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Sulfoluciferin is biosynthesized by a specialized luciferin sulfotransferase in fireflies. Timothy R Robert Fallon, Fu-Shuang Li, Maria Alejandra Vicent, and Jing-Ke Weng Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00402 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on May 26, 2016
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Biochemistry
Sulfoluciferin is biosynthesized by a specialized luciferin sulfotransferase in fireflies. Timothy R. Fallon†, ‡, Fu-Shuang Li†, Maria A. Vicent†,§ and Jing-Ke Weng*,†, ‡ †
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
‡Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA §
Department of Biology, Williams College, Williamstown, MA, 01267, USA
Supporting Information Placeholder ABSTRACT: Firefly luciferin is a specialized metabolite re-
stricted to fireflies (family Lampyridae) and other select families of beetles (order Coleoptera). Firefly luciferin undergoes luciferase-catalyzed oxidation to produce light, thereby enabling the luminous mating signals essential for reproductive success in most bioluminescent beetles. Although firefly luciferin and luciferase have become widely used biotechnological tools, questions remain regarding the physiology and biochemistry of firefly bioluminescence. Here we report sulfoluciferin to be an in vivo derivative of firefly luciferin in fireflies, and report the cloning of luciferin sulfotransferase (LST) from the North American firefly Photinus pyralis. LST catalyzes the production of sulfoluciferin from firefly luciferin and the sulfo-donor PAPS. Sulfoluciferin is abundant in several surveyed firefly genera as well as in the bioluminescent elaterid beetle Pyrophorus luminosus at a low level. We propose that sulfoluciferin could serve as a luciferin storage molecule in fireflies, and that LST may find use as a new tool to modulate existing biotechnological applications of the firefly bioluminescent system.
Bioluminescence is the production of light by a chemical reaction in a biological context. In well-described cases, such as fireflies,1 luminous ostracods,2 and dinoflagellates,3 the reaction consists of the oxidation of a small molecule, known as luciferin, by an enzyme, known as luciferase, with molecular oxygen. Despite the shared nomenclature of luciferin and luciferase, known bioluminescence consists of at least seven independently evolved systems with structurally unique luciferins and non-homologous luciferases.4 Firefly luciferin (hereinafter luciferin) was the first luciferin to be structurally characterized,1 and firefly luciferase (hereinafter luciferase) was the first luciferase gene to be cloned.5 As luciferase has no prosthetic groups and requires only Dluciferin, ATP, Mg2+, and O2 to produce light, the enzyme has been readily adapted to in vivo and in vitro applications, such as usage as a reporter gene,6 and quantification of ATP by
luminometry.7 Although extensive research exists on the biotechnological usage of firefly bioluminescence, key questions remain on the metabolic biochemistry of the firefly bioluminescent system. For example, it is unknown how luciferin is biosynthesized from primary metabolic precursors,8 and is unclear how accessory enzymes function to store excess luciferin or to recycle the luminescent reaction product oxyluciferin.9 ,10 Scheme 1. DL-luciferin is enzymatically interconverted to sulfoluciferin by luciferin sulfotransferase (LST) NH2 N N
O O
-O
S
O
O
O-
O
O
HO
P
HO
S
N S
DL-luciferin
O
OH
O-
O
HO
PAPS
N
N N
O P
HO
OO
N
O
N
O P
O
NH2 N
N
O
P
OH
O-
PAP
OH
luciferin sulfotransferase
O O
-
O O
S
O
N
N
S
S
OH
DL-sulfoluciferin
In an effort to elucidate firefly luciferin metabolism, we analyzed methanolic extracts of the posterior abdominal tissue containing the bioluminescent lantern (hereinafter referred to as “lantern” tissue) from the firefly Photinus pyralis by liquid-chromatography high-resolution accurate-mass massspectrometry (LC-HRAM-MS). Under positive ion mode, this analysis detected luciferin as one of the most dominant mass features in the total ion chromatogram (TIC) (Figure S1). We also noted an identical ion to the luciferin [M+H]+ ion with a well resolved retention time two minutes earlier than that of luciferin (Figure S2). In-depth analysis of the MS1 & MS2 scans revealed that the luciferin-matching ion was likely an in-source fragment ion from a [M+H]+ precursor ion with a mass-to-charge ratio (m/z) of 360.9614. The 360.9614 precursor ion also had a highly similar fragmentation pattern to luciferin (Figure S3). Given the constraint of the luciferin chemical formula (C11H8N2O3S2) and the high mass accuracy (
