Pyrrolysine (symbol Pyl or O; encoded by the 'amber' stop codon
UAG) is an ɑ-amino acid that is used in the biosynthesis of proteins
in some methanogenic archaea and Bacteria; it is not present in
humans. It contains an α-amino group (which is in the protonated
–+NH3 form under biological conditions), a carboxylic acid 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.
4 Catalytic function
5 Genetic coding
7 Potential for an alternative translation
9 Further reading
10 External links
Nearly all proteins are made using only 20 standard amino acid
building blocks. Two unusual genetically-encoded amino acids are
selenocysteine and pyrrolysine.
Pyrrolysine was discovered in 2002 at
the active site of methyl-transferase enzyme from a methane-producing
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.
As determined by X-ray crystallography and
MALDI mass spectrometry,
pyrrolysine is made up of 4-methylpyrroline-5-carboxylate in amide
linkage with the ϵN of lysine.
Pyrrolysine is synthesized in vivo by joining two molecules of
L-lysine. One molecule of lysine is first converted to
(3R)-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.
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 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 CH+
3 is transferred to the cofactor's cobalt atom with a change of
oxidation state from I to III. The methylamine-derived ammonia is then
released, restoring the original imine.
Unlike posttranslational modifications of lysine such as
hydroxylysine, methyllysine, and hypusine, pyrrolysine is incorporated
during translation (protein synthesis) as directed by the genetic
code, just like the standard amino acids. It is encoded in mRNA by the
UAG codon, which in most organisms is the 'amber' stop codon. 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, 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)
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 (mtmB), dimethylamine
methyltransferase (mtbB), and trimethylamine methyltransferase (mttB).
Homologs of pylS and pylT have also been found in an Antarctic
Methanosarcina barkeri and a
The occurrence in Desulfitobacterium is of special interest, because
bacteria and archaea are separate domains in the three-domain system
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 in the mRNA, forced
the incorporation of pyrrolysine instead of terminating translation 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.
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The encoded amino acid
Branched-chain amino acids (Valine
Positive charge (pKa)
Negative charge (pKa)
Aspartic acid (≈3.9)
Glutamic acid (≈4.1)
Amino acids types: Encoded (proteins)