Let's continue to discuss the action and evolution in the "
What's wrong with evolution?" thread.
More from agentrfr:
But I say mutations can only eliminate traits. They cannot produce new features.
Well, this is a bit difficult to hold. Let's see.
Nylon was first synthesized in 1935 by Wallace Carothers at DuPont, yet bacteria (Arthrobacter sp. KI7, formerly Flavobacterium sp.) can metabolize nylon (1). Therefore, one of two things are true:
1) The nylon breakdown capabilities of these bacteria were present before nylon was synthesized.
2) Or, new enzymes capable of nylon metabolism evolved (through whatever means).
So what is nylon?
Nylon-6 is produced from caprolactam by ring cleavage polymerization and consists of more than 100 units of 6-aminohexanoate (Ahx). During the polymerization reaction, some molecules fail to polymerize and remain as linear oligomers, while others undergo head-to-tail condensation to form cyclic oligomers (Figure 1). These cyclic and linear oligomers (called nylon oligomers) are the byproducts from nylon factories and become nylon bug food. Three enzymes, 6-aminohexanoate-cyclic dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII) and endo-type 6-aminohexanoate oligomer hydrolase (EIII), are found to be responsible for the degradation of the nylon oligomers (Figure 2) (2). The genes responsible for coding these proteins are located on plasmid pOAD2 (45,519 bp) in strain KI72 (not only K172 though) (3).
Figure 1: 6-aminohexanoate and associated dimers
Figure 2: Breakdown of nylon oligomers by 6-Aminohexanoate dimer Hydrolases
So what is the mechanism of nylon degradation? Amide hydrolysis...
Negoro et al. (2007) wrote an interesting article and proposed a possible mechanism of how the 6-aminohexanoate linear dimer (Ald) is broken down by the E2 enzyme (4). See figure 5.
First, a little background information:
1) Asp181 and Tyr170 form a hydrogen bond in the unbound state of E2 (Figure 3b). Asp181, and probably Ser112 and Ile345, are crucial in accepting the 6-aminohexaonate substrate, as upon binding, 6-aminohexaonate forms a stable electrostatic interaction with Asp181 and causes structural alterations that localizes Tyr170 to the catalytic center (Figure 3).
2) The important amino acid residues involved in catalyzing the hydrolysis (catalytic center) of Ald into two 6-aminohexanoate molecules are (Fig 4):
Tyr170
Asp181
Ile345
Ser112
Tyr215 and/or Lys115
Water molecules within the catalytic centre also play an important role.
Figure 3: Unbound conformation of EII. (1wyc.pdb)
Figure 4: Bound conformation of EII. (2dcf.pdb)
The mechanism:
1) Upon binding, a conformational change occurs as a result of an electrostatic interaction between the Ald-amine and Asp181, causing in the Tyr170 to come into contact with the amide bond of Ald (1-2 in figure). Tyr170(phenolic hydroxyl) protonates the amide nitrogen (2 in figure)
2) A nucleophilic attack, facilitated by deprotonation of Ser112 by Ile345 and/or Lys115, to Ald by Ser112 results in the formation of a tetrahedral (3 in figure) intermediate and subsequent hydrolysis as a result a nucleophilic attack from water on the carbonyl carbon results in the hydrolysis of the amide bond and release of 1 Ald molecule (4 in figure).
3
) Subsequent deacylation releases the second Ald molecule and influx of water molecules restores the activity of the unbound E2 (its more complicated than this, but it is covered in the paper) [[vi]] (5-8 in figure).
Figure 5: Mechanism of nylon break down
And now for the important part. Where did the information come from for this novel adaptation?
Amide hydrolysis for other amides present in nature is quite common. Beta-lactamases, the enzymes responsible for the breakdown of… you guessed it… beta-lactams are present in many types of bacteria. Beta-lactamase breaks the 4-membered heteroatomic ring structure (three carbon atoms and one nitrogen atom) open by hydrolyzing the amide bond of a beta-lactam (Figure 6).
See
figure
Figure 6: Amide bonds of Ald and betalactams
EII’ (nylB’) is an enzyme also encoded on plasmid OAD2 of Arthrobacter sp. KI72. The enzyme has B-lactamase folds and is also able to catalyze the breakdown of Ald. EII’ is a classical carboxylesterase with high activity towards carboxylesters with short acyl chains (5). The accession code for the the nylB' (EII') amino acid sequence is
P07062 and the FASTA sequence can be used to search for similar sequences in other bacterial, archael and eukaryotic genomes
at this site (with the BlastP program). After doing so, it can be seen that proteins with beta-lactam folds with 6-aminohexanoate-dimer hydrolytic activity (non-specific Ald amide hydrolysis) is spread throughout the bacterial and archaeal kingdoms.
EII’ is, therefore a pre-existing 6-aminohexanoate-dimer hydrolase with low activity (0.5% that of EII (nylB)) towards Ald that gained an increase in activity towards the Ald through amino acid substitutions in the catalytic cleft containing the “Ser-X-X-Lys” motive (6). The information needed to metabolize 6-aminohexanoate for energy was already present (presumably the lysine degradation pathway) and the useful esterase with B-lactam folds with minimal Ald hydrolytic activity allowed the bacteria to survive under stressful conditions where the sole energy source was Ald.
To answer the question of whether the nylon breakdown capabilities of these bacteria were present before nylon was synthesized or, new enzymes capable of nylon metabolism evolved (through whatever means)... The answer is a bit of both...
The evidence points to (and seems like the simplest explanation - Occam's razor) a
pre-existing classical carboxylesterase with B-lactamase folds (NylB'/EII') with low activity (0.5% that of EII (nylB) towards nylon oligomers, gained an increase in activity towards the the oligomers through amino acid substitutions in the catalytic cleft containing the “Ser-X-X-Lys” motive without affecting the activity towards its original substrates.
Pre-existing nylon digesting enzymes were just optimized with a few mutations. Therefore mutations can produce new traits.
References:
1. Prijambada ID, Negoro S, Yomo T, Urabe I. Emergence of nylon oligomer degradation enzymes in Pseudomonas aeruginosa PAO through experimental evolution. Appl Environ Microbiol. 1995 May;61(5):2020-2.
2. Negoro S. Biodegradation of nylon oligomers. Appl Microbiol Biotechnol. 2000 Oct;54(4):461-6.
3. Kato K, Ohtsuki K, Koda Y, Maekawa T, Yomo T. et al. A plasmid encoding enzymes for nylon oligomer degradation: nucleotide sequence and analysis of pOAD2. Microbiology. 1995 Oct;141 ( Pt 10):2585-90.
4. Negoro S, Ohki T, Shibata N, Sasa K, Hayashi H et al. Nylon-oligomer degrading enzyme/substrate complex: catalytic mechanism of 6-aminohexanoate-dimer hydrolase. J Mol Biol. 2007 Jun 29;370(1):142-56.
5. Ohki T, Wakitani Y, Takeo M, Yasuhira K, Shibata N, Higuchi Y, et al. Mutational analysis of 6-aminohexanoate-dimer hydrolase: relationship between nylon oligomer hydrolytic and esterolytic activities. FEBS Lett. 2006 Sep 18;580(21):5054-2058.
6. Negoro S, Ohki T, Shibata N, Mizuno N, Wakitani Y et al. X-ray crystallographic analysis of 6-aminohexanoate-dimer hydrolase: molecular basis for the birth of a nylon oligomer-degrading enzyme. J Biol Chem 2005 Nov 25;280(47):39644-52
