Living where nobody wants to live and how to do it

October 24, 2011

Always have been interested in what it would be like to live inside a hot and poisonous volcano? Then this is something for you:

In the following I want to describe a paper which was published by, among others, members of the Department of Microbiology at Radboud University Nijmegen where I did my BSc internship. The team of researchers was headed by Marjan Smeulders from Nijmegen and Thomas Barends working at the Max Planck Institute for Medical Research in Heidelberg. I am glad that their work on a archeal CS2 hydrolase ended up in a fine piece of work in Nature just last week. This enzyme is essential part of the energy metabolism of a certain species of archeon which was originally extracted from a volcanic region near Naples, Italy. We are thus surely talking about an extremophilic organism here! Next to the evolutionary and ecological implications of the paper, I am also fascinated by the wide range of genetic, biochemical, computational and biophysical methods which they used. I will not describe these methods here, but rather concentrate on the results and the conclusions which can be drawn from these results. For more information please refer to the actual paper which is named at the end.

I will begin with an introduction of the 3D structure of CS2 hydrolase, which is essential to the paper. Later I will also describe the functional consequences that the evolution of this enzyme had over time. Enjoy!

Fig. 1: X-ray chrystallographic structure of an archeal CS2 hydrolase and overview of the tertiary and quaternary structure of the enzyme. A more detailed description can be found in the text.

In the top left corner the basic dimer structure of the enzyme is depicted. Due to interactions of the N- and C-terminal tails of the monomeric amino acid chains four dimers can combine to assemble a square shaped octamer, which consists out of eight parts in total (four dimers, consisting out of two basic amino acid chains each). This construct is displayed in the top right corner where the colors denote the eight individual basic parts. The bottom left corner shows the final so-called quaternary structure of the hydrolase enzyme which consists out of two intertwined octameric (eight pieced) square “rings”. When summing up the total enzyme is thus a hexadecameric complex which is constructed out of sixteen amino acid chains (primary structure) each possessing a N- and C-terminus. The graph in the bottom right corner is the result of a small-angle X-ray scattering (SAXS) experiment. It shows the X-ray intensity (y-axis) plotted against the scattering angle (x-axis) of a sample containing all bigger and smaller pieces described above. Since the resolution of this method is relatively low, the resulting total scattering curve has to be matched to more accurate sub X-ray or NMR curves of the structures of the individual parts which are in the sample. The scattering of the X-rays can thus give first clues about whether the  tertiary (eight pieced in this case) or the quaternary structure (sixteen pieced) is predominant in a given sample. In this case the red line describes a mix of 83% of the sixteen pieced total structure and 17% of the smaller eight pieced subpart.

Fig. 2: Model of the CS2 hydrolase active site and the channel structure which defines the nature of the molecules which can gain access to the enzymes catalytic site. Again, a more detailed explanation can be found in the text.

The left part of Fig. 2 pictures a detailed model of the active site of the enzyme. One histidine and two cysteine amino acids can clearly be seen. The physiochemical characteristics of these three active site residues hold the catalytic zinc ion in place which is essential for the hydrolysis of CS2 into H2S and CO2. The blue, orange and green cages around the molecules and atoms represent the electron density which confirms the positions of the compounds within the structure. The single zinc ion could be located because its electron density is significantly different from its surroundings. On the right half of Fig. 2 the active site is pictured again, but now more in relation to the overall structure. This enzyme structure allows only one single passage way which compounds can take in order to arrive at the catalytic site (red circle) and is called “tunnel” (green). Large non-polar and uncharged amino acids like the predominant phenylalanine give this tunnel very hydrophobic characteristics which together with the diameter dramatically restricts the amount of compounds which could theoretically reach the catalytic site. In red, blue and grey the association of the hydrophobic amino acids to the different monomers (Fig. 1) is described.

Fig. 3: Mutational sites in the tunnel leading to the active site and the effects of these respective mutations on the hydrolases reaction speed and affinity.

In order to check whether the size and characteristics of the tunnel really influence the catalytic site in terms of specificity or this is purely based on the build-up of the active site itself, seven different mutations where one at a time introduced into the tunnel. The right-handed portion of Fig. 3 denotes the position and sort of these mutations. From the graph on the left it can be concluded that mutations which introduce smaller amino acids or delete one and thus lead to a widening of the tunnel, also lead to increased reaction speed. Inversely, obstructing the tunnel by larger-than-normal amino acids or blocking the active site (position 78) decreases the ability of the enzyme to process its substrates.

In addition a genetic comparison of the present enzyme with related enzymes that catalyze different or the same chemical reactions had shown that the active site of all of these enzymes is relatively conserved (for example between carbonic anhydrase and CS2 hydrolase). The specificity of the active site itself thus seems to be low and the activity universal. So the specificity of such a CS2 hydrolase must originate from somewhere else.

With this paper the authors deliver a possible answer to this question and bring up more evidence that nature (evolution) does not have to change active sites at high costs all the time, but that it is also possible to modify quaternary structures of enzymes in order to make them more specific for another compound which might be more favorable for an organism. By modifying non-catalytic  site residues (tunnel residues in this case) and allowing the passage of other chemical compounds it is therefore possible redesign the functional properties of an enzyme.

Smeulders et al. Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archeon. Nature 478, 412-416 (2011).

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