexts for biotechnological applications.

scholarly journals Reprogramming bacterial protein organelles as a nanoreactor for hydrogen production

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tianpei Li ◽  
Qiuyao Jiang ◽  
Jiafeng Huang ◽  
Catherine M. Aitchison ◽  
Fang Huang ◽  
...  
Keyword(s):  
Hydrogen Production ◽  
Self Assembly ◽  
Rational Design ◽  
Carbon Fixation ◽  
Catalytic Performance ◽  
Enzyme Activation ◽  
Production Of Hydrogen ◽  
Catalytically Active ◽  
Oxygen Sensitive ◽  
Protein Shell

Abstract Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biological elements and reactions. The carboxysome is a specialized bacterial organelle that encapsulates enzymes into a virus-like protein shell and plays essential roles in photosynthetic carbon fixation. The naturally designed architecture, semi-permeability, and catalytic improvement of carboxysomes have inspired rational design and engineering of new nanomaterials to incorporate desired enzymes into the protein shell for enhanced catalytic performance. Here, we build large, intact carboxysome shells (over 90 nm in diameter) in the industrial microorganism Escherichia coli by expressing a set of carboxysome protein-encoding genes. We develop strategies for enzyme activation, shell self-assembly, and cargo encapsulation to construct a robust nanoreactor that incorporates catalytically active [FeFe]-hydrogenases and functional partners within the empty shell for the production of hydrogen. We show that shell encapsulation and the internal microenvironment of the new catalyst facilitate hydrogen production of the encapsulated oxygen-sensitive hydrogenases. The study provides insights into the assembly and formation of carboxysomes and paves the way for engineering carboxysome shell-based nanoreactors to recruit specific enzymes for diverse catalytic reactions.

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

2004 ◽  
Vol 186 (3) ◽  
pp. 623-630 ◽  
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.

scholarly journals Selective molecular transport through the protein shell of a bacterial microcompartment organelle

2015 ◽  
Vol 112 (10) ◽  
pp. 2990-2995 ◽  
Author(s):  
Chiranjit Chowdhury ◽  
Sunny Chun ◽  
Allan Pang ◽  
Michael R. Sawaya ◽  
Sharmistha Sinha ◽  
...  
Keyword(s):  
Small Molecules ◽  
Carbon Fixation ◽  
Molecular Diffusion ◽  
Molecular Transport ◽  
Phenotypic Data ◽  
Bacterial Microcompartments ◽  
Shell Protein ◽  
Bacterial Microcompartment ◽  
Selective Permeability ◽  
Protein Shell

Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.

scholarly journals Characterization of the Carboxysomal Carbonic Anhydrase CsoSCA from Halothiobacillus neapolitanus

2006 ◽  
Vol 188 (23) ◽  
pp. 8087-8094 ◽  
Author(s):  
Sabine Heinhorst ◽  
Eric B. Williams ◽  
Fei Cai ◽  
C. Daniel Murin ◽  
Jessup M. Shively ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Catalytic Efficiency ◽  
Biochemical Properties ◽  
Bisphosphate Carboxylase ◽  
Shell Protein ◽  
Dehydration Reactions ◽  
Chemolithotrophic Bacteria ◽  
Halothiobacillus Neapolitanus ◽  
Protein Shell

ABSTRACT In cyanobacteria and many chemolithotrophic bacteria, the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO2, through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO2 hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO2 that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO2 trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.

scholarly journals Structural analysis of cross α-helical nanotubes provides insight into the designability of filamentous peptide nanomaterials

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fengbin Wang ◽  
Ordy Gnewou ◽  
Charles Modlin ◽  
Leticia C. Beltran ◽  
Chunfu Xu ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Synthetic Peptide ◽  
Quaternary Structure ◽  
De Novo ◽  
Rational Approach ◽  
Lateral Interactions ◽  
Protein Assemblies ◽  
Self Assembling ◽  
Arginine Residues ◽  
Insight Into

AbstractThe exquisite structure-function correlations observed in filamentous protein assemblies provide a paradigm for the design of synthetic peptide-based nanomaterials. However, the plasticity of quaternary structure in sequence-space and the lability of helical symmetry present significant challenges to the de novo design and structural analysis of such filaments. Here, we describe a rational approach to design self-assembling peptide nanotubes based on controlling lateral interactions between protofilaments having an unusual cross-α supramolecular architecture. Near-atomic resolution cryo-EM structural analysis of seven designed nanotubes provides insight into the designability of interfaces within these synthetic peptide assemblies and identifies a non-native structural interaction based on a pair of arginine residues. This arginine clasp motif can robustly mediate cohesive interactions between protofilaments within the cross-α nanotubes. The structure of the resultant assemblies can be controlled through the sequence and length of the peptide subunits, which generates synthetic peptide filaments of similar dimensions to flagella and pili.

The effect of amino substituents on the interactions of quinazolone derivatives with c-KIT G-quadruplex: insight from molecular dynamics simulation study for rational design of ligands

RSC Advances ◽  
2015 ◽  
Vol 5 (93) ◽  
pp. 76642-76650 ◽  
Author(s):  
Kiana Gholamjani Moghaddam ◽  
Seyed Majid Hashemianzadeh
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
Simulation Study ◽  
Rational Design ◽  
Ligand Design ◽  
Dynamics Simulation ◽  
Rational Ligand Design ◽  
G Quadruplex ◽  
Ligand Interactions ◽  
Insight Into

Our study provides insight into the effect of different substituents on the G-quadruplex–ligand interactions which helps us rational ligand design.

scholarly journals KBE009: A Bestatin-Like Inhibitor of the Trypanosoma cruzi Acidic M17 Aminopeptidase With In Vitro Anti-Trypanosomal Activity

Life ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1037
Author(s):  
Jorge González-Bacerio ◽  
Irina Arocha ◽  
Mirtha Elisa Aguado ◽  
Yanira Méndez ◽  
Sabrina Marsiccobetre ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Rational Design ◽  
Specific Effect ◽  
Dermal Fibroblasts ◽  
Aminopeptidase Activity ◽  
Geometrical Shape ◽  
Starting Point ◽  
Insight Into ◽  
Leucyl Aminopeptidase

Chagas disease, caused by the kinetoplastid parasite Trypanosoma cruzi, is a human tropical illness mainly present in Latin America. The therapies available against this disease are far from ideal. Proteases from pathogenic protozoan have been considered as good drug target candidates. T. cruzi acidic M17 leucyl-aminopeptidase (TcLAP) mediates the major parasite’s leucyl-aminopeptidase activity and is expressed in all parasite stages. Here, we report the inhibition of TcLAP (IC50 = 66.0 ± 13.5 µM) by the bestatin-like peptidomimetic KBE009. This molecule also inhibited the proliferation of T. cruzi epimastigotes in vitro (EC50 = 28.1 ± 1.9 µM) and showed selectivity for the parasite over human dermal fibroblasts (selectivity index: 4.9). Further insight into the specific effect of KBE009 on T. cruzi was provided by docking simulation using the crystal structure of TcLAP and a modeled human orthologous, hLAP3. The TcLAP-KBE009 complex is more stable than its hLAP3 counterpart. KBE009 adopted a better geometrical shape to fit into the active site of TcLAP than that of hLAP3. The drug-likeness and lead-likeness in silico parameters of KBE009 are satisfactory. Altogether, our results provide an initial insight into KBE009 as a promising starting point compound for the rational design of drugs through further optimization.

scholarly journals The cryoelectron microscopy structure of the human CDK-activating kinase

2020 ◽  
Vol 117 (37) ◽  
pp. 22849-22857 ◽  
Author(s):  
Basil J. Greber ◽  
Juan M. Perez-Bertoldi ◽  
Kif Lim ◽  
Anthony T. Iavarone ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rational Design ◽  
Control Cell ◽  
3D Structure ◽  
Structural Basis ◽  
Pol Ii ◽  
Insight Into ◽  
Cdk Activating Kinase

The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II (Pol II) subunit RPB1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell cycle progression. Here, we have determined the three-dimensional (3D) structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and to aid in the rational design of therapeutic compounds.

The Workspace of a General Geometry Planar Three-Degree-of-Freedom Platform-Type Manipulator

1993 ◽  
Vol 115 (2) ◽  
pp. 269-276 ◽  
Author(s):  
G. R. Pennock ◽  
D. J. Kassner
Keyword(s):  
Degree Of Freedom ◽  
Arbitrary Orientation ◽  
End Effector ◽  
The Absolute ◽  
Position Problem ◽  
General Geometry ◽  
Three Degree Of Freedom ◽  
Insight Into

This paper focuses on the direct workspace problems of a general geometry fully-parallel-actuated, planar three-degree-of-freedom platform-type manipulator. A set of equations are presented that determine the workspace as a function of the platform orientation. The formulation is governed by the solution to the inverse position problem of the manipulator. The reachable positions of the end-effector point, for a specified platform orientation, are analyzed. To illustrate the concepts, a practical example is included where the end-effector is required to move a cup filled with water. Then the platform orientation, for a specified location of the end-effector point, is studied. If an arbitrary orientation is possible, the specified location of the end-effector point is said to be within the primary workspace. The paper includes a detailed discussion of the total primary workspaces of the manipulator. The approach adopted here is to regard the manipulator as a combination of three planar, three-revolute open chains. For the sake of completeness, the influence of special manipulator geometry on the workspace is also discussed. Finally, the paper includes the conditions that cause stationary configurations of the manipulator. Insight into these undesirable configurations is provided by a study of the location of the absolute instant center of the platform.

scholarly journals GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

2009 ◽  
Vol 420 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Helge C. Dorfmueller ◽  
Vladimir S. Borodkin ◽  
Marianne Schimpl ◽  
Daan M. F. van Aalten
Keyword(s):  
Active Site ◽  
Cell Biology ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Acetyl Group ◽  
Nanomolar Range ◽  
Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

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