Supplementary Material s74

Supplementary Material

IpFPPS MF-SMMACRNRPCREWLKEFRRNISKTSTDKNSDAIIRSQDKMQHASKTHEVAFEKNLSY 59 DjFPPS MF-SMKLCRNRSCREFLREARRTISKTSTDKNSGAISRAPDHKLN------VESDSTGSY 53 TfFPPS MFPSVKICVNRTSRDILRKFQRHISKTSSVPNSDAISRQQDNTLKVN-HDLEAYNRNKSS 59 CfFPPS1 MF-STTRSLEKIVQSYKNELRRQISKTTSVTNSDAMAPRLDQPANKT----PTNEETGLP 55 MpFPPS1 MY------KTLTTFTRALSRRTAFSLCPAVAAG------27 *: . * :*: :: . *:

IpFPPS TRWMKQMQHNNIRALSTIQQTLVRPQQSSSLASKEQSRDFMAQFPDIIRELTEVGRNQEL 119 DjFPPS SRWKKQMHHNNIRALSTIQQSMIRPVQSSALVTKEQSRDFMALFPDLVRELTEVGKSQEL 113 TfFPPS IKWAKLSRHNNIRALSTIQTKVKPPVTSTPLVSKEESREFMAIFPDIVRDLTDAGRHTDI 119 CfFPPS1 KKLLKLQKYH--RFLSTLTPQQMPLAARGLAVSKDQSREFMACFPDIVRDLTETSKHVDV 113 MpFPPS1 ------RENHFRSMSVILPPPVTSVITGALVSKDELGNFMAVFPDIVKDLTDTSLQLNK 80 : : * :*.: .:*:: :*** ***::::**:.. :

IpFPPS PEVMKRYARVLQYNTPNGKKTRGLITISTYKMLEDPAKITTENLKKAGILGWCIEMLHTY 179 DjFPPS HDVMRRFARVLQYNTPTGKKNRGLIILSTYRMLEDPEKLTPENIRLASILGWCAEMVHAY 173 TfFPPS PEVTKRFAKVLQYNVPTGKKTRGLSCVTAYKILEKPENLTPENVKLANILGWCIELLRGF 179 CfFPPS1 PEASRWLAKLLQYNVPNGKKNRGLATVLAYKMLEKPENLTPENVHLANVMGWCTEMFHTH 173 MpFPPS1 PDVTKWLENLLQYNVPGGKNNRGLILVSSFKMLSSPSYLTDENLRLSYILGWCVEILQAY 140 :. : .:****.* **:.*** : ::::*..* :* **:: : ::*** *:.: .

IpFPPS FLIIDDIIDHSDTRRGAICWYRQPGIGLTAVYDAVMMENGVYLLLKRHFKDHPMYTNIIE 239 DjFPPS VLILDDIMDGSETRRGALCWFRQSGIGLTAVNDAVMIENAVYLLIKRHLKDHPMYVPLME 233 TfFPPS ELIVDDIVDNAETRRNAPCWFRKDNVGYFAIGDSILLESSVYSILRKYFSTLPCYVPMME 239 CfFPPS1 QLLLNDIMEGTEMRRGAPAWHRRPDVGLSSINDAILVQSAMYSTLKKHFHAKPYYKNVIE 233 MpFPPS1 QLVMDDLMDNAITRRGRPCWYRHNDVGLMAVNDGILLEQAIYQLLKKYFKDKPYYTHILE 200 *:::*::: : **. .*.*: .:* :: *.::::..:* ::::: * * ::*

IpFPPS LFHDMALKTSLGQSLDTMCLNDDGKPKLDIHNE-QDTSIVNYKTSYYSFYLPVAAAMYLL 298 DjFPPS LFHEGNLKTTLGQSLDAMCLDTNGKPKLDMFTMSRYTSIVKYKTAFYSFQMPVAIAMYLA 293 TfFPPS LVHDVTLKTAMGKSLEGLIMQ-GGKPNLDKFTMKNYNLMTKYKVGYYTFQLPVALAMYFA 298 CfFPPS1 MFNEMLLKCSTGQFLEKAMAK-TDKPDLSQFTMEKYLTIAKYKTAYHTFQMPVGLALLMS 292 MpFPPS1 LFYDVTMKSAMGQCLDMLTAKSFKSKKLEKYTMENYKAIVKYKTAYYSFVLPVCLAMRMT 260 :. : :* : *: *: . . .*. .. . :.:**..:::* :** *: :

IpFPPS GMDDPEQHRQARTILLDMGQFFQIQDDFLDCFGDPNVTGKLGTDIQDGKCSWLAVMALQR 358 DjFPPS GMSDEEQHRQAKTILMEMGQFFQIQDDFLDCFGDPTVTGKVGTDIQDGKCSWLAVVALQR 353 TfFPPS NMFDAEQHRQAKTILLEMGQFLQIQTDFLDCFGDP--AGKIGNDIQAGRCSWLAIVALQR 356 CfFPPS1 GVDDLETHRQAKTILLEMGQFFQIQDDFLDCFGDPAVTGKNGSDIQDGKCTWLAVVALQR 352 MpFPPS1 NVNDQEIFRQAKVILLDMGQFFQIQDDYLDCYGNPEITGKIGTDIEDGKCSWLAVKALQK 320 .: * * .***:.**::****:*** *:***:*:* :** *.**: *:*:***: ***:

IpFPPS STPAQRKIMEEHYGRSEPESVAIIKQLYEDLTLPNTYAIYEEESFNIIKTHIQQISKDCV 418 DjFPPS ASPAQRKIMEEHYGRPEPESIARIKNLYVDLCLPNTYAIYEEESFNIIKTHIQQISKGLR 413 TfFPPS ANPAQRKLMEDYYGMPGAQAGQIIKMLYEELSLPATYAAYEEESYNIINTHIQQISKGLP 416 CfFPPS1 ASPAQRHVLEEHYGSSKPEDVEKIKELYETLQLPHTYSVYEDATYDLLRTQIQQVTRGLP 412 MpFPPS1 VTTEQKKIMEDNYGIDDQFNVAVIKDLYQQLKLQNTFLLYEEESYNLIRTCIQNFSPGLS 380 .. *::::*: ** ** ** * * *: **: :::::.* **:.: .

IpFPPS T-IFSKHYGEAFKRES--- 433 DjFPPS HDLFLKIMENIYKREC--- 429 TfFPPS HELFFTLMEKLYRINEYDC 435 CfFPPS1 HDLFFKILDNIFRRNI--- 428 MpFPPS1 QDMFFKLLEKIYKRTL--- 396 :* . : ::

Supplementary Figure 1. ClustalW alignment of TfFPPS with two other coleopteran FPPSs [Ips pini FPPS (IpFPPS); Dendroctonus jeffreyi FPPS (DjFPPS), as well as with a lepidopteran FPPS1 [Choristoneura fumiferana FPPS1 (CfFPPS1)] and an aphid FPPS1 [Myzus persicae FPPS1 (MpFPPS1)]. The sequence shaded in gray is the first aspartate-rich motif (FARM). 2

↓ FARM DmFPPS FFIISDDVMD ApFPPS YQLVLDDIMD MpFPPS YQLVLDDIMD TfFPPS FELIVDDIVD

Supplementary Figure 2. Amino acid alignment of the FARM region (DDxxD) of TfFPPS with the corresponding residues in Drosophila melanogaster FPPS (DmFPPS) and two aphid FPPSs [Acyrthosiphon pisum FPPS (ApFPPS) and Myzus persicae FPPS (MpFPPS)]. The arrow points to the residue at position -4 relative to the FARM, which is known to play an important role on controlling product chain length in short-chain prenyltransferases such as FPPS. In most eukaryotes, this residue is aromatic (F/Y) but a glutamine (Q) substitutes for F/Y in aphid FPPSs, which can produce both C10 (geranyl diphosphate) and C15 (FPP) products (Vandermoten et al. 2008). Site-directed mutagenesis experiments have shown that the replacement of F/Y by Q was responsible for the dual GPP/FPP synthase activity of aphid FPPSs (Vandermoten et al. 2009). The Glu residue at the same position in TfFPPS is expected to have a similar impact on product chain length; it is therefore possible that this enzyme displays the same dual activity observed for aphid FPPS. However, the recombinant TfFPPS produced using a procedure used for other FPPSs (Cusson et al., 2006; Sen et al., 2007; Vandermoten et al., 2008) did not display prenyltransferase activity under in vitro conditions.

CUSSON, M., BÉLIVEAU, C., SEN, S.E., VANDERMOTEN, S., RUTLEDGE, R.J., STEWART, D., FRANCIS, F., HAUBRUGE, É., REHSE, P., HUGGINS, D.J., DOWLING, A.P.G., and GRANT, G.H. 2006. Characterization and tissue-specific expression of two lepidopteran farnesyl diphosphate synthase homologs: implications for the biosynthesis of ethyl-substituted juvenile hormones. Proteins 65:742-758.

SEN, S.E., TROBAUGH, C., BÉLIVEAU, C., RICHARD, T. and CUSSON, M. 2007. Cloning, expression and characterization of an insect farnesyl diphosphate synthase. Insect Biochem. Mol. Biol. 37:1198-1206.

VANDERMOTEN, S., CHARLOTEAUX, B., SANTINI,, S., SEN, S.E., BÉLIVEAU, C., VANDENBOL, M., FRANCIS, F., BRASSEUR, R., CUSSON, M., and HAUBRUGE, É. 2008. Characterization of a novel aphid prenyltransferase displaying dual geranyl/farnesyl diphosphate synthase activity. FEBS Letters 582:1928-1934. 3

VANDERMOTEN, S., SANTINI, S., HAYBRUGE, É., FRANCIS, F., BRASSEUR, R., CUSSON, M., and CHARLOTEAUX, B. 2009. Structural features conferring dual GPP/FPP synthase activity to an aphid prenyltransferase. Insect Biochem. Mol. Biol. 39:707-716.