Now and then you stumble upon a straightforward self-explaining stereotypical paper. In this case it bears the clear name “Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon”(Smeulders et al., 2011) and written by the Microbiology department of Radboud University Nijmegen in The Netherlands. It extensively describes how a hydrolase (which converts CS2 to H2S and CO2) was likely to be evolved from a β-carbonic anhydrase (which converts CO2 in HCO3–). But the work preceding this conclusion and the enzyme itself are actually much more interesting…
It al started in an old sauna in the picturesque city of Naples. Here the strain Acidianus A1-3 was isolated and brought back to the lab. Here it was cultured at 70°C by bubbling the highly toxic CS2 gas through the media. They showed that the cell extract of the Acidianus archaeon was able to convert the gas CS2 into the gas responsible for the rotten egg smell; hydrogen sulfide (H2S). Then the use of classical ion exchange chromatography was used to separate the enzyme from the cell extract. By using trypsin digestion and a mass spectrometer the peptide sequence was resolved. Based on the amino acid sequences primers were designed to amplify the original DNA from the Acidianus by using degenerate PCR. Resulting in the 615 base pair DNA sequence of the carbon disulfide hydrolase. Writing this down only takes 4 sentences, the description in the supplementary information already took 3 pages, imaging how much time it took to conduct this work.
The following two reactions are believed to be catalyzed by the CS2 hydrolase from the Acidianus strain:
CS2 + H2O -> COS + H2S
COS + H2O -> CO2 + H2S
COS is the abbreviation of carbonyl sulfide, obviously with the carbon in the middle, so formally written as OCS. COS plays an role in the formation of peptides form single amino acids (Leman, Orgel, & Ghadiri, 2004) and thus a key element in the “origin of life”)
The β-carbonic anhydrase enzyme (for example from the Pisum sativum) catalyzes the following reaction:
CO2 + H2O -> HCO3 -+ H+
With the DNA sequence in hand the work horse of to days biotechnology can be used; E. coli. The gene of the CS2 hydrolase was transformed into E. coli and the purified proteins were crystallized. This resulted in a very interesting and unique crystal structure (Figure 1). The fold is called a catenane of dimer of dimer of dimers; are you still with me? First back to the basic biochemistry: the CS2 hydrolase is carrying out a reaction which is quite homologues with the hydration of CO2 to HCO3– by carbonic anhydrases. Quantum chemical calculations showed that CS2 could also be converted by carbonic anhydrases. If we check out the crystal structure of a β-carbonic anhydrase of the Pisum sativum (Kimber & Pai, 2000), the plant responsible in daily life for producing pea’s, we already can conclude it is quite an interesting structure (Figure 1). The authors call the anhydrase of the pea plant an “octamer with a novel dimer of dimers of dimers arrangement”. Indeed the biologic assembly of this pea protein shows a compact interlocking octamer with 8 active sites and a small hole in the middle.
Now back to the CS2 hydrolase from the sauna in Naples. SAXS measurement and ultracentrifugation already showed this protein is in a hexadecamer (16 proteins making up the biological unit) state. So simple math says 2 x 8 = 16, but how is this math done in 3D? Well the solution is quite elegant, remember the hole in the octamer? In the hexadecamer this hole is used to interlock the two octamers as depicted in Figure 2, this is called a catenane. So the final assembly is a catenane of dimer of dimer of dimers. The authors claim they were only able to find two occurrences of known natural catenane proteins (bovine mitochondrial peroxiredoxin III and the gp5 capsid protein of bacteriophage HK97). On the other hand, synthetic catenanes are not uncommon (Blankenship & Dawson, 2003).
The question arises why this protein prevails itself in such an elegant appearance. The authors can only speculate but they think a catenane assembly results in a more tight packing of the protein and thus a higher density of active sites can be achieved. This is advantages given the gaseous nature of the COS intermediate, thus there is more change of converting the intermediate before it diffuses away. Another result of this catenane assembly of the CS2 hydrolase is the active sites are now only become accessible by a 15 Å long hydrophobic tunnel compared to β-carbonic anhydrase where the active site is readily available (for cool pictures check out the original paper). This makes sense since CS2 is an order of magnitude more hydrophobic then CO2 (the substrate used by the octomeric β-carbonic anhydrase). So in this case the catenane assembly gives rise to a specificity filter: an nice example of evolution taking place at the quaternary structure level instead of at primary level by an active site mutation.
Too bad this article only got cited 5 times…
Check out the article (Smeulders et al., 2011) if you want to read more on how the CS2 hydrolase evolved from the β-carbonic anhydrase and with which straightforward experiments they showed the importance of this 15 Å long hydrophobic tunnel.
Smeulders, M. J., Barends, T. R. M., Pol, A., Scherer, A., Zandvoort, M. H., Udvarhelyi, A., Khadem, A. F., et al. (2011). Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon. Nature, 478(7369), 412–6. doi:10.1038/nature10464 (Featured article)
Blankenship, J. W., & Dawson, P. E. (2003). Thermodynamics of a Designed Protein Catenane. Journal of Molecular Biology, 327(2), 537–548. doi:10.1016/S0022-2836(03)00115-3
Kimber, M. S., & Pai, E. F. (2000). The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases. The EMBO journal, 19(7), 1407–18. doi:10.1093/emboj/19.7.1407
Leman, L., Orgel, L., & Ghadiri, M. R. (2004). Carbonyl sulfide-mediated prebiotic formation of peptides. Science (New York, N.Y.), 306(5694), 283–6. doi:10.1126/science.1102722