Structural and inhibition insights into carbonic anhydrase CDCA1 from the marine diatom Thalassiosira weissflogii

Carbonic anhydrases (CAs) catalyze with high efficiency the reversible hydration of carbon dioxide, an essential reaction for many biological processes, such as photosynthesis, respiration, renal tubular acidification, and bone resorption. Diatoms, which are one of the most common types of phytoplankton and are widespread in oceans, possess CAs fundamental for acquisition of inorganic carbon. Recently, in the marine diatom Thalassiosira weissflogii a novel enzyme, CDCA1, naturally using Cd in its active site, has been isolated and categorized in a new CA class, namely zeta-CA. This enzyme, which consists of three repeats (R1, R2 and R3), is a cambialistic carbonic anhydrase that can spontaneously exchange Zn or Cd at its active centre, presumably an adaptative advantage for diatoms that grow fast in the metal-poor environment of the surface ocean. In this paper we completed the characterization of this enzyme, reporting the X-ray structure of the last repeat, CDCA1-R3 in its cadmium-bound form, and presenting a model of the full length protein obtained by docking approaches. Results show that CDCA1 has a quite compact not symmetric structure, characterized by two covalently linked R1-R2 and R2-R3 interfaces and a small non-covalent R1-R3 interface. The three dimensional arrangement shows that most of the non-conserved aminoacids of the three repeats are located at the interface regions and that the active sites are far from each other and completely accessible to the substrate. Finally, a detailed inhibition study of CDCA1-R3 repeat in both cadmium- and zinc- bound form has been performed with sulfonamides and sulfamates derivatives. The results have been compared with those previously reported for other CA classes, namely alpha- and beta-classes, and correlated with the structural features of these enzymes.     

Regulation of carbonic anhydrase expression by zinc, cobalt, and carbon dioxide in the marine diatom Thalassiosira weissflogii

TWCA1 is the major Zn-requiring isoform of carbonic anhydrase (CA) in the marine diatom Thalassiosira weissflogii. We have examined the roles that trace metals and CO(2) play in the regulation of TWCA1 expression over ranges of concentrations that bracket those encountered in the marine environment. Both steady-state levels of TWCA1 and the kinetics of induction were measured by western analysis. TWCA1 levels correlated well with cellular CA activity levels. TWCA1 was induced at a low CO(2) concentration but the level of induction, as determined by western analysis, was dependent on the availability of Zn. Co effectively substituted for Zn in regulating TWCA1 expression and promoting TWCA1 activity. Upon shift from low to high CO(2), the concentration of TWCA1 decreased. The expression of TWCA1 is diel cycle regulated, and cellular TWCA1 decreased during the dark phase. These results provide the basis for studying the expression of CA in field populations and, taken together with previous radiolabeling studies, provide strong evidence of in vivo metal substitution of Co for Zn in a CA. Our data also support the conclusion that TWCA1 plays a central role in carbon acquisition in T. weissflogii.     

The R-state proinsulin and insulin hexamers mimic the carbonic anhydrase active site

The cobalt(II)-substituted proinsulin and insulin hexamers have been studied in solution via electronic absorption spectroscopy. Hexameric proinsulin is shown to undergo the phenol-induced T6 to R6 conformational transition in a manner analogous to that previously established for insulin. In the absence of coordinating anions, the coordination spheres of the Co(II) ions in the proinsulin and insulin R6 hexamers comprise identical pseudotetrahedral arrangements of 3 histidine residues and 1 hydroxide ion. At alkaline pH, the visible absorption spectrum of the phenol-induced R6 Co(II) center is strikingly similar to the distinctive spectrum of the alkaline form of Co(II)-carbonic anhydrase. Exogenous ligands may coordinate to the Co(II) ions of the R6 proinsulin and insulin hexamers via replacement of the hydroxide ion, forming pseudotetrahedral adducts possessing characteristic spectra. The binding affinity of such ligands is shown to be strongly pH-dependent. The data presented establish that, although the Co(II)-substituted proinsulin and insulin R6 hexamers lack enzyme-like activity, these species duplicate spectrochemical characteristics of the Co(II)-carbonic anhydrase active site that are believed to be important signatures of carbonic anhydrase catalytic function.     

Entrapment of carbon dioxide in the active site of carbonic anhydrase II

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO(2)-pressurized, cryo-cooled crystals to capture the first step of CO(2) hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO(2), for both the holo and the apo (without zinc) enzyme to 1.1A resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO(2) and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675-3679). These structures provide insight into the long hypothesized binding of CO(2) in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO(2). This method may also have a much broader implication for the study of other enzymes for which CO(2) is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.     

Kinetic analysis of multiple proton shuttles in the active site of human carbonic anhydrase

We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His(7) in HCA III. Using an (18)O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His(7) and His(64) within the active site cavity, shows the conformation of the side chain of His(64) moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His(7). This suggests a cause of decreased proton transfer in catalysis.     

Role of hemoglobin in proton transfer to the active site of carbonic anhydrase.

The binding of bovine oxyhemoglobin to bovine carbonic anhydrase with a dissociation constant between 10(-5) and 10(-7) M has been determined by countercurrent distribution using aqueous, biphasic polymer systems. This result provides an explanation for the very efficient proton transfer between hemoglobin and carbonic anhydrase, a transfer which enhances the catalytic activity of carbonic anhydrase as measured by 18O exchange between bicarbonate and water at chemical equilibrium (Silverman, D. N., Tu, C. K., and Wynns, G. C. (1978) J. Biol. Chem, 253, 2563-2567). Two rate constants describing 18O exchange activity of carbonic anhydrase at pH 7.5 show saturation behavior when plotted against hemoglobin concentration consistent with a dissociation constant of 2.5 X 10(-6) M between bovine hemoglobin and carbonic anhydrase. Interpretation of these rate constants in terms of a two-step model for 18O exchange indicates that hemoglobin enhances the rate of exchange from carbonic anhydrase of water containing the oxygen abstracted from bicarbonate, but does not affect the catalytic interconversion of CO2 and HCO3- at chemical equilibrium.     

A novel carbonic anhydrase II binding site regulates NHE1 activity

Carbonic anhydrase II (CAII) binds to and regulates transport by the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. We localized and characterized the CAII binding region on the C-terminal tail of the Na(+)/H(+) exchanger. CAII did not bind to acidic sequences in NHE1 that were similar to the CAII binding site of bicarbonate transporters. Instead, by expressing a variety of fusion proteins of the C-terminal region of the Na(+)/H(+) exchanger, we demonstrated that CAII binds to the penultimate group of 13 amino acids of the cytoplasmic tail. Within this region, site-specific mutagenesis demonstrated that amino acids S796 and D797 form part of a novel CAII binding site. Phosphorylation of the C-terminal 26 amino acids by heart cell extracts did not alter CAII binding to this region, but phosphorylation greatly increased CAII binding to a protein containing the C-terminal 182 amino acids of NHE1. This suggested that an upstream region of the cytoplasmic tail acts as an inhibitor of CAII binding to the penultimate group of 13 amino acids. The results demonstrate that a novel phosphorylation-regulated CAII binding site exists in distal amino acids of the NHE1 tail.     

Nanoscale enzyme inhibitors: Fullerenes inhibit carbonic anhydrase by occluding the active site entrance

We investigated a series of derivatized fullerenes possessing alcohol, amine, and amino acid pendant groups as inhibitors of the zinc enzymes carbonic anhydrases (CAs, EC 4.2.1.1). We discovered that fullerenes bind CAs with submicromolar—low micromolar affinity, despite the fact that these compounds do not possess moieties normally associated with CA inhibitors such as the sulfonamides and their isosteres, or the coumarins. The 13 different mammalian CA isoforms showed a diverse inhibition profile with these compounds. By means of computational methods we assessed the inhibition mechanism as being due to occlusion of the active site entrance by means of the fullerene cage (possessing dimension of the same order of magnitude as the opening of the enzyme cavity, of 1 nm). The pendant moieties to the fullerene cage make interactions with amino acid residues from the active site, among which His64, His94, His96, Val121, and Thr200. Fullerenes thus represent a totally new class of nanoscale CA inhibitors which may show applications for targeting physiologically relevant isoforms, such as the dominant CA II and the tumor-associated CA IX.     

Effects of two ionizing groups on the active site of human carbonic anhydrase B.

Investigation of some pH-dependent properties of human erythrocyte carbonic anhydrase B indicate that the active site is influenced by at least two charged groups. The properties studied include the pH dependence of inhibition of native, monocarboxamidomethyl, and monocarboxymethyl enzymes by iodide ion and the pH dependence of the visible spectra of the cobalt derivatives of these enzymes. One ionizing group has a pKa of about 7.3 in the native enzyme, 8.2 in the carboxyamidomethyl enzyme, and 9.0 in the carboxymethyl enzyme. It has a major influence on activity and anion inhibition, and on the visible spectra of the cobalt enzymes. A second group has a pKa of about 6.1 in native and modified enzymes. When zinc is at the active site, the secondary group in its acidic form decreases the Ki for I-. With the carboxyamidomethyl and carboxymethyl enzymes, the Ki decreases by about an order of magnitude. However, if cobalt is substituted for zinc in the modified enzymes, this group does not influence the Ki for I- and the binding of I- does not influence the pKa of the spectral transitions caused by ionization of this secondary group. In the case of nonalkylated Co2+-enzyme, another ionizing group with a pK of about 6.2 prevents the binging of I- at low pH. These results show that the active site is altered when cobalt is substituted for zinc in carbonic anhydrase B.     

A "Reverse burst" active site titration procedure for human carbonic anhydrase B.

Between pH 7 and pH 10.5, p-nitrophenyl $\text{p}$-sulfamyl benzoate (PNP-SAB) binds very strongly to human carbonic anhydrase B (dissociation constant on the order of 10^?9 M or less at pH 7.5), but is not hydrolyzed by the enzyme. Because the binding is essentially stoichiometric under readily accessible conditions, this ester may be used as an active site titrant, by measuring the rapid hydrolysis of excess unbound PNP-SAB catalyzed by an added nucleophile (“reverse burst”).     

An inhibitor-like binding mode of a carbonic anhydrase activator within the active site of isoform II

The 2,4,6-trimethylpyridinium derivative of histamine is an effective activator of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). However, unlike other CA activators, which bind at the entrance of the active site cavity, an X-ray crystal structure of hCA II in complex with the 1-[2-(1H-imidazol-4-yl)-ethyl]-2,4,6-trimethylpyridinium salt evidenced a binding mode never observed before either for activators or inhibitors of this enzyme, with the 2,4,6-trimethylpyridinium ring pointing towards the metal ion deep within the enzyme cavity, and several strong hydrophobic interactions stabilizing the adduct. Indeed, incubation of the activator with the enzyme for several days leads to potent inhibitory effects. This is the first example of a CA activator which after a longer contact with the enzyme behaves as an inhibitor.     

The influence of cell size on the growth rate of Thalassiosira weissflogii

Growth rate and average cell volume were measured throughoutauxospore formation in two populations of Thalassiosira weissflogii(Hustedt). In both cases, the entire population shifted fromrelatively small (800 µm³) to large cells (2800 µm³)over a 5 day interval. This shift was accompanied by a dramaticincrease in the average growth rate of the populations from1.6 to 3.4 doublings/day.     

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump.