Crystal structure of pentameric shell protein CsoS4B of Halothiobacillus neapolitanus α-carboxysome.

Carboxysome, encapsulating an enzymatic core within an icosahedral-shaped semipermeable protein shell, could enhance CO2 fixation under low CO2 conditions in the environment. The shell of Halothiobacillus neapolitanus α-carboxysome possesses two 38% sequence-identical pentameric proteins, namely CsoS4A and CsoS4B. However, the functions of two paralogous pentameric proteins in α-carboxysome assembly remain unknown. Here we report the crystal structure of CsoS4B at 2.15 Šresolution. It displays as a stable pentamer, each subunit of which consists of a ß-barrel core domain, in addition to an insertion of helix α1 that forms the central pore. Structural comparisons and multiple-sequence alignment strongly indicate that CsoS4A and CsoS4B differ from each other in interacting with various components of α-carboxysome, despite they share a similar overall structure. These findings provide the structural basis for further investigations on the self-assembly process of carboxysome.

Engineering a thermostable Halothermothrix orenii ß-glucosidase for improved galacto-oligosaccharide synthesis.

Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining ß-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a ß-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (-1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent k cat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower k cat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

Modularity of a carbon-fixing protein organelle.

Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO(2)-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO(2). Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO(2) in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures.

Uncovering the roles of the scaffolding protein CsoS2 in mediating the assembly and shape of the α-carboxysome shell.

Carboxysomes are proteinaceous organelles featuring icosahedral protein shells that enclose the carbon-fixing enzymes, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), along with carbonic anhydrase. The intrinsically disordered scaffolding protein CsoS2 plays a vital role in the construction of α-carboxysomes through bridging the shell and cargo enzymes. The N-terminal domain of CsoS2 binds Rubisco and facilitates Rubisco packaging within the α-carboxysome, whereas the C-terminal domain of CsoS2 (CsoS2-C) anchors to the shell and promotes shell assembly. However, the role of the middle region of CsoS2 (CsoS2-M) has remained elusive. Here, we conducted in-depth examinations on the function of CsoS2-M in the assembly of the α-carboxysome shell by generating a series of recombinant shell variants in the absence of cargos. Our results reveal that CsoS2-M assists CsoS2-C in the assembly of the α-carboxysome shell and plays an important role in shaping the α-carboxysome shell through enhancing the association of shell proteins on both the facet-facet interfaces and flat shell facets. Moreover, CsoS2-M is responsible for recruiting the C-terminal truncated isoform of CsoS2, CsoS2A, into α-carboxysomes, which is crucial for Rubisco encapsulation and packaging. This study not only deepens our knowledge of how the carboxysome shell is constructed and regulated but also lays the groundwork for engineering and repurposing carboxysome-based nanostructures for diverse biotechnological purposes. IMPORTANCE: Carboxysomes are a paradigm of organelle-like structures in cyanobacteria and many proteobacteria. These nanoscale compartments enclose Rubisco and carbonic anhydrase within an icosahedral virus-like shell to improve CO2 fixation, playing a vital role in the global carbon cycle. Understanding how the carboxysomes are formed is not only important for basic research studies but also holds promise for repurposing carboxysomes in bioengineering applications. In this study, we focuses on a specific scaffolding protein called CsoS2, which is involved in facilitating the assembly of α-type carboxysomes. By deciphering the functions of different parts of CsoS2, especially its middle region, we provide new insights into how CsoS2 drives the stepwise assembly of the carboxysome at the molecular level. This knowledge will guide the rational design and reprogramming of carboxysome nanostructures for many biotechnological applications.

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme.

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.

Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation.

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism by facilitating high CO2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.

Deciphering molecular details in the assembly of alpha-type carboxysome.

Bacterial microcompartments (BMCs) are promising natural protein structures for applications that require the segregation of certain metabolic functions or molecular species in a defined microenvironment. To understand how endogenous cargos are packaged inside the protein shell is key for using BMCs as nano-scale reactors or delivery vesicles. In this report, we studied the encapsulation of RuBisCO into the α-type carboxysome from Halothiobacillus neapolitan. Our experimental data revealed that the CsoS2 scaffold proteins engage RuBisCO enzyme through an interaction with the small subunit (CbbS). In addition, the N domain of the large subunit (CbbL) of RuBisCO interacts with all shell proteins that can form the hexamers. The binding affinity between the N domain of CbbL and one of the major shell proteins, CsoS1C, is within the submicromolar range. The absence of the N domain also prevented the encapsulation of the rest of the RuBisCO subunits. Our findings complete the picture of how RuBisCOs are encapsulated into the α-type carboxysome and provide insights for future studies and engineering of carboxysome as a protein shell.

The murein of Thiobacillus neapolitanus.

Amino acid analysis of pure murein isolated from cells of T. neapolitanus revealed the typical constituents of most mureins form Gram-negative bacteria. i.e. glutamic acid, alanine and diaminopimelic acid, but the molecular ratio ot these was unusual, being approximately 1: 1: 1. The reduced amount of alanine was explained by the absence of monomers containing tetrapeptide side chains, as revealed by h. p. 1. c. analysis, [(3)H]glutamic acid, [(3)H]diaminopimelic acid and [(3)H]N-acetylglucosamine were incorporated into the murein and allowed to determine the degree of its crosslinkage (28%) and the occurrence of turnover.