Glucosylglycerate phosphorylase, a novel enzyme specificity involved in compatible solute metabolism

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1 Supplementary Information for Glucosylglycerate phosphorylase, a novel enzyme specificity involved in compatible solute metabolism Jorick Franceus, Denise Pinel, Tom Desmet Corresponding author: Tom Desmet, Centre for Synthetic Biology tom.desmet@ugent.be Website:

2 M. silvanus glucosylglycerate phosphorylase ATG GGC GGT AGC CAT CAT CAC CAC CAC CAC GGT ATG GCT AGC ATG TCG TCC CTG ACC CCG GAA CTG CGT CAA TCT ATT CTG GAA CAC CTG GGC TTT CTG TAT GGT GAA CGT GCA CCG GCT GTT CTG GGC CGC CTG GAA GAA ATT TGC AGC GGT TTC CCG GCA CAG CGT CGC GAA GGC GGT TGG TCT GAA AAA GAT GCG CTG CTG ATT ACC TAT GGC GAC CAG ATC CAC GCC GAA GGT GAA CCG CCG CTG CAA ACG CTG TAT GAT TTT CTG TAC GAA CGT CTG CGC GGC GTC TTT AGT GGT GTG CAT CTG CTG CCG TTC TAC CCG AGT ACC TCC GAT GAC GGC TTT TCC GTG GTT GAT TTC CAG CGT GTG GAC CCG GAA CTG GGT ACC TGG ACG GAT ATT CGT ATT ATC GCA CAG GAT TTT CGC CTG ATG GCT GAC CTG GTT TGT AAT CAT GTC AGT GCG AGC TCT CCG TGG TTT CAG GGC TTC CTG CAA GAT GAC CCG CAG TAT CAA GGC TTT TTC ATC ACC GTC GAT CCG GGT ACC GAC CTG AGC ACG GTG TTT CGT CCG CGT GCA CTG CCG CTG CTG ACC CCG TTC CAG ACG CCG TCA GGT GAA AAA CTG GTT TGG ACC ACG TTT TCG CCG GAT CAA ACC GAC CTG AAC TAT GCA AAT CCG GAA GTG CTG CTG GAA GTT ATT GAA GCT CTG CTG TGC TAC GTT CGT AAC GGT GCA GGT CTG ATC CGT CTG GAT GCA GTG GGC TTC ATT TGG AAA GAA ATC GGT ACC TCC TGT ATG CAT CTG GAA GGC GCC CAC CGT ATT GTG AAA CTG ATG CGT CTG GTC CTG GAT GCA GTG GCA CCG CAT GTG CTG CTG GTT AGC GAA ACG AAC GCA CCG CAC CGC GAA AAT ATC TCT TAT TTT GGC AAC GGT CAT GAT GAA GCG CAG CTG GTT TAC CAA TTT CCG CTG CCG CCG CTG GTC ATG CAC ACC TTC CGT ACG GGT GAC GCG TCT AAA CTG GCC GGT TGG GCG GCC GGT CTG ACC CTG CCG AGT GAA CGC ACC ACG TTT TTC AAT TTT CTG GCA TCC CAT GAT GGT ATT GGT GTC GTG CCG GCG GGC GGC ATT CTG CAA CCG GAA GAA ATC GCA GCT CTG GTG CGT CAA GCG CTG GAA CAT GGC GGT CGC GTT AAC CAC AAA GAT ACC CCG GAC GGC CCG GTC CCG TAT GAA CTG TGC CTG ACG CTG TTT GAT GCC CTG AGC AAC CCG AAC AGC GAT GAA GCG GAA GAC CTG AAA ATT GCC CGT TTC CTG GCG GCC AAT GTT ATC CTG CTG TCA CTG CAA GGC ATT CCG GGT GTG TAC ATC CAT TCG CTG TTT GGT TCA CCG TCG GAT CAC GCC GGC TTC GAA GAA TCA GGT ATT CCG CGT CGC CTG AAC CGC CAC AAA TTT ACC AAA GCA GAA CTG GAA GAA CGT CTG GCA GAC CCG GCT TCG CGT GCA GCT AAA ATC CTG GCG GCC TAT AGT CAT CTG CTG CGT GTG CGC TCC ATG CAT CCG GCA TTC CAC CCG AAT GCT CCG CAG CGT ATT CTG CCG AGC ACC GAA GTT CTG CGT ATC GTC CGT GGT GAA GGT GAT CAG GCA GTG GGT TGT TAC ATT AAT GTT ACG GAC CGT CCG CAA GTT GTC TCT CGC ATC GGT AAA AAC CTG ATC ACC GGC CAG TGG TTC ACG GGC GTT CTG AAA CCG TAT CAA GCG GCA TGG ATT ATC GAC TAA S. thermophila glucosylglycerate phosphorylase ATG GGC GGC TCT CAC CAC CAC CAC CAC CAC GGT ATG GCT AGC ATG GAA CCG GTT GAT CGT ATG CGC GAA CTG CTG TCA TTT ATC TAT GGC CCG GAT ACC GGT CGC GAC ACG TAT GAA CAT CTG CGT GCA CTG CTG GAA CGT TGG CGC AGT CGT CTG CCG GCT CCG GAT GAA GAA TAT GTG TCC GGT CGT CTG CCG CTG GAT CAC ACC GAC GCG GTT CTG ATT ACG TAC GGC GAC CAG TTT CGT CGC GAA GGT GAA GCA CCG CTG GCC ACC CTG GGC GAA TTT CTG CGT GAA TAC CTG AGT GGC ACC ATG AAA GGT GTG CAT ATT CTG CCG TTT TTC CAC TAT AGC TCT GAT GAC GGT TTT TCC GTG ATC GAT TAC CGC ACC GTT AAC CCG GAA TGG GGC ACG TGG GAA GAA GTT CGT CGC ATT AGC GGT GAA TTT CGT CTG ATG GTT GAT CTG GTC CTG AAT CAT TGC AGC GCA AAA TCT GAA TGG TTT CGT CGT TTT CTG CAA GGC GAT CCG GCT TAT GAA GAC TTT TTC ATC ACC GTT GAA CCG GGC ACG GAT CTG TCC GGT GTC TTT CGT CCG CGT GCA CTG CCG CTG GTG CAC GAA TTT GAA TCA CCG AAA GGT CCG GTC CTG GTG TGG ACC ACG TTT TCG CGC GAT CAA GTT GAC CTG AAC TAC GCG AAT CCG CGT GTC CTG CTG GAA ATG ATC GAT ATC TTT CTG TTC TAT GTG AGC CAG GGC GCC CAA ATT ATC CGC CTG GAC GCA ATT GCT TAC CTG TGG AAA GAA CTG GGT ACC CCG TGT ATC CAT CAC CCG AAA ACG CAT GCG GTG GTT AAA CTG TTT CGT GCC ATT TGC GAA GAA GTC TGT CCG TGG GTG CTG ATT ATC ACC GAA ACG AAC GTG CCG CAT GAA GAA AAT ATC TCG TAT TTC GGT GAT ATG GAC GAA GCA CAC CTG GTG TAC CAG TTT GCT CTG CCG CCG CTG GTT CTG GAT GCG TTC CTG CGC AAA GAC GTC AGC TAT CTG CGC GAA TGG GCC CGT ACC ATT GAT ACG TAT GGC GGT AAA GTG AGC TAC TTT AAC TTC CTG GCG TCT CAT GAC GGT ATT GGT GTT CTG CCG GCA CGT GGC ATC CTG CCG GAT GAA CAC ATT GAC AGC ATG ATC GAA GCC GTC AAA GAT CGT GGC GGT CTG ATC AGT TAT AAA TCC ACC CCG GAA GGT GAA GTT CCG TAT GAA CTG AAC ATT AAT TAC CTG TCA GCG ATC TCG GAA GCA CAT CTG GAT CGT CCG ACC CGT GCA CGT AAA TTT CTG GCA TCA CAG GCT GTT ATG CTG TCG CTG GTC GGC ATG CCG GGT ATT TAC GTT CAC AGT CTG CTG GGC TCC GAA AAC TGG CGT GAA GGC GTC GAA AAA ACC GGT ATG AAC CGC ACG ATC AAC CGT CAA AAA TGC TCT TAC GAT GAA ATC CTG CGC GAA CTG GAA GAC CCG GAA AGC CTG CGT TCT ATG GTG TTT AAA GGC TAC CTG GAT ATG CTG GCG GCC CGT CGC AAA AGT CGC GCG TTC GAT CCG CGT GGT ACC CAG GAA GTC CTG GAC GCA CCG GAA ACG GTG CTG GCT CTG ATG CGT CGC AGC CAT GAT GCA ACC GAA GAA GTG CTG TGC CTG ATT AAT GTG TCT CAC GTT GAA CAA GAA TGT GTC TTT CCG AGT TCC ATC TTC GCT TCA TCG CCG GAA GTG CAC CTG TTT ACC GAA CTG ACG AGC GGT GAT ACC CTG GTT CCG TAT CGC GAA GAT GAA GAC CGT TTC TCC ATT TCG CTG GGC GGC TAT GAA GTC CTG TGG CTG ACC CCG TAT CGT GAC CGT GAT TAA

3 E. coli glucosylglycerate phosphorylase ATG GGC GGC TCT CAC CAC CAC CAC CAC CAC GGT ATG GCT AGC ATG AAA CAG AAA ATT ACG GAT TAC CTG GAC GAA ATC TAC GGT GGA ACA TTT ACC GCA ACT CAT TTA CAG AAA CTT GTA ACG CGT CTT GAG AGT GCG AAA CGA TTA ATT ACA CAG CGA CGT AAA AAA CAC TGG GAT GAA AGT GAT GTC GTG TTA ATT ACC TAT GCC GAT CAA TTT CAC AGC AAT GAT TTA AAA CCA TTA CCC ACA TTT AAT CAG TTT TAC CAT CAA TGG CTG CAA AGC ATT TTT TCA CAT GTT CAT TTG TTG CCG TTT TAT CCA TGG TCA TCT GAT GAT GGC TTT TCG GTA ATT GAT TAT CAT CAG GTC GCC AGT GAA GCG GGG GAG TGG CAG GAT ATT CAG CAA CTC GGT GAA TGC AGT CAT TTA ATG TTT GAT TTT GTC TGC AAC CAT ATG TCG GCA AAA AGT GAA TGG TTT AAA AAC TAT TTA CAA CAG CAT CCA GGT TTT GAA GAT TTT TTT ATT GCC GTT GAC CCG CAA ACC GAT CTC AGC GCC GTC ACT CGC CCG CGT GCG TTA CCG TTA TTA ACG CCA TTC CAG ATG CGC GAT CAT TCA ACG CGC CAT TTA TGG ACC ACC TTT AGT GAC GAT CAA ATT GAC CTG AAT TAC CGT AGC CCT GAA GTG TTG CTG GCG ATG GTG GAT GTT TTA CTC TGT TAC CTT GCG AAA GGT GCT GAG TAT GTC CGC CTG GAT GCC GTT GGC TTT ATG TGG AAA GAG CCG GGA ACA AGC TGC ATC CAT CTG GAA AAA ACA CAT CTG ATT ATC AAA CTG TTA CGG TCG ATT ATT GAT AAC GTT GCG CCA GGT ACA GTG ATC ATT ACC GAG ACC AAT GTT CCG CAT AAA GAC AAC ATT GCT TAC TTT GGC GCA GGC GAT GAC GAA GCA CAT ATG GTG TAC CAG TTC TCG CTG CCG CCA CTG GTG CTG CAT GCG GTG CAA AAA CAG AAC GTT GAG GCG CTT TGT GCG TGG GCG CAA AAC CTG ACA CTA CCT TCC AGC AAC ACC ACC TGG TTT AAC TTC CTC GCC TCT CAC GAT GGC ATC GGG CTA AAC CCG CTA CGG GGC TTG TTG CCT GAA AGC GAA ATA TTA GAG CTG GTC GAG GCG TTA CAG CAG GAA GGT GCA TTA GTA AAC TGG AAA AAT AAT CCC GAC GGT ACA CGC AGT CCG TAT GAA ATA AAT GTG ACC TAT ATG GAT GCG TTA AGC CGC CGT GAG AGT AGC GAT GAA GAA CGT TGC GCC AGG TTT ATC CTT GCC CAT GCG ATT TTG TTA AGT TTC CCC GGT GTG CCA GCG ATA TAT ATT CAA AGT ATT CTT GGC TCG CGT AAT GAT TAC GCA GGT GTC GAA AAA CTC GGA TAT AAC CGT GCG ATT AAC CGT AAA AAA TAT CAC AGT AAA GAG ATA ACC CGA GAA CTG AAC GAT GAA GCT ACA TTA AGG CAT GCG GTA TAT CAT GAG TTG TCG CGT TTA ATT ACA CTT CGT CGC AGC CAT AAC GAG TTT CAT CCG GAT AAT AAT TTT ACC ATT GAT ACG ATT AAT TCA TCC GTA ATG CGT ATT CCA AGA AGT AAC GCT GAT GGT AAT TGT CTG ACT GGA TTG TTT AAT GTC AGT AAA AAT ATT CAG CAT GTA AAT ATT ACT AAT CTG CAT GGT CGG GAT CTG ATT AGT GAA GTT GAT ATA TTG GGT AAT GAA ATA ACG CTG CGC CCC TGG CAG GTT ATG TGG ATT AAA GGC GGC TCT CAC CAT CAC CAT CAC CAT TAA Fig. S1 Nucleotide sequences for the glucosylglycerate phosphorylases from Meiothermus silvanus, Spirochaeta thermophila and Escherichia coli. Sequences from M. silvanus and S. thermophila were codon-optimised for E. coli. Codons changed by the optimisation algorithm (GeneArt) are marked in bold. The His 6 -tag and linker sequences are underlined. For the sequence from E. coli, a C-terminal or N-terminal His 6 -tag was tried (both possible tags are shown in the sequence).

4 100 pcxp34h ptrc99a pet21a Relative activity (%) Constitutive / 20 C Constitutive / 37 C 1.0 mm IPTG / 37 C 0.1 mm IPTG / 37 C 1.0 mm IPTG / 20 C 0.1 mm IPTG / 20 C 1.0 mm IPTG / 37 C 0.1 mm IPTG / 37 C 1.0 mm IPTG / 20 C 0.1 mm IPTG / 20 C Fig. S2 Expression of. The enzyme activity in cell extracts obtained using different expression vectors (pcxp34h, ptrc99a, pet21a), IPTG concentrations (0.1 mm or 1 mm) and expression temperatures (37 C or 20 C) is compared relative to the highest activity (100% = 2.8 U/mL of cell extract) Expression at each condition was performed in duplicate and the resulting activities had a CV of < 5%.

5 Fig. S3 SDS-PAGE analysis of recombinantly expressed glucosylglycerate phosphorylase from M. silvanus. Lane 1, insoluble fraction of the cell extract; 2, insoluble fraction after mild heat purification; 3, soluble fraction of the cell extract; 4, initial flow-through of the Ni-NTA column; 5-7, wash fractions during Ni-NTA purification; 8, purified.

6 Specific activity (U/mg) Specific activity (U/mg) Concentration Glc1P (mm) Concentration D-Glycerate (mm) 0,8 1,0 Specific activity (U/mg) 0,6 0,4 0,2 Specific activity (U/mg) 0,8 0,6 0,4 0,2 0, , Concentration glucosylglycerate (mm) Concentration inorganic phosphate (mm) Fig. S4 Michaelis-Menten curves for M. silvanus glucosylglycerate phosphorylase for α-d-glucose 1-phosphate, D-glycerate, glucosylglycerate and phosphate at 42 C in 50 mm MES, ph 6.5 (synthesis direction) or ph 6.0 (degradation direction). In synthesis direction, either 20 mm D-glycerate or 100 mm α-d-glucose 1-phosphate was used as cosubstrate, while either 100 mm phosphate or 20 mm glucosylglycerate was used as cosubstrate in the degradation direction.

7 MK NK VQ LITY A D R LG DG...T IK...MK NK VQ LITY A D R LG DG...T LS...MEIQ NK AM LITY A D S LG K...N LK...MPII NK TM LITY A D S LG K...N LK MSSLTPELRQSIL E H L GF LYG ERAPAVL.GR L EEICSGF...PAQR...REGG W SEK D AL LITY G D Q IHAEGEP PL Q...MEPVDRMR E L L SF IYG PETGRDTHEA L HALLDGWRGRLPSPDEEYASGRLP L DHT D AV LITY G D Q FGRKGEA PL A...MKQKIT D Y L DE IYG GTFTATHLQK L VTRLES...AKRLITQRRKKH W DES D VV LITY A D Q FHSNDLK PL P TT SMTDI L RTR F DGVYD G VH I LPF F TPFDGA D A GF D P I D H TK V D ERL G S W D D V AE L S K T H N I M V D AI V NH M S W E S KQ FQD VL SMADI L RTR F DGVYD G VH I LPF F TPFDGA D A GF D P I D H TK V D ERL G S W D D V AE L S K T H N I M V D AI V NH M S W E S KQ FQD VL DVHQV L KED I GDAIG G VH L LPF F PS..TG D R GF A P A D Y TR V D AAF G D W A D V EA L G E E Y Y L M F D FM I NH I S R E S VM YQD FK ELNEN I ENY F GDAVG G VH L LPF F PS..TG D R GF A P I D Y HE V D SAF G D W D D V KC L G E K Y Y L M F D FM I NH I S R Q S KY YKD YQ T LYD F LY E R L RGVFSG VH L LPF YP S..T S D D GF SV V D F Q R V DP E L G T W T D IR I I A Q DFR L M A D L VC NH V S A S S P WF QG FL T LGE F LR E Y L SGTMKG VH I LPF FP Y..S S D D GF SV M D Y R R V NP E W G T W D D VR R I S E DFR L M V D L VL NH C S A K S E WF RR FL T FNQ F YH Q W L QSIFSH VH L LPF YP W..S S D D GF SV I D Y H Q V AS E A G E W Q D IQ Q L G E CSH L M F D F VC NH M S A K S E WF KN YL TT TT A K G E E S E Y YPM F L TMSSV FP N...GA TE E D LAG IYR P R PGL P FTHYK F.A G K T RLV W VS F TP Q Q V D ID TDSDKGW EYL M E K G E E S E Y YPM F L TMSSV FP N...GA TE E D LAG IYR P R PGL P FTHYK F.A G K T RLV W VS F TP Q Q V D ID TDSDKGW EYL M K N H D D S K Y KDF F I RWEKF WA KAGENRP TQ A D VDL IYK R K DKA P TQEIT F DD G T T ENL W NT F GE E Q I D ID VNSAIAK EFI K E K H E A S A Y KDL F L NWDKF WP K...NRP TQ E D VDL IYK R K DRA P KQEIQ F AD G S V EHL W NT F GE E Q I D LD VTKEVTM DFI R Q DD.. P Q YQ G F F I T VD... P G T D LS T V F RPRALPL LTP FQ TPS.GEKLV W TT F S P D Q T D LNY A NP E VLL E VI Q GD.. P E YE D F F I T VE... P G T D LS G V F RPRALPL VHE FE SAK.GPVLV W TT F S R D Q V D LNY A NP R VLL E MI Q QH.. P G FE D F F I A VD... P Q T D LS A V T RPRALPL LTP FQ MRDHSTRHL W TT F S D D Q I D LNY R SP E VLL A MV TT S IF D Q M AASHVSY I RLDA VG Y GA K E AG T S C F M.T PK T F KL I SRL R E EGVK..RGL EIL I E VH S YY KK...QV EI ASKVD S IF D Q M AASHVSY I RLDA VG Y GA K E AG T S C F M.T PK T F KL I SRL R E EGVK..RGL EIL I E VH S YY KK...QV EI ASKVD T TL E D M VKHGANL I RLDA FA Y AV K K VD T N D F FVE PE I W DT L NEV R E ILTP..LKA EIL P E IH E HY SI...PK KI NDHGY S TI E N L AANGCDL I RLDA FA Y AV K K LD T N D F FVE PE I W TL L DKV R D IAAV..SGA EIL P E IH E HY TI...QF KI ADHDY E AL L C Y VRN GA GL I RLDA V G F I W K E I G T S C M H L.EGA H R IVKL M R L V L D A V A P HVL LVS E TN A PHRENI S YFG NGH DEAQ D IF L F Y VSQ GA QI I RLDA I A Y L W K E L G T P C I H H.PKT H A VVKL F R A I C E E V C P WVL IIT E TN V PHKENI S YFG D.M DEAH D VL L C Y LAK GA EY V RLDA V G F M W K E P G T S C I H L.EKT H L IIKL L R S I I D N V A P GTV IIT E TN V PHKDNI A YFG AGD DEAH TT TT RV Y D F A LP PLL LH A L ST G HVEPV A H W TDIR... P NNAV T V L DT HDGIG VID IGSDQL D RS L KGLVPD E DVDNLVNT I H RV Y D F A LP PLL LH S L FT G HVEPV A H W TEIR... P NNAV T V L DT HDGIG VID IGSDQL D RS L KGLVPD E DVDNLVNT I H FT Y D F A LP MTT LY T L YS G KTNQL A K W LKMS... P MKQF T T L DT HDGIG VVD ARDILT D DE I DY.ASE Q...LYK V G YV Y D F A LP MVT LY S L YS S KVDRL A K W LKMS... P MKQF T T L DT HDGIG VVD VKDILT D EE I TY.TSN E...LYK V G LV Y Q F P LP PLVM HTFRTG D ASK L AG W A AG L TLPSERT T F FNF L AS HDGIG VV P AG GIL QPEE I AA LV RQA...LEH G LV Y Q F A LP PLVL DAFLRK D VSY L RE W A RT I DTYGGKV S Y FNF L AS HDGIG VL P AR GIL PDEY I DA MI EAV...KDR G MV Y Q F S LP PLVL HAVQKQ N VEA L CA W A QN L TLPSSNT T W FNF L AS HDGIG LN P LR GLL PESE I LE LV EAL...QQE G AN THGESQAATG A AA SNLDL Y QVNSTYYS A LG C N... D QH Y IA AR AV Q F F L P G V P QV Y YVG A LAG K ND ME LL RK AN THGESQAATG A AA SNLDL Y QVNSTYYS A LG C N... D QH Y LA AR AV Q F F L P G V P QV Y YVG A LAG R ND ME LL RR AN VK.KTYSS.. A SY NNLDI Y QINSTYYS A LG N D... D AA Y LL SR VF Q V F A P G I P QI Y YVG L LAG E ND IA LL ES AN VN.RKYST.. A EY NNLDI Y QINSTYYS A LG D D... D QK Y FL AR LI Q A F A P G I P QV Y YVG F LAG K ND LE LL ES GR VN H K D...T P D G P.V P Y EL CLTLFD A LS NPNSDEAEDL K IA RFL AANV ILLS LQ G I P G V Y IHSL F GS P S DHA G F EE GL IS Y K S...T P Q G E.V P Y EL NINYLS A IS ESHLDRP..T R AR KFL ASQA VMLS LV G M P G I Y VHSL L GS E N WRE G V EK AL VN W K N...N P D G TRS P Y EI NVTYMD A LS RRESSD..EE R CA RFI LAHA ILLS FP G V P A I Y IQSI L GS R N DYA G V EK T NN G R DI NR HYYS TA EI DENL KRP... VV KA L NA L AK F R NE LD A F D GTFSYTT D DD T SI S FTWRGE..TSQATLTFE T NN G R DI NR HYYS TA EI DENL ERP... VV KA L NA L AK F R NE LP A F D GEFSYEV D GD T SI T FRWTAADGTSTAALTFE T KE G R NI NR HYYT RE EV KSEV KRP... VV AN L LK L LS W R NE SP A F D LAGSITV D TP T DT T IVVTRQDENGQNKAVLT T KE G R NI NR HYYS SE EI AKEV KRP... VV KA L LN L FT Y R NQ SA A F D LDGRIEV E TP N EA T IVIERQNKDGSHIAKAE S G IP R R L NR H K FTKAELEER L A D PASRAAK I LAAYSH LL RV R SMHPA F H P NAPQR I LPSTEVLR. I V R G.. E GDQAVG.. T G MN R T I NR Q K LSYEGVLEE L R D PESLRSM V FEGYLD ML AA R RKSRA F D P RGMQE V LEAPETVFA L L R RSP D ATEEVL.. L G YN R A I NR K K YHSKEITRE L N D EATLRHA V YHELSR LI TL R RSHNE F H P DNNFT I DTINSSVMR I P R SNA D GNCLT TT.. TT TTT PKRGLG...V DN TTP V AMLEW..EDSAGDHRSDDLIANPPVVA... PGRGLG...T DN ATP V ASLAW..SDAAGDHETRDLLANPPIADID... ADAANKTFEIV EN GQT V MS...SDN...LTQN... INLQDMTYRVT EN DQT I SF...E......CYI NV TDRP Q VVSR...IG K...N L IT...GQWFTGV L KP YQ AA WI ID......CLI NV SHIE Q ECVFPSSIF R TAPDAHLFTE L TSGDTLVPYREDEDRFSIS L GG YE VL WL TPYRDKG...GLF NV SKNI Q HVNITNLHG R...D L ISE...VDILGNEIT L RP WQ VM WI K... Fig. S5 Multiple sequence alignment of several sucrose phosphorylases (SP) and glucosylglycerate phosphorylases (GGaP) (: Bifidobacterium adolescentis SP, UniProt ID Q84HQ2; : Bifidobacterium longum SP, Q84BY1; : Leuconostoc mesenteroides SP, Q59495; : Streptococcus mutans SP, P10249; : Meiothermus silvanus GGaP, D7BAR0; : Spirochaeta thermophila GGaP, G0GBS4; : Escherichia coli GGaP, P76041). Sequence similarities are indicated for the SP and the GGP groups (red box/white character: conserved across all sequences; green box/red character: conserved within group, but different between groups; red character: similarity within group). BLOSUM62 similarity score matix was used for similarity determination. Numbering is according to, and the secondary structure depicted above sequences is derived from the crystal structure of (PDB entry 2GDU). Residues and comprise loop B and loop A, respectively. Black circles, donor site residues; black stars, catalytic residues. This figure was created with ESPript (1).

8 Table S1 Expression vectors used in this study and their properties. Vector Resistance Promotor Induction Source pcxp34h ampicillin custom constitutive Developed in-house (2) pet21a(+) ampicillin T7 IPTG Novagen ptrc99a ampicillin trc IPTG Amersham Pharmacia Biotech

9 Table S2 Substrate specificity for glucosylglycerate phosphorylase from M. silvanus, S. thermophila and E. coli. Reactions were performed in 50 mm MES buffer ph 6.5 with 100 mm Glc1P and 100 mm of the acceptor substrate at 42 C (M. silvanus and S. thermophila) or 30 C (E. coli). M. silvanus GGaP Specific activity (U/mg) S. thermophila GGaP E. coli GGaP Acids D-glyceric acid* 110 ± 4 80 ± 6 + L-glyceric acid 7.5 ± ± 0.6 n.d. glycolic acid - - n.d. L-lactic acid - - n.d. gluconic acid - - n.d. 3-hydroxypropionic acid - - n.d. D-malic acid - - n.d. L-malic acid - - n.d. Monosaccharides D-glucose D-arabinose D-xylose D-ribose D-lyxose D-fructose D-psicose D-tagatose D-galactose D-mannose L-glucose L-arabinose L-ribose deoxy-L-ribose L-fucose L-sorbose Disaccharides trehalose kojibiose nigerose maltose isomaltose sucrose maltulose leucrose - n.d. n.d. palatinose - n.d. n.d. Sugar alcohols L-xylitol D-xylitol D-mannitol D-arabitol D-sorbitol myo-inositol Oligosaccharides Maltotriose Maltotetraose 1.3 ± Maltopentaose 0.8 ± Isomaltotriose Panose* - - n.d. Starch# Inulin # - - n.d. Pullulan # - - n.d. Dextran # - - n.d. Cyclodextrins α-cyclodextrin* - - n.d. β-cyclodextrin* - - n.d. Substituted sugars D-glucosamine glucuronic acid - - n.d. galacturonic acid - - n.d. N-acetylglucosamine N-acetylgalactosamine* - - n.d. Other Pyrogallol* - - n.d. L-Alanine - - n.d. - : No activity detected (< 0.5 U/mg) + : Significant activity detected n.d. : Not determined * : Acceptor concentration of 20 mm # : Acceptor concentration of 10 mg/ml

10 References 1. Robert X, Gouet P Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42: Aerts D, Verhaeghe T, De Mey M, Desmet T, Soetaert W A constitutive expression system for high-throughput screening. Eng Life Sci 11:10 19.