Flexible peptide assembly

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A jack of all trades is a master of none, as the saying goes. But a protein has been discovered that shuns specialism, and that multitasks to give flexibility to its biosynthetic repertoire.

The biological machinery used by microorganisms to synthesize complex, antibiotic peptides is often compared to Henry Ford's car assembly line: different protein modules line up, each with their own specific synthetic job1,2,3, and the molecule under construction is passed from one module to the next, accruing new parts until the desired compound is finally made. This is a sophisticated approach for constructing tailor-made peptides, but building large, multi-protein factories requires quite an investment of resources from the microorganisms involved. This raises the question of whether alternative, more efficient approaches have been adopted by nature — for example, the use of compact protein factories in which one module can execute several operations.

Hamano and colleagues4 report in Nature Chemical Biology the first discovery of a protein that does exactly that. They find that the antibiotic polypeptide poly-ɛ-lysine (ɛ-PL) is constructed by a single protein structure that selects the correct building blocks, activates them and then couples them together.

As a potent antibiotic, ɛ-PL has garnered much attention, and is used in some countries as a food preservative5. Constructed from lysine amino acids, its distinctive characteristic is that the lysines are coupled together through their side chains, rather than through groups in their main chains (as occurs between amino acids during normal ribosomal protein synthesis). This mode of coupling suggests the involvement of an enzyme assembly line — a non-ribosomal peptide synthetase (NRPS), to use the technical jargon.

Another quirk of ɛ-PL is that it is actually a cocktail of peptides, the components of which differ in the number of lysines that make up each molecule. The normal range of chain lengths is 25–35 amino acids. The origin of this chain-length diversity is a topic of debate: it could be the result of degradation of longer ɛ-PL chains, or the effect of an unusually flexible NRPS synthesis. To solve the mystery, Hamano and colleagues4 analysed different cell extracts from an ɛ-PL-producing strain of the bacterium Streptomyces albulus. They found one insoluble fraction in which ɛ-PL was formed, and from this they purified a single active enzyme, which they named ɛ-PL-synthetase (Pls).

Using an extensive biochemical-characterization procedure, Hamano and colleagues identified within Pls several different domains that are involved in peptide synthesis (Fig. 1). One of these domains is responsible for selecting lysine amino acids (rather than any of the many other naturally occurring amino acids), and for 'activating' the carboxylic acid group in the lysine so that it can form a peptide bond. The authors dubbed this the A domain, after the analogous region in traditional NRPS enzymes. The activated lysine molecule is then connected to another domain, which holds it in the right position for coupling to the nascent ɛ-PL molecule. Again, by analogy with NRPSs, the authors designated this the T domain.

Figure 1: Antibiotic assembly.

Hamano and colleagues4 have identified the enzyme that synthesizes the polypeptide antibiotic ɛ-PL, which consists of many lysine amino acids linked together through their side chains. The authors propose the following mechanism for its synthesis. a, The first part of the enzyme (the A domain) specifically selects lysine as a building block for ɛ-PL, and activates the amino acid so that it is ready to react. b, The activated lysine is transferred to the next part of the enzyme (the T domain), which holds it in the correct orientation for reaction with the nascent ɛ-PL. c, ɛ-PL fragments are held in an adjacent tunnel-shaped cavity, in such a way that the side-chain amino group (red), rather than the main-chain amino group (blue), reacts with the activated lysine. d, The extended ɛ-PL molecule may be released from the cavity, or can remain in place, ready to couple with another lysine molecule. For simplicity, the product shown contains three lysine units, but ɛ-PL actually contains molecules that have 25–35 lysines.

The recognizable A and T domains comprise the first half of Pls, but the remaining part of the protein acts differently from normal peptide assembly lines. Once activated, lysines cannot be passed on to a neighbouring protein module (as they would be in an NRPS), because there is only one protein involved in the synthesis. Instead, they react with an ɛ-PL molecule, which is positioned in such a way that the activated lysine can couple only with the side-chain amine of ɛ-PL. This part of the process is still not completely understood. The most plausible mechanism is that the second part of the Pls protein forms a long, slender tunnel or cavity, which is occupied by the growing ɛ-PL molecule. Unlike in NRPSs, the nascent peptide is not covalently connected to the protein. After the coupling step with the activated lysine, the extended ɛ-PL peptide can either be released or remain bound in the cavity. This means that the number of lysines coupled together is not strictly controlled, and would explain the diversity of chain lengths found in ɛ-PL.

When the working mechanisms of traditional NRPS complexes and the Pls protein are compared, some important differences can be observed. First, each NRPS assembly line allows the synthesis of a complex and perfectly defined peptide, composed of many different amino acids that can form bonds using groups in their main chains or in their side chains. The biological machinery required to create such complex structures is finely tuned, and isn't flexible enough to incorporate amino acids into the product other than those it has evolved to accept. This fine tuning also makes it difficult to modify NRPSs to make analogues of naturally occurring antibiotic peptides (which is desirable for drug discovery).

But the peptide synthesized by Pls lacks the structural complexity of traditional NRPS peptides, because it is composed solely of lysines connected through their side chains. Pls therefore needs only one amino-acid selection domain and one amino-acid activation domain (the A and T domains, respectively). Hamano and colleagues4 also show that there is some limited flexibility in substrate selection by the enzyme: it can incorporate a few amino acids other than lysine. Furthermore, because the peptide sequence and general domain architecture of Pls are known, it should be possible to change its selectivity even further by protein engineering. Thus, we might one day be able to exploit the biosynthetic flexibility of Pls to make polypeptides with interesting biological activity — not only antibiotics, but potentially also molecules for drug delivery and gene therapy.

So there is more to Pls than meets the eye. At first glance, it might seem less impressive than NRPSs — after all, to stretch the analogy with assembly lines further, if NRPSs produce the peptide equivalent of sophisticated cars, then Pls manufactures relatively simple bicycles. But NRPSs can make only black Model T Fords; Pls meanwhile can produce everything from unicycles to tandems, and, as Hamano and colleagues have shown, might also offer a greater choice of colours.


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    Yamanaka, K., Maruyama, C., Takagi, H. & Hamano, Y. Nature Chem. Biol. doi:10.1038/nchembio.125 (2008).

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    Oppermann-Sanio, F. & Steinbuchel, A. Naturwissenschaften 89, 11–22 (2002).

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van Hest, J. Flexible peptide assembly. Nature 456, 186–187 (2008) doi:10.1038/456186a

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