Luciferase Activity of Insect Fatty Acyl-CoA Synthetases with Synthetic Luciferins

Abstract

Long-chain fatty acyl-CoA synthetases (ACSLs) are homologues of firefly luciferase but are incapable of emitting light with firefly luciferin. Recently, we found that an ACSL from the fruit fly Drosophila melanogaster is a latent luciferase that will emit light with the synthetic luciferin CycLuc2. Here, we have profiled a panel of three insect ACSLs with a palette of >20 luciferin analogues. An ACSL from the nonluminescent beetle Agrypnus binodulus (AbLL) was found to be a second latent luciferase with distinct substrate specificity. Several rigid luciferins emit light with both ACSLs, but styryl luciferin analogues are light-emitting substrates only for AbLL. On the other hand, an ACSL from the luminescent beetle Pyrophorus angustus lacks luciferase activity with all tested analogues, despite its higher homology to beetle luciferases. Further study of ACSLs is expected to shed light on the features necessary for bioluminescence and substrate selectivity.

Figure 1

Fatty acyl-CoA synthetases and firefly luciferase catalyze similar, two-step reactions. (a) Firefly luciferase catalyzes the formation of an activated adenylate of D-luciferin followed by oxidation to an excited state oxyluciferin that is responsible for light emission. (b) Fatty acyl-CoA synthetases catalyze the formation of an activated adenylate of a free fatty acid followed by displacement of the adenylate by CoASH to form the acyl-CoA product. (c) A subset of synthetic luciferins emit light with CG6178 and/or AbLL in addition to firefly luciferase.

Figure 2

ACSL luciferase activity. (a) Identification of emissive substrates for ACSLs. Each enzyme (20 nM) was treated with the indicated luciferin analog (250 μM). Significant flux over no-substrate control was observed (p < 0.05) unless marked “ns”. (b) Burst kinetics profiles of each ACSL with D-luciferin, CycLuc2, and their pre-activated adenylates. Purified enzyme (100 nM) was rapidly injected into substrate (100 μM). Light emission was recorded every 0.5 s for 1 s pre-injection and 120 s post-injection. Background luminescent signal in the absence of enzyme is shown for reference (in gray). All assays were performed in triplicate, are represented as the mean ± SEM, and are presented on the same log scale.

Figure 3

ACSL activity in live CHO cells. (a) Live CHO cells expressing the indicated ACSL treated with either high or low dose of each luciferin analogue. (b) Dose-response curves of live CHO cells expressing the indicated enzyme treated with D-luciferin or CycLuc2 up to 2.5 mM. The assays were performed in triplicate and are represented as the mean ± SEM.

Figure 4

Photon flux from live CHO cells expressing FLuc or AbLL after treatment with 100 μM of D-luciferin or the indicated styryl luciferin analogue. The assays were performed in triplicate and are represented as the mean ± SEM.