Eric B. Williams

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

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, CO(2) 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 CO(2)-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 (epsilon-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 CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.

The Structure of beta-carbonic anhydrase from the carboxysomal shell reveals a distinct subclass with one active site for the price of two.

CsoSCA (formerly CsoS3) is a bacterial carbonic anhydrase localized in the shell of a cellular microcompartment called the carboxysome, where it converts \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} to CO2 for use in carbon fixation by ribulose-bisphosphate carboxylase/oxygenase (RuBisCO). CsoSCA lacks significant sequence similarity to any of the four known classes of carbonic anhydrase (α, β, γ, or δ), and so it was initially classified as belonging to a new class, ϵ. The crystal structure of CsoSCA from Halothiobacillus neapolitanus reveals that it is actually a representative member of a new subclass of β-carbonic anhydrases, distinguished by a lack of active site pairing. Whereas a typical β-carbonic anhydrase maintains a pair of active sites organized within a two-fold symmetric homodimer or pair of fused, homologous domains, the two domains in CsoSCA have diverged to the point that only one domain in the pair retains a viable active site. We suggest that this defunct and somewhat diminished domain has evolved a new function, specific to its carboxysomal environment. Despite the level of sequence divergence that separates CsoSCA from the other two subclasses of β-carbonic anhydrases, there is a remarkable level of structural similarity among active site regions, which suggests a common catalytic mechanism for the interconversion of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} and CO2. Crystal packing analysis suggests that CsoSCA exists within the carboxysome shell either as a homodimer or as extended filaments.

CO2 Fixation Kinetics of Halothiobacillus neapolitanus Mutant Carboxysomes Lacking Carbonic Anhydrase Suggest the Shell Acts as a Diffusional Barrier for CO2

The widely accepted models for the role of carboxysomes in the carbon-concentrating mechanism of autotrophic bacteria predict the carboxysomal carbonic anhydrase to be a crucial component. The enzyme is thought to dehydrate abundant cytosolic bicarbonate and provide ribulose 1.5-bisphosphate carboxylase/oxygenase (RubisCO) sequestered within the carboxysome with sufficiently high concentrations of its substrate, CO2, to permit its efficient fixation onto ribulose 1,5-bisphosphate. In this study, structure and function of carboxysomes purified from wild type Halothiobacillus neapolitanus and from a high CO2-requiring mutant that is devoid of carboxysomal carbonic anhydrase were compared. The kinetic constants for the carbon fixation reaction confirmed the importance of a functional carboxysomal carbonic anhydrase for efficient catalysis by RubisCO. Furthermore, comparisons of the reaction in intact and broken microcompartments and by purified carboxysomal RubisCO implicated the protein shell of the microcompartment as impeding diffusion of CO2 into and out of the carboxysome interior.

Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome

The carboxysome is a bacterial organelle that functions to enhance the efficiency of CO2 fixation by encapsulating the enzymes ribulose bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. The outer shell of the carboxysome is reminiscent of a viral capsid, being constructed from many copies of a few small proteins. Here we describe the structure of the shell protein CsoS1A from the chemoautotrophic bacterium Halothiobacillus neapolitanus. The CsoS1A protein forms hexameric units that pack tightly together to form a molecular layer, which is perforated by narrow pores. Sulfate ions, soaked into crystals of CsoS1A, are observed in the pores of the molecular layer, supporting the idea that the pores could be the conduit for negatively charged metabolites such as bicarbonate, which must cross the shell. The problem of diffusion across a semiporous protein shell is discussed, with the conclusion that the shell is sufficiently porous to allow adequate transport of small molecules. The molecular layer formed by CsoS1A is similar to the recently observed layers formed by cyanobacterial carboxysome shell proteins. This similarity supports the argument that the layers observed represent the natural structure of the facets of the carboxysome shell. Insights into carboxysome function are provided by comparisons of the carboxysome shell to viral capsids, and a comparison of its pores to the pores of transmembrane protein channels.

Characterization of the Carboxysomal Carbonic Anhydrase CsoSCA from Halothiobacillus neapolitanus

In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-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, CO(2), 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 CO(2) 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 CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) 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.

Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component

The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

Measurable and Influential Parameters That Influence Corrosion Performance Differences between Multiwall Carbon Nanotube Coating Material Combinations and Model Parent Material Combinations Derived from Epoxy-Amine Matrix Materials

Protective coatings are often erroneously thought of as perfect environmental barriers for metal substrates; however, a host of corrosion inducing environmental contaminants permeate through defect-free coatings. Carbon nanotubes are high aspect ratio nanofillers with unique mechanical, electrical, and polymer interaction properties with well-established yet, for practical reasons, often unrealized potential. The research objective was to quantify and understand the influential effects and relationships between low concentration levels of multiwall carbon nanotubes (MWCNT) dispersed into epoxy-amine matrix materials and the different water hydrogen bonding interactions on corrosion rates of steel substrates. We hypothesize that when water directly hydrogen bonds with polymer, substrate and/or MWCNTS, the localized waters capacity to transfer environmental contaminants through the coating, i.e., to and from the substrate, diminishes due to a reduced potential to contribute to the formation of water hydration shells and therefore aid in diminishing the corrosion rate. We measured the absolute pre-exposure water content, and monitored to delineate between the ratio and shifting ratio of in situ free versus bound water hydrogen bonding interactions at the coating/air interface using ATR-FTIR spectroscopy in a 5% NaCl fog environment in an attempt to correlate these differences with experimental corrosion rates. Free water content was reduced from ∼20% to <1% of the total water concentration when 1.0 wt % MWCNTs was dispersed into the parent polymer network. Concurrently, the bound water content was measured to shift from ∼2% to >80% with the same MWCNT concentration. The MWCNT bound water resulted in 25% less corrosion for the same steel substrates albeit the measured water vapor diffusivity was the same for each material combination evaluated. Interestingly, the measured pre-exposure bound water content was predictive of which material would corrode slowest and fastest, i.e., the ratio of starting water states seems to be mechanistically related to the corrosion process and the values have potential to predict corrosion rates for a variety of samples evaluated.

Understanding Scribe Profile and Tool Type Effects on Visual Corrosion Assessments

Visual analysis is the most common evaluation of accelerated corrosion performance after exposure using a scribed protective coating on a metal substrate. Although the scribed-coating visual inspection is standardized, the specificity of initial conditions relative to the final performance has never been unified between laboratories, scribing tools, nor by published testing standards. To validate the initial state of the coating performance based on the selected tool, salt spray corrosion tests ASTM B117 and GM14872 were performed on 60 epoxy-amine primer coated UNS 10080/10100 steel test panels with nine different scribes (3 test panels each with 3 non-scribed control panels). The width of the initial scribe greatly impacted the visual corrosion assessment of the test results during ASTM B117, while the initial scribe width had little impact during GM14872. These results suggest that correlating the initial scribe with the final corrosion evaluation of the coating defect resulted in a better inter-comparable evaluation of coatings subjected to visual analysis by accelerated corrosion or electrolyte corrosion.

A rapid quantitative protocol for measuring carbon nanotube degree of dispersion in a waterborne epoxy–amine matrix material

The available literature makes it very clear that accurate measurements of carbon nanotube dispersion quality are very complicated and the typical characterization is neither simple nor reliable. Most methods to quantify carbon nanotube dispersion reported in the literature require investigator-chosen assumptions or software interpretations that are impractical at best and misleading at worst for facile application. Herein, we report on the use of visible light absorption-based method(s) and validate that these were quantitative for discerning dispersibility differences for MWCNTs with three distinct surface chemistry modifications and concentration levels blended with polymeric materials. Ultimately, the dispersion quality was quantified via the trendline slope of the thickness-normalized absorbance values as a function of MWCNT concentration. Extremely poor dispersions were represented by statistically insignificant slope trendlines. Our data revealed that hydroxyl surface modification increased MWCNT dispersibility by a factor of ~2.8 and ~2.6 compared to the as-received MWCNT formulations via the absorption and the blackness methods, respectively. These results support and quantifiably validate that simple optical blackness values directly measured the degree of dispersion for MWCNTs in coatings applied to substrates, and our data support that this is a simple and effective quality control metric.