Specific features for competent binding of substrates at the FMN adenylyltransferase site of FAD synthase from Corynebacterium ammoniagenes

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1 Appendix A. Supplementl Mteril Specific fetures for competent inding of sustrtes t the FMN denylyltrnsferse site of FAD synthse from Corynecterium mmonigenes Soni Arill-Lun 1,, An Serrno 1,2, nd Milgros Medin 1, * 1 Deprtment of Biochemistry nd Moleculr nd Cellulr Biology, Fculty of Sciences, nd Institute of Biocomputtion nd Physics of Complex Systems (Joint Units: BIFI-IQFR nd GBsC-CSIC), University of Zrgoz, Spin; rilun2@gmil.com; mmedin@unizr.es 2 Centro de Investigciones Biológics, CSIC, Rmiro de Meztu 9, E Mdrid, Spin; nserr1979@gmil.com * Correspondence: mmedin@unizr.es These two uthors hve eqully contriuted to the study nd must oth e considered s first uthors.

2 Supplementry Figures. Alignment of with other FADSs MtFADS LmFADS-I LmFADS-II SpFADS CuFADS BoFADS MDIWYGTAAVPKDLDNSAVTIGVFDGVHRGHQKLINATVEKAREVGAKAIMVTFDPHPVSVFLPRRA MTLYTALSEVPAGYGPSVVTIGNFDGVHRGHARVISRVVSLAEEHGLSSIAVSFDPHPMQVHRPEAA MKTIYLHHPITTDEWTSIKK--VMALGFFDGVHLGHQAVIKKAKQIAEQKGLQTAVLTFDPHPSVVLSNIRK MEVSHVTLAPNKDSRPAVLTIGKFDGVHIGHQTILNTALSIKKENE-ILTAISFSPHPLWALKQIEI MIITIPIKNQKDIGTPSDSVVVLGYFDGIHKGHQELFRVANKAARKDLLPIVVMTFNESPKIALEPYHP MSHGASLWVKIPNVSIWYGLDRVPHNLEGAAVTIGVFDGVHRGHQELINRAVAKAKELGVPAVMFTFDPHPTVVFQPESV MKKSSFQRLTGTEGLPPALHDCVVAIGNFDGVHRGHQAVLERALELAERESRPAVVLTFEPHPRSFFK-QDQ MtFADS LmFADS-I LmFADS-II SpFADS CuFADS BoFADS PLGITTL--AERFALAESFGIDGVLVIDFTRELSGTSPEKYVEFLLEDTLHASHVVVGANFTFGENAAGTADSLRQICQS 145 HHDIMGQ--GSRRYFMCLLGLNDYLLLNYNLEFAAQTPEEFVKSTFVDALNARFVVIGDDVRFGKNNSGDLNTMRELGEK 145 QVKYLTP-LEDKAEKMAELGVDIMYVVRFTTQFSELSPQSFVDNYLVA-LNVEHVVAGFDYSYGKKGEGKMTDLAQYADG 148 YREMLTP-RMEKERWLAHYGVDHLIETAFTPRYAETTPEEFVRDHLTN-LNLSHIVVGSEFNFGKGRDSDVDLLRDLCKP 144 DLFLHILNPAERERKLKREGVEELYLLDFSSQFASLTAQEFFA-TYIKAMNAKIIVAGFDYTFGSDKK-TAEDLKDYFDG 147 PKLLGTV--EERAQLAMDLGIDHVVVQAFTPEIASWSPEEYIDRALLDTLRAKHVVVGENFTFGHKASGTPDTLREVSQN 158 PVDRLTD-AAEKAEILRLMGFDAVMEQPFTAEFSQRSAEDFVQHILVEKLRASRVVTGYDFHFGKGRRGTPEFLCEAGKK 150. Alignment of with NTs PPAT E.coli GCT B. sutilis NMNAT M. thermoutotrophicum NMNAT M. jnnschii PPAT T. thermophilus MDIWYGTAAVPKDLDNSAVTIGVFDGVHRGHQKLINATVEKAREVGAKAIMVTFDPHPVSVFLPRRAPLG MQKRAIYPGTFDPITNGHIDIVTRATQMFDHV-ILAIAASPSKKP---MFTLEERVA MKKVITYGTFDLLHWGHIKLLERAKQLGDYL-VVAIST DEFN MMTMRGLLVGRMQPFHRGHLQVIKSILEEVDEL-IICIGSAQLSHSIRDPFTAGERVM MRGFIIGRFQPFHKGHLEVIKKIAEEVDEI-IIGIGSAQKSHTLENPFTAGERIL MHVVYPGSFDPLTNGHLDVIQRASRLFEKV-TVAVLENPSKRGQY-LFSAEERLA PPAT E.coli GCT B. sutilis NMNAT M. thermoutotrophicum NMNAT M. jnnschii PPAT T. thermophilus ITTLAERFALA ESFGIDGVLVIDFTRELSGTSPEKYVEFLLEDTLHASHVVVGANFTFGEN LAQQATAHLGN VEVVGFSDLMANFARNQH ATVLIRGLRAVADFEYEMQLAHMN LQKQKKAYHSY EHRKLILETIRYVD EVIPEKNWEQK------K MLTKALSENGIPASRYYIIPVQDIECNALWVGHIKMLTPPFDRVYSGNPLVQRLFSEDGYEVTAPPLFYR MITQSLKDYDL---TYYPIPIKDIEFNSIWVSYVESLTPPFDIVYSGNPLVRVLFEERGYEVKRPEMFNR IIREATAHLAN VEAATFSGLLVDFVRRVG AQAIVKGLRAVSDYEYELQMAHLN Figure S1. Multiple sequence lignments of () the FMNAT module in different prokryotic FADSs (MtFADS, FADS from Mycocterium tuerculosis; LmFADS-I nd LmFADS-II, FADSs from Listeri monocytogenes type I nd type II; SpFADS, FADS from Streptococcus pneumonie; CuFADS, FADS from Corynecterium urelyticum; nd BoFADS, FADS from Brucell ovis) nd () the FMNAT module of with severl nucleotidyltrnsferses (PPAT, phosphopntetheine denylyltrnsferse; GCT, CTP:glycerol-3-phosphte citidilyltrnsferse; NMNAT, nicotinmide mononucleotide denylyltrnsferse). Residues shded in lck nd grey show respectively 80 % of identity nd similrity. The highly conserved motifs re underlined in green. Residues here studied re mrked with red str.

3 0 30 MER (deg cm 2 dmol -1 ) P56W L98W P58W L89K P56A/P58A L98A WT Wvelength (nm) ME (deg cm 2 dmol -1 ) Wvelength (nm) Figure S2. Circulr dichroism spectr (molr ellipticity) () in the fr-uv region (per residue) nd () in the ner-uv region for the different vrints. Spectr were recorded respectively in 5 mm nd 20 mm PIPES, 10 mm MgCl 2, ph 7.0 t 25 C. ε (µm -1 cm -1 ) WT (45µM) P56W (90µM) P58W (180µM) P56A/P58A (45µM) Wvelength (nm) Wvelength (nm) Figure S3. Visile difference spectr elicited upon titrtion of () WT, P56 nd P58 nd, () WT, nd L98 vrints (4-6 µm) with sturting FMN concentrtions (indicted in prenthesis for ech vrint). Spectr recorded in 20 mm PIPES, 10 mm MgCl2, ph 7.0 t 25 C WT L98A (45µM) L98K (45µM) L98W (135µM) ε (µm -1 cm -1 ) Wvelength (nm) WT (20µM) P56W (40µM) P58W (20µM) P56A/P58A (20µM) WT (20µM) L98A (40µM) L98K(20µM) L98W(40µM) Wvelength (nm) Figure S4. Visile difference spectr elicited upon titrtion of () WT, P56 nd P58 nd, () WT, L98 vrints (4-6 µm) with sturting FAD concentrtions (indicted in prenthesis for ech vrint). Spectr recorded in 20 mm PIPES, 10 mm MgCl2, ph 7.0 t 25 C.

() L98W vrints in silico models. Mutted K nd W residues re CPK coloured with C in white.

4 α4n L110 F109 L6n L98 I92 E97 T6 L1n K98 W98 c Figure S5. Crtoon detil of the environment of position 98 (shown s spheres) in () WT s well s in () L98K nd () L98W vrints in silico models. Mutted K nd W residues re CPK coloured with C in white. T6, I92, E97, F109 nd L110 side chins surrounding L98 re shown in sticks. Sustrtes re docked ccording to [1]. Rest of colour codes s in Figures 1 nd 6.

5 Supplementry Tles Tle S1. Extinction coefficients t 279 nm nd 25 C (n=3, men ± SD) for the vrints in 20 mm PIPES, 10 mm MgCl2, ph 7.0. ε 279 in Gdn/HCl (mm -1 cm -1 ) ε 279 in PIPES (mm -1 cm - 1 ) (ε 279 PIPES - ε 279 Gdn/HCl)/ ε 279 PIPES WT ± (%) P56W ± P58W ± P56A/P58A ± L98A ± L98K ± L98W ± Theoreticl vlue sed on the mino cid sequence ccording to [2]. Difference etween the theoreticl vlue under denturing conditions nd for the wild-type folded enzyme vlue is round 0.4 %. For the vrints this difference increses up to %. The extinction coefficient under denturing conditions only hs into ccount the numer of tryptophn, tyrosine nd cysteine (in its reduced stte) residues. However, the extinction coefficient under ntive conditions is influenced y other fctors, such s the protein folding round these residues, the uffer (ph nd ionic strength), or the prticulr electronic environment of ech residue contriuting to it. This is why the clculted extinction coefficient for the ntive protein does not mtch with the theoreticl vlue tht corresponds to n unfolded protein.

6 Tle S2. Thermodynmic prmeters for the interction of vrints with FAD, FMN in 20 mm PIPES, 10 mm MgCl2, ph 7.0, nd ATP in 20 mm PIPES, ph 7.0. (n=3, men ± SD). WT P56W P58W P56A/P58A L98A Lignd ΔH (kcl/mol) ΔG (kcl/mol) -TΔS (kcl/mol) FMN -22 ± ± ± 1 FAD -26 ± ± ± 1 ATP -44 ± ± ± 6 FMN -1.4 ± ± ± 0.1 FAD -0.9± ± ± 0.1 ATP -16 ± ± ± 2 FMN -1.3 ± ± ± 0.1 FAD ± ± ± 0.1 ATP -2.7 ± ± ± 0.1 FMN -1.8 ± ± ± 0.1 FAD ± ± ± 0.1 ATP -2.8 ± ± ± 0.2 FMN -1.2 ± ± ± 0.1 FAD n.d. n.d. n.d. ATP -20 ± ± ± 2 FMN n.d. n.d. n.d. L98K FAD n.d. n.d. n.d. ATP -39 ± ± ± 19 FMN -1.5 ± ± ± 0.1 L98W FAD -0.6 ± ± ± 0.1 ATP -32 ± ± ± 6 n.d. indictes tht no interction profile ws detected. REFERENCES 1. Lns, I., Seco, J., Serrno, A., Burno, R., Cossio, P., Dz, M.C., nd Medin, M. (2018). The Dimer-of-Trimers Assemly Prevents Ctlysis t the Trnsferse Site of Prokryotic FAD Synthse. Biophys J 115, Gill, S.C., nd von Hippel, P.H. (1989). Clcultion of protein extinction coefficients from mino cid sequence dt. Anl Biochem 182,

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