Detour: Fireflies and the Non-Ribosomal Code

Deduction of partial non-ribosomal code using fireflies

When Marahiel started his groundbreaking work on decoding the non-ribosomal code in the late 1990s, 160 A-domains had already been sequenced, and the amino acids that they encode had been experimentally identified. However, it was still not clear exactly how each A-domain encoded a specific amino acid.

We showed an alignment of three A-domain intervals in the main chapter, but Marahiel actually aligned all of the 160 identified A-domains in order to reveal the conservative core. Yet it still remained unclear which columns in this alignment defined the non-ribosomal signatures.

Help came from an unusual ally: Photinus pyralis, the most common species of firefly. Fireflies produce an enzyme called luciferase that helps them emit light to attract mates at night. Different species have different glow patterns, and females respond to males from the same species by identifying the color, duration, and intensity of their flash.

What do fireflies have to do with the non-ribosomal code? Firefly luciferase belongs to a class of enzymes called adenylate-forming enzymes, which share similarities with adenylation domains. For this reason, when Peter Brick published the three-dimensional structure of firefly luciferase in 1996, Marahiel was quick to take notice. In 1997, he and Brick joined forces and used firefly luciferase as a scaffold to reconstruct the first three-dimensional structure of an A-domain, which coded for phenylalanine (Phe); it’s worth noting that this A-domain belonged to an NRP synthetase encoding Gramicidin Soviet, the first mass-produced antibiotic.

Marahiel and Brick actually constructed the three-dimensional structure of a larger complex containing both the A-domain and Phe. This three-dimensional structure provided information about amino acid residues in the A-domain located close to Phe, a hypothetical active pocket of the A-domain. Marahiel further demonstrated experimentally and computationally that amino acids in this active pocket define the non-ribosomal code, thus producing the 8 purple columns in the 3-way alignment that we showed at the beginning of the chapter.

The table below shows the partial non-ribosomal code deduced by Marahiel. Although he was able to deduce some signatures, the non-ribosomal code is very redundant, meaning that multiple mutated variants of a signature all code for the same amino acid. For some amino acids, this redundancy is pronounced, e.g., Marahiel identified three very different signatures AWMFAAVL, AFWIGGTF, and FESTAAVY coding for Val.