Dehydroluciferyl‐AMP is the main intermediate in the luciferin dependent synthesis of Ap₄A catalyzed by firefly luciferase

Abstract

It was previously assumed that EWLH P -AMP was the intermediate complex in the synthesis of Ap R A catalyzed by firefly luciferase (EC 1.13.12.7), when luciferin (LH P ) was used as cofactor. However, here we show that LH P is partly transformed, shortly after the onset of the luciferase reaction, to dehydroluciferin (L) with formation of an EWL-AMP complex which is the main intermediate for the synthesis of Ap R A. Formation of three more derivatives of LH P were also observed, related to the production of light by the enzyme. CoA, a known stimulator of light production, inhibits the synthesis of Ap R A by reacting with the EWL-AMP complex and yielding L-CoA.z 1998 Federation of European Biochemical Societies.

T4 DNA ligase synthesizes dinucleoside polyphosphates

“…The apparent /£,i, values determined for P:~ and GTP in the second step of the reaction (formation of p4A and Ap~G, respectively) are in the order of mM (results not shown). These results were reminiscent of those obtained with firefly luciferase [35] and other ligases [36,37] in which the K,n values for the AMP acceptor substrate (P:~, ATP, GTP, etc.) were in the mM range.…”

“…Firefly luciferase (although classified as an oxygenase, EC 1.13.12.7) catalyzes very effectively the synthesis of nucleoside 5'-polyphosphates (pHA, n=3-20), diadenosine polyphosphates (ApI,A, n =4-16), and a variety of dinucleoside tetraphosphates (Ap4N, N = any nucleoside) using luciferin, ATP and polyphosphates or NTP as cofactors [35]. Yeast acetylCoA synthetase [36] and Pseudomonas.fi'agi acyl-CoA synthetase [37] catalyze also, to a lesser extent, the synthesis of (di)nucleoside polyphosphates.…”

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

“…In such reaction the complex luciferaseÁD-LH 2 -AMP reacts with oxygen in a dark reaction pathway leading to the oxidized product dehydroluciferyl-AMP (L-AMP) [Eq. (3)] (23,(38)(39)(40). …”

“…As L is an oxidative product of LH 2 and ATP, through the oxidation of LH 2 -AMP to L-AMP and its posterior pyrophosphorolysis (23,(38)(39)(40), it was originally regarded as the light emitter and equivocally called oxyluciferin (19,20,23,38). Its chemical synthesis was achieved (18) and its role on the bioluminescent reaction, as well as of its adenylated analog L-AMP, has been clarified.…”

Mammalian-Based Bioreporter Targets: Protein Expression for Bioluminescent and Fluorescent Detection in the Mammalian Cellular Background

“…It has previously been shown that even though oxyluciferin is a natural product of the luciferase reaction, it is capable of remaining bound as an inhibitor to enzymatic turnover (Denburg et al, 1969). The same was found to be true of another potential byproduct, L-AMP, which can account for up to 16% of the product formed during the luminescent reaction (Fontes et al, 1998). This may, in part, explain how the addition of CoA to the luminescent reaction can result in improved performance.…”