Pyrrolysine
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Pyrrolysine (symbol Pyl or O; encoded by the 'amber'
stop codon In molecular biology (specifically protein biosynthesis), a stop codon (or termination codon) is a codon ( nucleotide triplet within messenger RNA) that signals the termination of the translation process of the current protein. Most codons in ...
UAG) is an α-amino acid that is used in the biosynthesis of
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respon ...
s in some
methanogen Methanogens are microorganisms that produce methane as a metabolic byproduct in hypoxic conditions. They are prokaryotic and belong to the domain Archaea. All known methanogens are members of the archaeal phylum Euryarchaeota. Methanogens ar ...
ic archaea and
bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were am ...
; it is not present in humans. It contains an α-amino group (which is in the protonated – form under biological conditions), a
carboxylic acid In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is or , with R referring to the alkyl, alkenyl, aryl, or other group. Carboxyl ...
group (which is in the deprotonated –COO form under biological conditions). Its pyrroline side-chain is similar to that of lysine in being basic and positively charged at neutral pH.


Genetics

Nearly all genes are translated using only 20 standard
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
building blocks. Two unusual genetically-encoded amino acids are selenocysteine and pyrrolysine. Pyrrolysine was discovered in 2002 at the active site of methyltransferase enzyme from a methane-producing archeon, '' Methanosarcina barkeri''. This amino acid is encoded by UAG (normally a stop codon), and its synthesis and incorporation into protein is mediated via the biological machinery encoded by the ''pylTSBCD'' cluster of genes.


Composition

As determined by
X-ray crystallography X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angle ...
and MALDI mass spectrometry, pyrrolysine is made up of 4-methyl pyrroline-5- carboxylate in
amide In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a compound with the general formula , where R, R', and R″ represent organic groups or hydrogen atoms. The amide group is called a peptide bond when it is ...
linkage with the εN of lysine.


Synthesis

Pyrrolysine is synthesized ''in vivo'' by joining two molecules of L-lysine. One molecule of lysine is first converted to (3''R'')-3-methyl-D-ornithine, which is then ligated to a second lysine. An NH2 group is eliminated, followed by cyclization and dehydration step to yield L-pyrrolysine.


Catalytic function

The extra pyrroline ring is incorporated into the active site of several methyltransferases, where it is believed to rotate relatively freely. It is believed that the ring is involved in positioning and displaying the methyl group of methylamine for attack by a corrinoid cofactor. The proposed model is that a nearby
carboxylic acid In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is or , with R referring to the alkyl, alkenyl, aryl, or other group. Carboxyl ...
bearing residue, glutamate, becomes protonated, and the proton can then be transferred to the imine ring nitrogen, exposing the adjacent ring carbon to nucleophilic addition by methylamine. The positively charged nitrogen created by this interaction may then interact with the deprotonated glutamate, causing a shift in ring orientation and exposing the methyl group derived from the methylamine to the binding cleft where it can interact with corrinoid. In this way a net is transferred to the cofactor's
cobalt Cobalt is a chemical element with the symbol Co and atomic number 27. As with nickel, cobalt is found in the Earth's crust only in a chemically combined form, save for small deposits found in alloys of natural meteoric iron. The free element, ...
atom with a change of oxidation state from I to III. The methylamine-derived
ammonia Ammonia is an inorganic compound of nitrogen and hydrogen with the formula . A stable binary hydride, and the simplest pnictogen hydride, ammonia is a colourless gas with a distinct pungent smell. Biologically, it is a common nitrogeno ...
is then released, restoring the original imine.


Genetic coding

Unlike posttranslational modifications of lysine such as hydroxylysine, methyllysine, and hypusine, pyrrolysine is incorporated during
translation Translation is the communication of the Meaning (linguistic), meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The ...
( protein synthesis) as directed by the
genetic code The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...
, just like the
standard amino acid Proteinogenic amino acids are amino acids that are incorporated biosynthetically into proteins during translation. The word "proteinogenic" means "protein creating". Throughout known life, there are 22 genetically encoded (proteinogenic) amino aci ...
s. It is encoded in mRNA by the UAG codon, which in most organisms is the 'amber'
stop codon In molecular biology (specifically protein biosynthesis), a stop codon (or termination codon) is a codon ( nucleotide triplet within messenger RNA) that signals the termination of the translation process of the current protein. Most codons in ...
. This requires only the presence of the ''pylT'' gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon, and the '' pylS'' gene, which encodes a class II aminoacyl-tRNA synthetase that charges the ''pylT''-derived tRNA with pyrrolysine. This novel tRNA-aaRS pair ("orthogonal pair") is independent of other synthetases and tRNAs in
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Esc ...
, and further possesses some flexibility in the range of amino acids processed, making it an attractive tool to allow the placement of a possibly wide range of functional chemical groups at arbitrarily specified locations in modified proteins. For example, the system provided one of two fluorophores incorporated site-specifically within calmodulin to allow the real-time examination of changes within the protein by FRET spectroscopy, and site-specific introduction of a photocaged lysine derivative. ''(See Expanded genetic code)''


Evolution

The ''pylT'' and ''pylS'' genes are part of an operon of '' Methanosarcina barkeri'', with homologues in other sequenced members of the ''Methanosarcinaceae'' family: ''M. acetivorans'', ''M. mazei'', and ''M. thermophila''. Pyrrolysine-containing genes are known to include
monomethylamine methyltransferase Methylamine-corrinoid protein Co-methyltransferase (, ''mtmB (gene)'', ''monomethylamine methyltransferase'') is an enzyme with systematic name ''monomethylamine:5-hydroxybenzimidazolylcobamide Co-methyltransferase''. This enzyme catalyses the fo ...
(mtmB), dimethylamine methyltransferase (mtbB), and
trimethylamine methyltransferase Trimethylamine-corrinoid protein Co-methyltransferase (, ''mttB (gene)'', ''trimethylamine methyltransferase'') is an enzyme with systematic name ''trimethylamine:5-hydroxybenzimidazolylcobamide Co-methyltransferase''. This enzyme catalyses the ...
(mttB). Homologs of ''pylS'' and ''pylT'' have also been found in an Antarctic archaeon, '' Methanosarcina barkeri'' and a Gram-positive
bacterium Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were a ...
, '' Desulfitobacterium hafniense''.Reviewed in The occurrence in ''Desulfitobacterium'' is of special interest, because bacteria and archaea are separate domains in the
three-domain system The three-domain system is a biological classification introduced by Carl Woese, Otto Kandler, and Mark Wheelis in 1990 that divides cellular life forms into three domains, namely Archaea, Bacteria, and Eukaryota or Eukarya. The key differenc ...
by which living things are classified. When use of the amino acid appeared confined to the ''Methanosarcinaceae'', the system was described as a "late archaeal invention" by which a 21st amino acid was added to the genetic code. Afterward it was concluded that "PylRS was already present in the last universal common ancestor" some 3 billion years ago, but it only persisted in organisms using methylamines as energy sources. Another possibility is that evolution of the system involved a horizontal gene transfer between unrelated microorganisms. The other genes of the Pyl operon mediate pyrrolysine biosynthesis, leading to description of the operon as a "natural genetic code expansion cassette". Some differences exist between the bacterial and archaeal systems studied. Homology to ''pylS'' is broken into two separate proteins in ''D. hafniense''. Most notably, the UAG codon appears to act as a stop codon in many of that organism's proteins, with only a single established use in coding pyrrolysine in that organism. By contrast, in methanogenic archaea it was not possible to identify any unambiguous UAG stop signal. Because there was only one known site where pyrrolysine is added in ''D. hafniense'' it was not possible to determine whether some additional sequence feature, analogous to the SECIS element for selenocysteine incorporation, might control when pyrrolysine is added. It was previously proposed that a specific downstream sequence "PYLIS", forming a
stem-loop Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence wh ...
in the mRNA, forced the incorporation of pyrrolysine instead of terminating
translation Translation is the communication of the Meaning (linguistic), meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The ...
in methanogenic archaea. However, the PYLIS model has lost favor in view of the lack of structural homology between PYLIS elements and the lack of UAG stops in those species.


Potential for an alternative translation

The tRNA(CUA) can be charged with lysine ''in vitro'' by the concerted action of the ''M. barkeri'' Class I and Class II Lysyl-tRNA synthetases, which do not recognize pyrrolysine. Charging a tRNA(CUA) with lysine was originally hypothesized to be the first step in translating UAG amber codons as pyrrolysine, a mechanism analogous to that used for selenocysteine. More recent data favor direct charging of pyrrolysine on to the tRNA(CUA) by the protein product of the ''pylS'' gene, leading to the suggestion that the LysRS1:LysRS2 complex may participate in a parallel pathway designed to ensure that proteins containing the UAG codon can be fully translated using lysine as a substitute amino acid in the event of pyrrolysine deficiency. Further study found that the genes encoding LysRS1 and LysRS2 are not required for normal growth on methanol and methylamines with normal methyltransferase levels, and they cannot replace pylS in a recombinant system for UAG amber stop codon suppression.


References


Further reading

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External links

* {{Amino acids Proteinogenic amino acids Pyrrolines Secondary amino acids