Presentation

10 mm Pontodrilus litoralis Ceder Key, Florida. 12 Aug 2014 Ceder Key, Florida. 12 Aug 2014 10 mm Pontodrilus litoralis Pontodrilus litoralis Evolution of firefly luciferase and Biosynthetic origin of firefly luciferin

Yuichi Oba Graduate School of Bioagricultural Sciences, Nagoya University, Japan [email protected]

2014 Aug 13, 14:20-14:45 Firefly Symposium 2014 Gainesville, FL, USA ✓Bioluminescence mecahnism is common in all four coleopeteran families.

Bioluminescence reaction in beetles

2+ N N C O O H ATP, Mg , O2 N N O + Light H O S S Beetle luciferase H O S S (> 46% AA similarity) Beetle luciferin Oxyluciferin

Lampyridae Phengodidae Rhagophthalmidae Elateridae (Aquatica lateralis) (Phengodes sp.) (Rhagophthalmus ohbai) (Pyrophorus angustus)

Photo of Phengodes sp.: http://www.flickriver.com/groups/1247869@N24/pool/interesting/ ✓Bioluminescence mecahnism is common in all four coleopeteran families. ✓Bioluminescence for each group was evolved independently. Bioluminescence reaction in beetles

2+ N N C O O H ATP, Mg , O2 N N O + Light H O S S Beetle luciferase H O S S (> 46% AA similarity) Beetle luciferin Oxyluciferin

Luciferins in other arthropods O H O H

O H N H H N O N N N a O 2C N H H N H N NH N H N NH 2 Lampyridae PhengodidaeH Rhagophthalmidae Elateridae N a O C O (Aquatica lateralis) (Phengodes sp.) (Rhagophthalmus ohbai) (Pyrophorus2 angustus)

Ostracoda Krill (Vargula hilgendorfii) (MeganyctiphanesKrill (Crustacean) norvegica) What was the firefly luciferase evoleved from ? Firefly MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTD-AHIEVNITYAEYFEM Vargula -MKIIILSVILAYCVTVNCQDACPVEAEPPSSTPTVPTSCEAKEGECIDTRCATCKRDIL American firefly Photinus pyralis luciferase . :. * .:: * :. . . ** : .: *:.. : .: :

Firefly SVRLAEAMK-RYGLNTNHRIVVCSENSLQFFMPVLGALFIG-----VAVAPANDIYNERE vs Vargula SDGLCENKPGKTCCRMCQYVIECRVEAAGYFRTFYGKRFNFQEPGKYVLARGTKGGDWSV * *.* : . : :: * :: :* .. * * .:* ... : ostracod Vargula hilgendorfii luciferase Firefly LLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLP Vargula TLTMENLDGQKGAVLTKTTLEVAGDVIDITQATADPITVNGGADPVIANPFTIGEVTIAV *. *:. . ..::*. *: :* 。 . * ::. :* :.: .:

Firefly PGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIP Vargula VEIPGFNITVIEFFK----LIVIDILGGRSVRIAPDTANKGLISGICGNLEMNDADDFTT : :::. .* : :::: *. .: . .:: :. : .::: .

Firefly DTAILSVVP-FHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLF Vargula DADQLAIQPNINKEFDGCPFYGNPSD----IEYCKGLMEPYRAVCRNNINFYYYTLSCAF *: *:: * ::: *. . * : : * : :: :. *

Firefly SFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILIT Vargula AYCMGGEERAKHVLFDYVETCA--APETRGTCVLSGHTFYDTFDKARYQFQGPCKELLMA :: 。*: * : * .: ** :: 。.: *: . : * : . . :*::

Firefly PEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALI Vargula ADCYWNTWDV-KVSHRDVESYTEVEKVTIRKQSTVVDLIVDGKQVKVGGVDVSIPYSSEN .: :. * ** . .:::. 。. :* * * : * *: . . .:

Firefly DKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHP-----NIFDA Vargula TSIYWQDGDILTTAILPEALVVKFNFKQLLVVHIRDPFDGKTCGICGNYNQDSTDDFFDA . * ... :: * :.: .:*.*: : : .: .* :: ::***

Firefly G-VAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGL Vargula EGACALTPNPPGCTEEQKPEAERLCNSLFDSSIDEKCNVCYKPDRIAR--CMYEYCLRGQ ...*. : .* *: . .. :* .. ...:: : : :*

Firefly TGKLDARKIREILIKAKKGGKSKL----- Vargula QGFCDHAWEFKKECYIKHGDTLEVPPECQ * * : *:*.. ::

(no significant homology) Firefly MEDAKNIKKGPAPFYPLEDGTAGEQL---HKAMKRYALVPGTIAFTDAHIEVNITYAEYF American firefly Photinus pyralis luciferase Parsley MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKVGDKSCLINGATGETFTYSQVE * *. *:。 * * ::* .::. : * .. .: :。.:**:: vs Firefly EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNS Parsley LLSRKVASGLNKLGIQQGDTIMLLLPNSPEYFFAFLGASYRGAISTMANPFFTSAEVIKQ Parsley 4-coumarate:CoA ligase :* ::*..::: *:: .. *:: ** ::*:..*** : *. : ** ::.. *:::.

Firefly MNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFN parsley LKASLAKLIITQACYVDKVKDYAAEK-NIQIICIDDAPQDCLHFSKLMEADESEMP---- :: * ..:::.. ::*: : : ** * * *: * *..: *.:*

Firefly EYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIP-DTA parsley ------EVVIDSDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYMHSEDV * . .. :. : ***:******* *.*: . .:: * * : : .

Firefly ILSVVPFHHGFGMFTTLGY-LICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFF parsley MICILPLFHIYSLNAVLCCGLRAGVTILIMQKFDIVPFLELIQKYKVTIGPFVPPIVLAI ::.::*:.* :.: :.* * .*. :::* :*: **. :*.**: . :**.:. :

Firefly AKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEG parsley AKSPVVDKYDLSSVRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGMTEAGPVLAMCLAF ***.::******.:: : **.***.**: :** :* . : ****:**: ..: :

Firefly DDKP-----GAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNA parsley AKEPYEIKSGACGTVVRNAEMKIVDPETNASLPRNQRGEICIRGDQIMKGYLNDPESTRT .:* ** *.** * *:** :*. :* *****:*:** **.**:*:**:*.:

Firefly LIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVA parsley TIDEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAPAELEALLLTHPTISDAAVV **::****:***.: *:*:.:*******.:*****:********::** **.* **.*.

Firefly GLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKL parsley PMIDEKAGEVPVAFVVRTNGFTTTEEEIKQFVSKQVVFYKRIFR-VFFVDAIPKSPSGKI : *:.***:*.*.** :* * **:** ::*:.**. *:: *.*** :**. :**:

Firefly DARKIREILIKAKKGGKSKL parsley LRKDLRAKIASGDLPK---- :.:* : ...

(34% amino acid identity)

Schröder J (1989) Nucleic Acid Res. 17, 460. Homologous gene of Firefly MED---AKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYF CG6178 MTSKLLPGNIVYG-GPVTERQAQDSRSLGQYILDKYKSFGDRTVLVDAVNGVEYSASFMH beetle luciferase in * . . ** * .*. : : . : :.:* . . .:.** *: : : .

Drosophila (CG6178) Firefly EMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNS CG6178 KSIVRLAYILQKLGVKQNDVVGLSSENSVNFALAMFAGLAVGATVAPLNVTYSDREVDHA : **** ::: *:: *. : :.****::* :.::..* :*.:*** * *.:**: ::

Firefly MNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFN CG6178 INLSKPKIIFASKITIDRVAKVASKNKFVKGIIALSGTS--KKFKNIYDLKELMEDEKFK :*:*:*.::*.** :::: :* .* ::: ** :...: : *:.:* : *:

Firefly -EYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTA CG6178 TQPDFTSPAANKDEDVSLIVCSSGTTGLPKGVQLTQMNLLATLDSQIQPTV--IPMEEVT : **.. : ::*: ::**: ***:******* *.: . . :. :* . : :.:

Firefly ILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFA CG6178 LLTVIPWFHAFGCLTLITTACVGARLVYLPKFEEKLFLSAIEKYRVMMAFMVPPLMVFLA :*:*:*:.*.** :* : * *:* : :***:*** :::.*:: *::**.*: *:*

Firefly KSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGD CG6178 KHPIVDKYDLSSLMVLLCGAAPLSRETEDQIKERIGVPFIRQGYGLSESTLSVLVQNDEF * .::******.* : .*.****:*. : : :*: :* *******:*:* ::*: :

Firefly DKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDG CG6178 CKPGSVGVLKVGIYAKVIDPDTGKLLGANERGELCFKGDGIMKGYIGDTKSTQTAI-KDG ***:** : : ***:* **** **.*:*****.:* **.**:.:.::*:: * ***

Firefly WLHSGDLAYWDEDEHFFIVGRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDD CG6178 WLHTGDIGYYDDDFEFFIVDRIKELIKYKGYQVPPAEIEALLLTNDKIKDAAVIGKPDEE ***:**:.*:*:* .****.*:*.*********.***:*::** : :* **.* * **::

Firefly AGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKRDARKIR CG6178 AGELPLAFVVKQANVQLTENEVIQFVNDNASPAKRLRGGVIFVDEIPKNPSGKILRRILR ***** *.** : . :**:*::::* .:.:.**:*****:****:**. :** * :*

Firefly EILIKAKKGGKSKL CG6178 EMLKKQK----SKL *:* * * *** (41% amino acid identity) Molecular phylogenetic tree of beetle luciferases and the homologues (Bayesian tree)

Photinus pyralis (AB644228) Lucidina biplagiata (AB535101) 1.00/100 Luciola cruciata LcLuc1 (AB220162) 1.00/- 1.00/97 Aquatica lateralis LlLuc1 (AB644226) Luciola italica (DQ138966) 1.00/100 1.00/99 Luciola mingrelica (S61961) 0.56/64 Luciolinae 0.88/86 Luciola parvula (AB644227) 1.00/94 Luciola unmunsana (AF420006) 0.99/- 0.93/- 1.00/100 Lampyroidea maculata (DQ137139)

Luciola terminalis (EU302126)* LAMPYRIDAE Photuris pennsylvanica (D25416) 1.00/100 Photurinae Cyphonocerus ruficollis (AB604789) Psilocladinae 0.52/99 Drilaster axillaris (AB604790) 0.52/- Stenocladius azumai azumai (AB644225) Ototretinae Photuris pennsylvanica (U31240) Photurinae 1.00/100 1.00/100 Luciola cruciata LcLuc2 (AB490793) 1.00/100 Luciolinae Aquatica lateralis LlLuc2 (AB693934) luciferase Beetle Phrixothrix hirtus head (AF139645) 1.00/100 Phrixothrix vivianii lateral (AF139644) 1.00/100 Phrixothrix hirtus lateral PHENGODIDAE 1.00/100 Brasilocerus sp. lateral (FJ545728)* 1.00/100 (AF139645)* 1.00/88 Taximastinocerus sp. lateral (GU062703)* Rhagophthalmus ohbai (AB255748) 1.00/64 RHAGOPHTHALMIDAE 1.00/100 Pyrophorus plagiophthalmus dGR (AF543413)* 1.00/- Pyrophorus mellifluus dGR (AF545854)* 1.00/98 Pyrophorus plagiophthalmus vOR (AF543373)* 1.00/-Pyrophorus mellifluus vOR (AF545853)* 0.81/- 1.00/66 Pyrophorus angustus dPaLuc (AB479112) ELATERIDAE 1.00/100 0.81/- Pyrearinus termitilluminans (AF116843) Photophorus jansonii (AB767301) 1.00/95 Pyrophorus angustus vPaLuc (AB479113) 0.99/66 Agrypnus binodulus AbLL (AB353886) 1.00/98 Pyrophorus angustus PaLL (AB479114) Luciferase-like 0.67/94 Luciola cruciata LcLL2 (AB196456) Tribolium castaneum TmLL-1 (AB098615) acyl-CoA synthetase 0.67/- 1.00/100 Tribolium castaneum TmLL-2 (AB098616) 0.97/- Luciola cruciata LcLL1 (AB196455) Drosophila melanogaster CG6178 (NM_142964) Oba, Y (2014) Insect Bioluminecence in the Post-Molecular Biology Tribolium castaneum TmLL-3 (AB098617) Era. In: Hoffmann KH (ed.) Insect Molecular Biology and Ecology, Parsley 4-coumarate:CoA ligase 1 CRC Press. (P14912) Rat long-chain fatty-acyl CoA synthetase 1 (P18163) Asterisks (*) indicate the luciferase genes for which luminescence activities of the recombinant protein were not examined. RESULT 1．CG6178 (homologous gene of firefly luciferase in fruit fly Drosophila) is a "fatty acyl-CoA synthetase". Oba et al. (2004) Gene

Bioluminescence reaction (oxygenation)

2+ N N C O O H ATP, Mg , O2 N N O + Light H O S S CG6178 H O S S Firefly luciferin Oxyluciferin

Fatty acyl-CoA synthetic reaction (thioesterification)

ATP, Mg2, CoA C O O H C O - S - C o A CG6178 Fatty acid Fatty acyl-CoA

Lauric acid is the C12:0 best substrate

C10:0

C14:0 C20:4

C18:1

C16:0 1.00/99 0.1 changes Diaphanes pectinealis (DQ408300)* 0.97/99 Lampyris turkestanicus (AY742225) Nyctophila cf. caucasica (DQ072141)* 1.00/89 1.00/100 Lampyris noctiluca (X89479) 1.00/100 Pyrocoelia rufa (AF328553) 0.98/99 1.00/100 Pyrocoelia miyako (L39928) Lampyrinae Pyrocoelia pectoralis (EF155570)* 1.00/100 Pyrocoelia pygidialis (ACF61063) Cratomorphus distinctus (AY633557)

Luciola terminalis (EU302126)* LAMPYRIDAE Photuris pennsylvanica (D25416) 1.00/100 Photurinae Cyphonocerus ruficollis (AB604789) Psilocladinae 0.52/99 Drilaster axillaris (AB604790) 0.52/- Stenocladius azumai azumai (AB644225) Ototretinae Photuris pennsylvanica (U31240) Photurinae 1.00/100 1.00/100 Luciola cruciata LcLuc2 (AB490793) 1.00/100 Luciolinae Aquatica lateralis LlLuc2 (AB693934) luciferase Beetle Phrixothrix hirtus head (AF139645) 1.00/100 Phrixothrix vivianii lateral (AF139644) 1.00/100 Phrixothrix hirtus lateral PHENGODIDAE 1.00/100 Brasilocerus sp. lateral (FJ545728)* 1.00/100 (AF139645)* 1.00/88 Taximastinocerus sp. lateral (GU062703)* Rhagophthalmus ohbai (AB255748) 1.00/64 RHAGOPHTHALMIDAE 1.00/100 Pyrophorus plagiophthalmus dGR (AF543413)* 1.00/- Pyrophorus mellifluus dGR (AF545854)* 1.00/98 Pyrophorus plagiophthalmus vOR (AF543373)* 1.00/-Pyrophorus mellifluus vOR (AF545853)* 0.81/- 1.00/66 Pyrophorus angustus dPaLuc (AB479112) ELATERIDAE 1.00/100 0.81/- Pyrearinus termitilluminans (AF116843) Photophorus jansonii (AB767301) 1.00/95 Pyrophorus angustus vPaLuc (AB479113) 0.99/66 Agrypnus binodulus AbLL (AB353886) 1.00/98 Pyrophorus angustus PaLL (AB479114) Luciferase-like 0.67/94 Luciola cruciata LcLL2 (AB196456) Tribolium castaneum TmLL-1 (AB098615) acyl-CoA synthetase 0.67/- 1.00/100 Tribolium castaneum TmLL-2 (AB098616) 0.97/- Luciola cruciata LcLL1 (AB196455) Drosophila melanogaster CG6178 (NM_142964) Oba, Y (2014) Insect Bioluminecence in the Post-Molecular Biology Tribolium castaneum TmLL-3 (AB098617) Era. In: Hoffmann KH (ed.) Insect Molecular Biology and Ecology, Parsley 4-coumarate:CoA ligase 1 Photinus pyralis CRC Press. Luciola cruciata (P14912) Rat long-chain fatty-acyl CoA synthetase 1 (P18163) Asterisks (*) indicate the luciferase genes for which luminescence activities of the recombinant protein were not examined. RESULT 2．Firefly luciferases possess the enzymatic activity for fatty acyl-CoA synthesis (bifunctional). Oba et al. (2003) FEBS Lett. Oba et al. (2005) Biosci. Biotech. Biochem.

Bioluminescence reaction (oxygenation)

2+ N N C O O H ATP, Mg , O2 N N O + Light H O S S Firefly uciferase H O S S Firefly luciferin Oxyluciferin

Fatty acyl-CoA synthetic reaction (thioesterification)

ATP, Mg2, CoA C O O H C O - S - C o A Firefly luciferase Fatty acid Fatty acyl-CoA

Lauric acid is the best substrate C12:0

C14:0

C10:0 C20:4 C16:0 C18:1 Photinus pyralis Luciola cruciata RESULT 1．CG6178 (homologous gene of firefly luciferase in fruit fly Drosophila) is a "fatty acyl-CoA synthetase".

RESULT 2．Firefly luciferases possess the enzymatic activity for fatty acyl-CoA synthesis.

These results suggest that firefly luciferase arose from a fatty acyl-CoA synthetase*, with retaining the original function.

* "Fatty acyl-CoA synthetase" is an enzyme for fatty acid metabolism commonly occurring in all organisms. Luciferase homologue in nonluminous click beetle Agrypnus binodulus (AbLL) is a fatty acyl-CoA synthtase, but not luciferase.

Luciola terminalis (EU302126)* LAMPYRIDAE Photuris pennsylvanica (D25416) 1.00/100 Photurinae Non luminous beetle Cyphonocerus ruficollis (AB604789) Psilocladinae Agrypnus binodulus 0.52/99 Drilaster axillaris (AB604790) 0.52/- Stenocladius azumai azumai (AB644225) Ototretinae Photuris pennsylvanica (U31240) Photurinae 1.00/100 1.00/100 Luciola cruciata LcLuc2 (AB490793) 1.00/100 Luciolinae Aquatica lateralis LlLuc2 (AB693934) luciferase Beetle Phrixothrix hirtus head (AF139645) 1.00/100 Phrixothrix vivianii lateral (AF139644) 1.00/100 Phrixothrix hirtus lateral PHENGODIDAE 1.00/100 Brasilocerus sp. lateral (FJ545728)* 1.00/100 (AF139645)* 1.00/88 Taximastinocerus sp. lateral (GU062703)* Rhagophthalmus ohbai (AB255748) 1.00/64 RHAGOPHTHALMIDAE 1.00/100 Pyrophorus plagiophthalmus dGR (AF543413)* 1.00/- Pyrophorus mellifluus dGR (AF545854)* 1.00/98 Pyrophorus plagiophthalmus vOR (AF543373)* 1.00/-Pyrophorus mellifluus vOR (AF545853)* 0.81/- 1.00/66 Pyrophorus angustus dPaLuc (AB479112) ELATERIDAE 1.00/100 0.81/- Pyrearinus termitilluminans (AF116843) Photophorus jansonii (AB767301) 1.00/95 Pyrophorus angustus vPaLuc (AB479113) 0.99/66 Agrypnus binodulus AbLL (AB353886) 1.00/98 Pyrophorus angustus PaLL (AB479114) Luciferase-like 0.67/94 Luciola cruciata LcLL2 (AB196456) Tribolium castaneum TmLL-1 (AB098615) acyl-CoA synthetase 0.67/- 1.00/100 Tribolium castaneum TmLL-2 (AB098616) 0.97/- Luciola cruciata LcLL1 (AB196455) Drosophila melanogaster CG6178 (NM_142964) Oba, Y (2014) Insect Bioluminecence in the Post-Molecular Biology Tribolium castaneum TmLL-3 (AB098617) Era. In: Hoffmann KH (ed.) Insect Molecular Biology and Ecology, Parsley 4-coumarate:CoA ligase 1 CRC Press. (P14912) Rat long-chain fatty-acyl CoA synthetase 1 (P18163) Asterisks (*) indicate the luciferase genes for which luminescence activities of the recombinant protein were not examined. AbLL ---MSKESNIVYGPVGAAPVLESTAGKQLFDSLKRHGHL-PQAIIDYQTKQSISYKNLFE 56 Pyrophorus --MMKREKNVVYGPEPIHPLEDLTAGEMLFRALRKHSHL-PQALVDVYGEEWISYKEFFE 57 Luciola MENMENDENIVVGPKPFYPIEEGSAGTQLRKYMERYAKLGAIAFTNAVTGVDYSYAEYLE 60 M394 *..:.*:* ** *: : :** * :.::.:* . *: : ** : :*

AbLL ATCKLAHSLEEYGLKQNDVIAICSENNLNFYKPVCAALYCGIVIAPLNDSYSEGEYVNAL 116 T344 Pyrophorus ATCLLAQSLHNCGYKMSDVVSICAENNKRFFVPIIAAWYIGMIVAPVNEGYIPDELCKVM 117 Luciola KSCCLGKALQNYGLVVDGRIALCSENCEEFFIPVIAGLFIGVGVAPTNEIYTLRELVHSL 120 S345 :* *.::*.: * .. :::*:** .*: *: *. : *: :** *: * * : :

AbLL NISEPKLIFCSKKCLPRLVGLKARCSFIKGFVVIDSTEDINGNECLPNFILRNSDPNFDI 176 Pyrophorus GISRPQLVFCTKNILNKVLEVQSRTDFIKRIIILDAVENIHGCESLPNFISRYSDG--NI 175 Luciola GISKPTIVFSSKKGLDKVITVQKTVTTIQTIVILDSKVDYRGYQCLDTFIKRNTPPGFQA 180 .**.* ::*.:*: * ::: :: *: ::::*: : .* :.* .** * : : T343

AbLL EKYEPRVFNSNEQVAAILLSSGTTGFPKGVMLTHKNFSILFAHANDPVSGTQRIPGTTVL 236 Pyrophorus ANFKPLHYDPVEQVAAILCSSGTTGLPKGVMQTHQNICVRLIHALDPRAGTQLIPGVTVL 235 Luciola SSFKTVEVDRKEQVALIMNSSGSTGLPKGVQLTHENTVTRFSHARDPIYGNQVSPGTAVL 240 .::. : **** *: ***:**:**** **:* : ** ** *.* **.:**

AbLL SILPYFHGFGFITNISYIKSGIRVVMLQRFEPEAFLRAIEEYEVRSTITVPPILIFLAKS 296 Pyrophorus VYLPFFHAFGFSINLGYFMVGLRVIMLRRFDQEAFLKAIQDYEVRSVINVPAIILFLSKS 295 DLSA Luciola TVVPFHHGFGMFTTLGYLICGFRVVMLTKFDEGTFLKTLQDYKCTSVILVPTLFAILNKS 300 （luciferin analogue) :*:.*.**: .:.*: *:**:** :*: :**:::::*: *.* **.:: :* **

AbLL PIVDKYNLSSLKEIICGAAPSGREIVEAVVKRLKVSGIRYGYGLTECGLAICTTPPNNFK 356 Pyrophorus PLVDKYDLSSLRELCCGAAPLAKEVAEVAVKRLNLPGIRCGFGLTESTSANIHSLRDEFK 355 Luciola ELLNKYDLSNLVEIASGGAPLSKEVGEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDK 360 :::**:**.* *: .*.** .:*: *...:*:::.*:* *:**** * : .: *

AbLL IGSSGVVVPFMAVKIRDVESGKTLKPTQIGEICVKGDMLMKGYAGNEKATKEMIDEDGWL 416 Pyrophorus SGSLGRVTPLMAAKIADRETGKALGPNQVGELCVKGPMVSKGYVNNVEATKEAIDDDGWL 415 Luciola PGASGKVVPLFSAKVIDLDTKKSLGPNRRGEVCVKGPMLMKGYVNNPEATKELIDEEGWL 420 *: * *.*:::.*: * :: *:* *.: **:**** *: ***..* :**** **::***

AbLL HTGDIGYFDKDGHIYIVDRIKELIKYKGFQVPPAELEALLLHHPCVKDAAVIGIPDELAG 476 Pyrophorus HSGDFGYYDEDEHFYVVDRYKELIKYKGSQVAPAELEEILLKNPCIRDVAVVGIPDLEAG 475 Luciola HTGDIGYYDEEKHFFIVDRLKSLIKYKGYQVPPAELESVLLQHPNIFDAGVAGVPDPVAG 480 *:**:**:*:: *:::*** *.****** **.***** :**::* : *..* *:** **

AbLL ELPAAFIVKQHGKEVTEKEIVDYIAKQVSSAKHLRGGVRFIPDIPRTAAGKIQRNLLRNM 536 Pyrophorus ELPSAFVVKQPGKEITAKEVYDYLAERVSHTKYLRGGVRFVDSIPRNVTGKITRKELLKQ 535 Luciola ELPGAVVVLESGKSMTEKEVMDYVASQVSNAKRLRGGVRFVDEVPKGLTGKIDGRAIREI 540 ***.*.:* : **.:* **: **:*.:** :* *******: .:*: :*** . : :

AbLL IAKKKSKL 544 Fatty acyl-CoA synthetase in A. binodulus Pyrophorus LLEKSSKL 543 Click beetle Pyrophorus plagiophthalmus luciferase Luciola LKKPVAKM 548 Firefly Luciola cruciata luciferase : : :*: AbLL ---MSKESNIVYGPVGAAPVLESTAGKQLFDSLKRHGHL-PQAIIDYQTKQSISYKNLFE 56 Pyrophorus --MMKREKNVVYGPEPIHPLEDLTAGEMLFRALRKHSHL-PQALVDVYGEEWISYKEFFE 57 Luciola MENMENDENIVVGPKPFYPIEEGSAGTQLRKYMERYAKLGAIAFTNAVTGVDYSYAEYLE 60 M394 *..:.*:* ** *: : :** * :.::.:* . *: : ** : :*

AbLL PIVDKYNLSSLKEIICGAAPSGREIVEAVVKRLKVSGIRYGYGLTECGSAICTTPPNNFK 356 Pyrophorus PLVDKYDLSSLRELCCGAAPLAKEVAEVAVKRLNLPGIRCGFGLTESTSANIHSLRDEFK 355 Luciola ELLNKYDLSNLVEIASGGAPLSKEVGEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDK 360 :::**:**.* *: .*.** .:*: *...:*:::.*:* *:**** * : .: *

AbLL PIVDKYNLSSLKEIICGAAPSGREIVEAVVKRLKVSGIRYGYGLTECTLAICTTPPNNFK 356 Pyrophorus PLVDKYDLSSLRELCCGAAPLAKEVAEVAVKRLNLPGIRCGFGLTESTSANIHSLRDEFK 355 Luciola ELLNKYDLSNLVEIASGGAPLSKEVGEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDK 360 :::**:**.* *: .*.** .:*: *...:*:::.*:* *:**** * : .: *

AbLL PIVDKYNLSSLKEIICGAAPSGREIVEAVVKRLKVSGIRYGYGLTESGLAICTTPPNNFK 356 Pyrophorus PLVDKYDLSSLRELCCGAAPLAKEVAEVAVKRLNLPGIRCGFGLTESTSANIHSLRDEFK 355 Luciola ELLNKYDLSNLVEIASGGAPLSKEVGEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDK 360 :::**:**.* *: .*.** .:*: *...:*:::.*:* *:**** * : .: *

AbLL PIVDKYNLSSLKEIICGAAPSGREIVEAVVKRLKVSGIRYGYGLTESTLAICTTPPNNFK 356 Pyrophorus PLVDKYDLSSLRELCCGAAPLAKEVAEVAVKRLNLPGIRCGFGLTESTSANIHSLRDEFK 355 Luciola ELLNKYDLSNLVEIASGGAPLSKEVGEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDK 360 :::**:**.* *: .*.** .:*: *...:*:::.*:* *:**** * : .: *

AbLL PIVDKYNLSSLKEIICGAAPSGREIVEAVVKRLKVSGIRYGYGLTECTSAICTTPPNNFK 356 Pyrophorus PLVDKYDLSSLRELCCGAAPLAKEVAEVAVKRLNLPGIRCGFGLTESTSANIHSLRDEFK 355 Luciola ELLNKYDLSNLVEIASGGAPLSKEVGEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDK 360 :::**:**.* *: .*.** .:*: *...:*:::.*:* *:**** * : .: *

AbLL PIVDKYNLSSLKEIICGAAPSGREIVEAVVKRLKVSGIRYGYGLTESGSAICTTPPNNFK 356 Pyrophorus PLVDKYDLSSLRELCCGAAPLAKEVAEVAVKRLNLPGIRCGFGLTESTSANIHSLRDEFK 355 Luciola ELLNKYDLSNLVEIASGGAPLSKEVGEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDK 360 :::**:**.* *: .*.** .:*: *...:*:::.*:* *:**** * : .: *

AbLL PIVDKYNLSSLKEIICGAAPSGREIVEAVVKRLKVSGIRYGYGLTESTSAICTTPPNNFK 356 Pyrophorus PLVDKYDLSSLRELCCGAAPLAKEVAEVAVKRLNLPGIRCGFGLTESTSANIHSLRDEFK 355 Luciola ELLNKYDLSNLVEIASGGAPLSKEVGEAVARRFNLPGVRQGYGLTETTSAIIITPEGDDK 360 :::**:**.* *: .*.** .:*: *...:*:::.*:* *:**** * : .: *

AbLL IAKKKSKL 544 Fatty acyl-CoA synthetase in A. binodulus Pyrophorus LLEKSSKL 543 Click beetle Pyrophorus plagiophthalmus luciferase Luciola LKKPVAKM 548 Firefly Luciola cruciata luciferase : : :*:

rlu)

4 10

Luminescence Luminescence 10 (x activity 2

0 No AbLL C343C C343C C343S C343C C343S C343S C343S Enz -Wt G344G G344T G344T G344T G344G G344G G344T L345S L345S L345S L345L L345L L345S L345L Firefly luciferase is "readily evolvable" by a few mutations of fatty acyl-CoA Synthtease.

Oba et al. (2009) FEBS Lett.

rlu)

4 10

Luminescence Luminescence 10 (x activity 2

0 No AbLL C343C C343C C343S C343C C343S C343S C343S Enz -Wt G344G G344T G344T G344T G344G G344G G344T L345S L345S L345S L345L L345L L345S L345L Prado et al., 2011 Oba et al., 2009

Prado RA et al. (2011) Photochem. Photobiol. Sci. 10: 1226-1232.

Viviani VR et al. (2013) Biochemistry 52: 3963-3973. Zophobas morio (Tenebrionidae) Firefly luciferase is "readily evolvable" by a few mutations of fatty acyl-CoA Synthtease.

Oba et al. (2009) FEBS Lett.

rlu)

4 10

Luminescence Luminescence 10 (x activity 2

0 No AbLL C343C C343C C343S C343C C343S C343S C343S Enz -Wt G344G G344T G344T G344T G344G G344G G344T L345S L345S L345S L345L L345L L345S L345L Duality of firefly luciferase ✓ To date, luciferase gene in firefly (Lampyridae) have been isolated from over 30 species (Oba et al., 2012).

Cratomorphus distinctus (AY633557) Photinus pyralis (AB644228) Lucidina biplagiata (AB535101) Cyphonocerus ruficollis 1.00/100 Luciola cruciata LcLuc1 (AB220162) 1.00/- 1.00/97 Aquatica lateralis LlLuc1 (AB644226) Luciola italica (DQ138966) 1.00/100 1.00/99 Luciola mingrelica (S61961) 0.56/64 Luciolinae 0.88/86 Luciola parvula (AB644227) 1.00/94 Luciola unmunsana (AF420006) 0.99/- 0.93/- Lampyroidea maculata (DQ137139) 1.00/100 LAMPYRIDAE Luciola terminalis (EU302126)* Photuris pennsylvanica (D25416) 1.00/100 Photurinae Cyphonocerus ruficollis (AB604789) Psilocladinae Drilaster axillaris 0.52/99 Drilaster axillaris (AB604790) 0.52/- Stenocladius azumai azumai (AB644225) Ototretinae Photuris pennsylvanica (U31240) Photurinae 1.00/100 1.00/100 Luciola cruciata LcLuc2 (AB490793) 1.00/100 Luciolinae Aquatica lateralis LlLuc2 (AB693934) luciferase Beetle Phrixothrix hirtus head (AF139645) 1.00/100 Phrixothrix vivianii lateral (AF139644) 1.00/100 Phrixothrix hirtus lateral PHENGODIDAE 1.00/100 Brasilocerus sp. lateral (FJ545728)* 1.00/100 (AF139645)* 1.00/88 Taximastinocerus sp. lateral (GU062703)* Rhagophthalmus ohbai (AB255748) Stenocladius azumai 1.00/64 RHAGOPHTHALMIDAE 1.00/100 Pyrophorus plagiophthalmus dGR (AF543413)* 1.00/- Pyrophorus mellifluus dGR (AF545854)* 1.00/98 Pyrophorus plagiophthalmus vOR (AF543373)* 1.00/-Pyrophorus mellifluus vOR (AF545853)* 0.81/- 1.00/66 Pyrophorus angustus dPaLuc (AB479112) ELATERIDAE 1.00/100 0.81/- Pyrearinus termitilluminans (AF116843) Photophorus jansonii (AB767301) 1.00/95 Pyrophorus angustus vPaLuc (AB479113) 0.99/66 Agrypnus binodulus AbLL (AB353886) 1.00/98 Pyrophorus angustus PaLL (AB479114) Luciferase-like 0.67/94 Luciola cruciata LcLL2 (AB196456) Tribolium castaneum TmLL-1 (AB098615) acyl-CoA synthetase 0.67/- 1.00/100 Tribolium castaneum TmLL-2 (AB098616) 0.97/- Luciola cruciata LcLL1 (AB196455) Drosophila melanogaster CG6178 (NM_142964) Oba, Y (2014) Insect Bioluminecence in the Post-Molecular Biology Tribolium castaneum TmLL-3 (AB098617) Era. In: Hoffmann KH (ed.) Insect Molecular Biology and Ecology, Parsley 4-coumarate:CoA ligase 1 CRC Press. (P14912) Rat long-chain fatty-acyl CoA synthetase 1 (P18163) Asterisks (*) indicate the luciferase genes for which luminescence activities of the recombinant protein were not examined. ✓ To date, luciferase gene in firefly (Lampyridae) have been isolated from over 30 species (Oba et al., 2012). ✓ Two different luciferases were isolated only from Photuris pennsylvanica (Kutuzov et al., 1996).

Cratomorphus distinctus (AY633557) Photinus pyralis (AB644228) Lucidina biplagiata (AB535101) 1.00/100 Luciola cruciata LcLuc1 (AB220162) 1.00/- 1.00/97 Aquatica lateralis LlLuc1 (AB644226) Luciola italica (DQ138966) 1.00/100 1.00/99 Luciola mingrelica (S61961) 0.56/64 Luciolinae 0.88/86 Luciola parvula (AB644227) 1.00/94 Luciola unmunsana (AF420006) 0.99/- 0.93/- Lampyroidea maculata (DQ137139) 1.00/100 LAMPYRIDAE Luciola terminalis (EU302126)* Photuris pennsylvanica Photuris pennsylvanica (D25416) 1.00/100 Photurinae Cyphonocerus ruficollis (AB604789) Psilocladinae 0.52/99 Drilaster axillaris (AB604790) 0.52/- Stenocladius azumai azumai (AB644225) Ototretinae Photuris pennsylvanica (U31240) Photurinae 1.00/100 1.00/100 Luciola cruciata LcLuc2 (AB490793) 1.00/100 Luciolinae Aquatica lateralis LlLuc2 (AB693934) luciferase Beetle Phrixothrix hirtus head (AF139645) 1.00/100 Phrixothrix vivianii lateral (AF139644) 1.00/100 Phrixothrix hirtus lateral PHENGODIDAE 1.00/100 Brasilocerus sp. lateral (FJ545728)* 1.00/100 (AF139645)* 1.00/88 Taximastinocerus sp. lateral (GU062703)* Rhagophthalmus ohbai (AB255748) 1.00/64 RHAGOPHTHALMIDAE 1.00/100 Pyrophorus plagiophthalmus dGR (AF543413)* 1.00/- Pyrophorus mellifluus dGR (AF545854)* 1.00/98 Pyrophorus plagiophthalmus vOR (AF543373)* 1.00/-Pyrophorus mellifluus vOR (AF545853)* 0.81/- 1.00/66 Pyrophorus angustus dPaLuc (AB479112) ELATERIDAE 1.00/100 0.81/- Pyrearinus termitilluminans (AF116843) Photophorus jansonii (AB767301) 1.00/95 Pyrophorus angustus vPaLuc (AB479113) 0.99/66 Agrypnus binodulus AbLL (AB353886) 1.00/98 Pyrophorus angustus PaLL (AB479114) Luciferase-like 0.67/94 Luciola cruciata LcLL2 (AB196456) Tribolium castaneum TmLL-1 (AB098615) acyl-CoA synthetase 0.67/- 1.00/100 Tribolium castaneum TmLL-2 (AB098616) 0.97/- Luciola cruciata LcLL1 (AB196455) Drosophila melanogaster CG6178 (NM_142964) Oba, Y (2014) Insect Bioluminecence in the Post-Molecular Biology Tribolium castaneum TmLL-3 (AB098617) Era. In: Hoffmann KH (ed.) Insect Molecular Biology and Ecology, Parsley 4-coumarate:CoA ligase 1 CRC Press. (P14912) Rat long-chain fatty-acyl CoA synthetase 1 (P18163) Asterisks (*) indicate the luciferase genes for which luminescence activities of the recombinant protein were not examined. ✓ To date, luciferase gene in firefly (Lampyridae) have been isolated from over 30 species (Oba et al., 2012). ✓ Two different luciferases were isolated only from Photuris pennsylvanica (Kutuzova, Hannah and Wood, 1996). ✓ Dr. John Day (UK) suggested the presence of two luciferases in various firefly species (Day et al. 2009).

Day JC et al. (2009) Mol. Phylogenet. Evol. 50: 93-101. All subfamilies of Lampyridae were included in Luc1 clade.

1.00/99 0.1 changes Diaphanes pectinealis (DQ408300)* 0.97/99 Lampyris turkestanicus (AY742225) Nyctophila cf. caucasica (DQ072141)* 1.00/89 1.00/100 Lampyris noctiluca (X89479) 1.00/100 Pyrocoelia rufa (AF328553) 0.98/99 1.00/100 Pyrocoelia miyako (L39928) Lampyrinae Luc1-type Pyrocoelia pectoralis (EF155570)* 1.00/100 Pyrocoelia pygidialis (ACF61063) One luciferase gene was already firefly luciferase Cratomorphus distinctus (AY633557) isolated from both Photinus pyralis (AB644228) Lucidina biplagiata (AB535101) species.(Tatsumi et al., 1989, 1.00/100 Luciola cruciata LcLuc1 (AB220162) 1992) 1.00/- 1.00/97 Aquatica lateralis LlLuc1 (AB644226) Luciola cruciata Luc2-type Luciola italica (DQ138966) 1.00/100 1.00/99 Luciola mingrelica (S61961) 0.56/64 Luciolinae firefly luciferase 0.88/86 Luciola parvula (AB644227) 1.00/94 Luciola unmunsana (AF420006) 0.99/- 0.93/- Lampyroidea maculata (DQ137139) 1.00/100 LAMPYRIDAE Luciola terminalis (EU302126)* Photuris pennsylvanica (D25416) 1.00/100 Photurinae Cyphonocerus ruficollis (AB604789) Psilocladinae 0.52/99 Drilaster axillaris (AB604790) 0.52/- Stenocladius azumai azumai (AB644225) Ototretinae Photuris pennsylvanica (U31240) Photurinae Aquatica lateralis 1.00/100 1.00/100 Luciola cruciata LcLuc2 (AB490793) 1.00/100 Luciola lateralis LlLuc2 (AB693934) Luciolinae Phrixothrix hirtus head (AF139645) 1.00/100 Phrixothrix vivianii lateral (AF139644) Second luciferase gene was 1.00/100 Phrixothrix hirtus lateral PHENGODIDAE Gene duplication occurred before 1.00/100 Brasilocerus sp. lateral (FJ545728)* 1.00/100 (AF139645)* isolated with full length ! diversification of Lampyridae. 1.00/88 Taximastinocerus sp. lateral (GU062703)* Rhagophthalmus ohbai (AB255748) 1.00/64 RHAGOPHTHALMIDAE 1.00/100 Pyrophorus plagiophthalmus dGR (AF543413)* 1.00/- Pyrophorus mellifluus dGR (AF545854)* 1.00/98 Pyrophorus plagiophthalmus vOR (AF543373)* 1.00/-Pyrophorus mellifluus vOR (AF545853)* 0.81/- 1.00/66 Pyrophorus angustus dPaLuc (AB479112) ELATERIDAE 1.00/100 0.81/- Pyrearinus termitilluminans (AF116843) Photophorus jansonii (AB767301) 1.00/95 Pyrophorus angustus vPaLuc (AB479113) 0.99/66 Agrypnus binodulus AbLL (AB353886) 1.00/98 Pyrophorus angustus PaLL (AB479114) Luciferase-like 0.67/94 Luciola cruciata LcLL2 (AB196456) Tribolium castaneum TmLL-1 (AB098615) acyl-CoA synthetase 0.67/- 1.00/100 Tribolium castaneum TmLL-2 (AB098616) 0.97/- Luciola cruciata LcLL1 (AB196455) Drosophila melanogaster CG6178 (NM_142964) Oba, Y (2014) Insect Bioluminecence in the Post-Molecular Biology Tribolium castaneum TmLL-3 (AB098617) Era. In: Hoffmann KH (ed.) Insect Molecular Biology and Ecology, Parsley 4-coumarate:CoA ligase 1 CRC Press. (P14912) Rat long-chain fatty-acyl CoA synthetase 1 (P18163) Asterisks (*) indicate the luciferase genes for which luminescence activities of the recombinant protein were not examined. Luminescence activity of Luc2 is comparable to that of Luc1, and Luminescence color for Luc2 luciferase is Green Most bluish among firefly luciferases !

Luminescence spectra of the recombinant LlLuc1 & LlLuc2

100 80 LlLuc2 (pH 7.9) lmax 539 nm 60 LlLuc1 (pH 7.5) lmax 550 nm 40 20

Relative intensity (%) intensity Relative 0 450 500 550 600 650 700 Aquatica lateralis Wavelength (nm) Ancestral luciferase in Lampyridae probably emitted with Green

568 nm Luciola parvula (Ohmiya et al., 1995) 566 nm Luciola italica (Branchini et al., 2006) 566 nm Luciola mingrelica (Koksharov and Ugarova, 2008) Luc1-type

Ancestral luciferase 545 nm Stenocladius azumai (Oba et al., 2011) in Lampyridae 545 nm Drilasiter axillaris (Oba et al., 2011) 538 nm Amydetes fanestratus (Viviani et al., 2011)

543 nm Luciola cruciata (Oba et al., 2010) 539 nm Luciola lateralis (Oba et al., 2013) Luc2-type 538 nm Photuris pennsylvanica (Kutuzova et al., 1997)

Luc2 is predominantly expressed in Eggs and Pupae and Female Adults

2.5 2.5 Luc1 Male Luc2 2.0 2.0 Luc2 was expressed only in ovary! 1.5 1.5 Female 1.0 Female 1.0 Male

0.5 0.5 Relative mRNA level RelativemRNA Relative mRNA level RelativemRNA 0 0

Larva Pupa Larva Pupa

Last instar larva Adult (male)

Eggs

1st instar larva 2-day-pupa (male) 6-day-pupa (male) Whole body of pupa, especially prothorax glow continuously, not enhanced by stimulation.

5 mm

Lumnescence of the adult lantern in pupa is enhanced by stimulation. Glow continuously, not enhanced by stimulation.

Eggs of L. lateralis and the Luminescence of L. lateralis pupa luminescence Luc2 is primarily expressed in Prothorax (T1 segment)

LlLuc1

7 Male

6 5 4 Luc1 is used for the luminescence of the H+T1 3 lantern in pupa 2 Female 1 T2+T3 Relative mRNA mRNA level Relative 0 H+T1 T2+T3 Abd

Abd LlLuc2 7 6 5 Female 4 Luc2 is used for the Male luminescence of the 3 pupal whole body, 2 especially prothorax 1

Relative mRNA mRNA level Relative 0 H+T1 T2+T3 Abd Luc2 Luc1 Luc2

Egg

Luc1 Larva Luc2 Fireflies have two differenct luciferases (Luc1 Pupa and Luc2). (in female ovary)

The gene was duplicated at the most basal position of the family Lampyridae.

Luc1-type luciferase is responsible for the on/off-capable yellowish luminescence of larval, pupal and adult lanterns. Luc1

Luc2-type luciferase is responsible for the Adult continuous greenish dim glow of eggs and whole pupal body.

Oba et al. (2013) Photochem. Photobiol. Sci. How firefly get luciferin molecule? Published on 01 January 1976. Downloaded by Nagoya Daigaku on 25/03/2014 08:39:03. View Article Online / Journal Homepage / Table of Contents for this issue

After injection of water (control)

100 281

Relative intensity (%) intensity Relative 0 280 282 284 286 288 290 292 294 m/z

・13C

After injection of water (control)

100 281

Relative intensity (%) intensity Relative 0 280 282 284 286 288 290 292 294 m/z

After injection of deuterium labeled hydroquinone and 13C labeled cysteine

100

+ /

50 +

Relative intensity (%) intensity Relative 0 280 282 284 286 288 290 292 294 m/z Biosynthetic pathway of firefly luciferin

1,4-Hydroquinone L-Cysteine L-Cysteine

Firefly luciferin Damaged skin by hydroquinone treatment No hydroquinone was detected in firefly extract. But after hydrolization, hydroquinone was detected.

Authentic hydroquinone

0 Firefly extract before hydrolization (1/10 specimen)

Firefly extract * after HCl treat (1/10 specimen)

0 2.00 4.00 6.00 8.00 10.00 [min] Red flour beetle Tribolium castaneum

Ejecting unpleasant secretion containing hydroquinone and benzoquinone when disturbed.

Arbutin Hydroquinone Benzoquinone

Arbutin: a glycosilated 'less toxic' hydroquinone found in several plants and insects. Arbutin is now used worldwide for 'safetly' whitening mosmetic. Happ GM (1978) J. Insect Physiol. 14: 1821-1837. Arbutin Hydroquinone Glucose

Authentic arbutin Authentic hydroquinone

Firefly extract Firefly extract after hydrolysis

* True biosynthetic pathway of firefly luciferin

Arbutin

1,4-Hydroquinone L-Cysteine L-Cysteine

Firefly luciferin Oba et al. (2013) PLoS ONE Conclusion

1 Firefly luciferase was originated from an enzyme involving in fatty acid metabolism (fatty acyl-CoA synthetase), by small numbers of amino acid substitutions.

2 Fireflies have two differenct luciferases (Luc1 and Luc2). The gene was duplicated at the most basal position of the family Lampyridae.

3 Firefly luciferin was biosynthesized from arbutin (glycosilated ‘less toxic’ hydroquinone) and L-cysteines in firefly.

N N CO OH ATP, Mg2+, O N N O 2 + Light H O S S Firefly luciferase H O S S Firefly luciferin

Biosynthesized from Evolved from an common arbutin and cysteines enzyme for fat metabolism "The presence of luminous organs in a few insects, belonging to different families and orders, offers a parallel case of difficulty."

In: The Origin of Species, Chapter VI 'Difficulties on theory' by C. Darwin

N N CO OH ATP, Mg2+, O N N O 2 + Light H O S S Firefly luciferase H O S S Firefly luciferin

Biosynthesized from Evolved from an enzyme arbutin and cysteines for fat metabolism Acknowledgements

Alumni: Makiko Ito, Mitsunori Sato, Reiko Sagegami-Oba, Yuichiro Ohta, Takeru Shintani, Kiichi Tanaka, Takanori Nakamura, Shinsuke Ohkubo, Koichiro Iida, Mizuho Kumazaki, Nanae Mori, Mayumi Yoshida, Mana Furuhashi, Naoki Yoshida (Firefly team of Oba's lab)

Grad. Students: Shusei Kanie (Firefly team of Oba's lab)

Corraborators: Darrin T Schultz (Fulbright fellow), Satoshi Inouye (JNC CO.), Haruyoshi Ikeya (Toin Gakuen High School), Makoto Ojika (Nagoya Univ.)

Advice: Marc A Branham (Florida Univ.), Xinhua Fu (Huazhong Agr. Univ.), Victor B Meyer-Rochow (Oulu Univ.), Nobuyoshi Ohba (Ohba Hotaru Institute), Keisuke Kawano (The Firefly Museum of Toyota City), John C Day (CEH, UK)

Japanese Woodblock "Koromo River near Tennouji Temple" by Kiyochika Kobayashi, 1880 (Oba's personal collection) 'second' lumious millipede, Paraspirobolus lucifugus, distributing in Japan Firefly luciferin is present in Lampyridae but not in the relatives

Luciola cruciata (Lampyridae) Aquatica lateralis (Lampyridae) Luciola parvula (Lampyridae) Lucidina biplagiata (Lampyridae)

Lycocerus suturellus (Cantharidae) Lycocerus vitellinus (Cantharidae) Lycostomus modestus (Lycidae) Plateros coracinus (Lycidae)

Oba et al. (2008) Biosci. Biotechnol. Biochem. 1.00/99 0.1 changes Diaphanes pectinealis (DQ408300)* 0.97/99 Lampyris turkestanicus (AY742225) Nyctophila cf. caucasica (DQ072141)* 1.00/89 1.00/100 Lampyris noctiluca (X89479) 1.00/100 Pyrocoelia rufa (AF328553) 0.98/99 1.00/100 Pyrocoelia miyako (L39928) Lampyrinae Pyrocoelia pectoralis (EF155570)* 1.00/100 Pyrocoelia pygidialis (ACF61063) Cratomorphus distinctus (AY633557)

Luciola terminalis (EU302126)* LAMPYRIDAE Photuris pennsylvanica (D25416) 1.00/100 Photurinae Cyphonocerus ruficollis (AB604789) Psilocladinae 0.52/99 Drilaster axillaris (AB604790) 0.52/- Stenocladius azumai azumai (AB644225) Ototretinae Photuris pennsylvanica (U31240) Photurinae 1.00/100 1.00/100 Luciola cruciata LcLuc2 (AB490793) 1.00/100 Luciolinae Aquatica lateralis LlLuc2 (AB693934) luciferase Beetle Phrixothrix hirtus head (AF139645) 1.00/100 Phrixothrix vivianii lateral (AF139644) 1.00/100 Phrixothrix hirtus lateral PHENGODIDAE 1.00/100 Brasilocerus sp. lateral (FJ545728)* 1.00/100 (AF139645)* 1.00/88 Taximastinocerus sp. lateral (GU062703)* Rhagophthalmus ohbai (AB255748) 1.00/64 RHAGOPHTHALMIDAE 1.00/100 Pyrophorus plagiophthalmus dGR (AF543413)* 1.00/- Pyrophorus mellifluus dGR (AF545854)* 1.00/98 Pyrophorus plagiophthalmus vOR (AF543373)* 1.00/-Pyrophorus mellifluus vOR (AF545853)* 0.81/- 1.00/66 Pyrophorus angustus dPaLuc (AB479112) ELATERIDAE 1.00/100 0.81/- Pyrearinus termitilluminans (AF116843) Photophorus jansonii (AB767301) 1.00/95 Pyrophorus angustus vPaLuc (AB479113) 0.99/66 Agrypnus binodulus AbLL (AB353886) 1.00/98 Pyrophorus angustus PaLL (AB479114) Luciferase-like 0.67/94 Luciola cruciata LcLL2 (AB196456) Tribolium castaneum TmLL-1 (AB098615) acyl-CoA synthetase 0.67/- 1.00/100 Tribolium castaneum TmLL-2 (AB098616) 0.97/- Luciola cruciata LcLL1 (AB196455) Drosophila melanogaster CG6178 (NM_142964) Oba, Y (2014) Insect Bioluminecence in the Post-Molecular Biology Tribolium castaneum TmLL-3 (AB098617) Era. In: Hoffmann KH (ed.) Insect Molecular Biology and Ecology, Parsley 4-coumarate:CoA ligase 1 CRC Press. Tenebrio molitor (P14912) Rat long-chain fatty-acyl CoA synthetase 1 (P18163) Asterisks (*) indicate the luciferase genes for which luminescence activities of the recombinant protein were not examined. RESULT 3．Homologous genes of firefly luciferase in flour beetle Tenebrio molitor (TmLL-1,2,3) are a "fatty acyl-CoA synthetase". Oba et al. (2006) Insect Mol. Biol.

Bioluminescence reaction (oxygenation)

2+ N N C O O H ATP, Mg , O2 N N O + Light H O S S TmLL-1,2,3 H O S S Firefly luciferin Oxyluciferin

Fatty acyl-CoA synthetic reaction (thioesterification)

ATP, Mg2, CoA C O O H C O - S - C o A TmLL-1,2,3 Fatty acid Fatty acyl-CoA

Tenebrio molitor