Pyrrolysine – the 22nd amino acid

November 22, 2011

For an amino acid, which are one of the building blocks of life, pyrrolysine was discovered quite recently. Just in 2002 it was discovered by mass spectrometric analysis and chrystallographic approaches. Until present pyrrolysine has been found in some enzymes of the methanogenic pathway called MtmB, MtbB, and MttB of archea belonging to the order of Methanosarcinales and two bacterial species. This rare amino acid is just regularly encoded by the natural genetic code. UAG, which is normally an amber-type stop codon on mRNA and stops translation, is the nucleotide code for pyrrolysine. Only a special nucleotide composition which surrounds the UAG triplets allows the interaction of the pyrrolysine carrying tRNA with its codon. In most other organisms this “exception from the rule” is not possible since the chemical forces of UAG do not allow tRNA binding of any kind. UAG thus functions as stop codon in the majority of all organisms. As its name already suggests it is similar to lysine and features a pyrroline ring at the lysine side chain (Fig. 1).

Fig 1: Chemical structure of pyrrolysine. A pyrrol ring is added to the lysine (right part of molecule) side chain (stretching from NH2 to NH).

Two genes are required in order to make out of the UAG stop codon a “normal” amino acid encoding codon. These genes are called pylT and pylS. Respectivly they encode a fitting, but unusual tRNA (of course with the antisense codon CUA) and an aminoacyl-tRNA synthetase which loads the pylT encoded tRNA with pyrrolysine. As a third requirement and as mentioned before also the nucleotide environment around UAG is important. Next to the two required genes also a special, so-called pyrrolysine insertion sequence (PYLIS sequence), which lies downstream of  UAG seems to be essential. Within the mRNA this sequence forms a stemloop and confers structural stability during tRNA binding.

After these genetic and molecular biology questions were (at least partly) answered, one question remained: How is pyrrolysine itself synthesized by the cell? Which pathway is employed and which enzymes take part in the catalyzed reactions?

A few weeks ago researchers from the Technical University of Munich shed some light on the biochemistry of pyrrolysine biosynthesis. In the following I want to explain briefly what they found out. However I want to keep it quite simple and will not mention which molecular clusters within the enzymes active site initiate and facilitate the electron transfer in order to catalyze pyrrolysine formation. Interested readers are referred to the original publication (1).

The researchers started out by overexpression of one of the genes which lies in the gene cluster which was already known to be responsible for pyrrolysine synthesis. Also a reaction pathway for the biosynthesis of pyrrolysine had been proposed by other researchers (2). Fig. 2 presents this pathway.

Fig. 2: Proposed route of pyrrolysine biosynthesis. In addition the names of the enzymes are mentioned which catalyze the reaction steps (adapted from (1), originally proposed by (2)).

Starting out from this foundation the TUM researchers purified the first step catalyzing PylB protein and determined its structure by X-ray crystallography. Fig. 3 depicts the result of this approach. A S-adenosylmethionine (SAM) cofactor, an iron-sulfur cluster, and the reaction product of the enzyme are found in the central cavity of the structure which at the same time seems to form the active site of the enzyme. To find a reaction product within the enzyme is actually a surprise since it tells a lot about the mechanisms by which the enzyme works. In this case the molecule number 2 from Fig. 2 was detected. The researchers concluded that this was really the catalysis product of the enzyme since impurities could be excluded. The compound is no metabolite of E. coli metabolism and was also not part of any of the buffers that was used during purification and structure determination. In addition the reaction had to be bound very tightly in order to “survive” the three-step purification and crystallization procedure. This fact also supported the catalysis product theory. Since the quality of the model’s active site region was quite good, it was possible to virtually insert a 3D molecular model of PylB’s substrate (lysine, Fig. 1 without the ring structure) into the cavity which helped to develop an actual model of how PylB catalyzes the first step of pyrrolysine biosynthesis. Knowledge of organic chemistry led the researchers to the possibility that abstraction of a hydrogen ion from one special position of the substrate leads to the formation of a radical (atom with one free electron, very reactive). This process does not occur spontaneously, but requires the action of PylB’s active site. Otherwise the energy barrier to abstract the hydrogen would be too high. Upon radical formation an isomerization occurs and the essential relocation of the methyl group (CH2) can occur which gives the reaction product its final structure. This sequential reaction probably only occurs because the substrate is located in close proximity to the SAM element within the active site which catalyzes the first step of the reaction.

Fig. 3: Stereoview of the crystal structure of PyB, the enzyme which catalyzes the first pyrrolysine biosynthesis step. The same enzyme is depicted on both sides, but stereoview is applied to enhance the 3D illusion without wearing special glasses. Important to note are the chemical groups which are present in the hypothetical active center of the enzyme. They can be seen in the top part of the enzyme. The SAM subunit is located between an iron-sulfur cluster and the reaction product which the enzyme delivers (compound 2 in Fig. 2). SAM together with the iron-sulfur cluster seem to initiate the catalyzing reaction (starting with regular lysine) by hydrogen abstraction and subsequent radical formation (adapted from (1)).

In addition the researchers also describe the potential dynamics of PylB which would enable it to release the reaction product. However, the proposed movement of a certain amino acid helix structure is only hypothetical. Still it is remarkably how this study, which combines molecular and structural biology with organic chemistry, elucidated a major mechanism behind the existence of the 22nd amino acid. The researchers also stress the importance of their work in the cadre of protein synthesis with non-natural amino acid substitutions for which the unusual incorporation method of pyrrolysine into proteins (described in the beginning) might be a suitable model.

(1) Quitterer, F., List, A., Eisenreich, W., Bacher, A. and Groll, M. (2011), Crystal Structure of Methylornithine Synthase (PylB): Insights into the Pyrrolysine Biosynthesis. Angewandte Chemie International Edition. doi: 10.1002/anie.201106765

(2) Gaston, M.A., Zhang, L., Green-Church, K.B. and Krzycki, J.A. (2011), The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine. Nature 471, 647-650. doi:10.1038/nature09918

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