A suicidal and extensively disordered luciferase with a bright luminescence
Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 [A] and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase. Significance statementEnzymes are typically characterized by an overall globular structure with a hydrophobic core and a defined cavity for binding of substrate, containing the active site amino acid residues. Gaussia Luciferase is a widely used luminescent reporter with a very strong, albeit short-lived, flash of light due to rapid auto-inactivation. We show, using solution NMR, that while this luciferase shows some secondary structure elements held together by disulfide bonds this highly unusual enzyme is extensively disordered with essentially no hydrophobic core. Although the enzymatic mechanism remains unknown, we have identified two essential arginine residues but, in the structure, these do not point into a common active site. In spite of this, the enzyme has high substrate specificity suggesting that it undergoes major structural rearrangements upon binding of substrate.