Supplemental Materials Supplemental Table 1: The strains and plasmids used in this study.

Strains Relevant properties Source S. parasanguinis FW213 Wide type (1) E.coli strain BL21-Gold(DE3) pET System host strain Stratagene E.coli strain Top10 Host for propagation of the recombinant plasmids Invitrogen GST-Gtf3Δ314-330 pGEX-Gtf3Δ314-330 transformed into Top10; Ampr This study gtf3 mutant Wild type; nss knockout; nss::aphA3; Kanr (2) fap1mutant Wide type; fap1::aphA3; Kanr (3) Plasmids pET-SUMO His fusion protein expression vector ; Kanr (Dr.Ma’s lab) pET-SUMO-Gtf3 gtf3 cloned in pET28-SUMO; Kanr This study pGEX-Gtf3 gtf3 cloned in pGEX-6P-1; Ampr (2) pGEX-Gtf3Δ314-330 gtf3 314-330aa deletion of pGEX-Gtf3 This study pGEX-R179A Gtf3 R179A mutation of pGEX-Gtf3 This study pGEX-Y211A Gtf3 Y211A mutation of pGEX-Gtf3 This stud pGEX-D214A Gtf3 D214A mutation of pGEX-Gtf3 This study pGEX-K246A Gtf3 K246A mutation of pGEX-Gtf3 This study pGEX-D249A Gtf3 D249A mutation of pGEX-Gtf3 This study pGEX-F314A Gtf3 F314A mutation of pGEX-Gtf3 This study pGEX-F315A Gtf3 F315A mutation of pGEX-Gtf3 This study pGEX-R318A Gtf3 R318A mutation of pGEX-Gtf3 This study pGEX-L320A Gtf3 L320A mutation of pGEX-Gtf3 This study pET-SUMO-F314A Gtf3 F314A mutation of pET-SUMO-Gtf3 This study pET-SUMO-F315A Gtf3 F315A mutation of pET-SUMO-Gtf3 This study pET-SUMO-R318A Gtf3 R318A mutation of pET-SUMO-Gtf3 This study pET-SUMO-L320A Gtf3 L320A mutation of pET-SUMO-Gtf3 This study pET-SUMO-K246A Gtf3 K246A mutation of pET-SUMO-Gtf3 This study pGEX-Gtf3 (GBS) gtf3 fragment from GBS cloned in pGEX-6P-1 This study pGEX-Gtf3(SK36) gtf3 fragment from SK36 cloned in pGEX-6P-1 This study pGEX-Gtf3(TIGR4) gtf3 fragment from TIGR4 cloned in pGEX-6P-1 This study pVPT-Gtf3 gtf3 cloned in pVPT-CHSV; Ermr (2) pVPT-CHSV E.coli-Streptococci shuttle vector; Ermr (4) pVPT-Gtf3 (GBS) gtf3 fragment from GBS cloned in pVPT-CHSV This study pVPT-Gtf3 (SK36) gtf3 fragment from SK36 cloned in pVPT-CHSV This study pVPT-Gtf3 (TIGR4) gtf3 fragment from TIGR4 cloned in pVPT-CHSV This study

Supplemental table 2: Primers used in this study.

Primer Sequence

Gtf3-HindIII 5’-GATCA-1F AAGCTTGCATGCGTGTATATATCACAAAT-3’ Gtf3-XhoI 5’-CATCA-987R CTCGAGArg179Ala-F

CTAATCACATATAGCTTGAAATAC-3’ 5’-TCATTTTCCAGGTAATCCCGAAGCTTTTAGTTTTGTGAAAGAGTGG-3’

Arg179Ala-R 5’-CCACTCTTTCACAAAACTAAAAGCTTCGGGATTACCTGGAAAATGA-3’ Lys246Ala-F 5’-GAATATCAATCATTGTACTGTTCTTATGCACTAGGAAGTTTTTTAGCAGC-3’ Lys246Ala-R 5’-GCTGCTAAAAAACTTCCTAGTGCATAAGAACAGTACAATGATTGATATTC-3’ Tyr211Ala-F 5’-GAATTACCTCAAAATGTTCATAAAATTAACGCTCGTCCAGACGAACAAC-3’ Tyr211Ala-R 5’-GTTGTTCGTCTGGACGAGCGTTAATTTTATGAACATTTTGAGGTAATTC-3’ Asp214Ala-F 5’-CATAAAATTAACTATCGTCCAGCCGAACAACTCTTAATGGAGATG-3’ Asp214Ala-R 5’-CATCTCCATTAAGAGTTGTTCGGCTGGACGATAGTTAATTTTATG-3’ Asp249Ala-F 5’-GTACTGTTCTTATAAACTAGGAGCTTTTTTAGCAGCAGGTATTCCTG-3’ Asp249Ala-R 5’-CAGGAATACCTGCTGCTAAAAAAGCTCCTAGTTTATAAGAACAGTAC-3’ Phe314Ala-F 5’-GTTTTAATCCTATTTTGCGTAAGGGTGCTTTTACAAGAAGATTGCTTAC-3’ Phe314Ala-R 5’-GTAAGCAATCTTCTTGTAAAAGCACCCTTACGCAAAATAGGATTAAAAC-3’ Phe315Ala- F 5’-GTTTTAATCCTATTTTGCGTAAGGGTTTTGCTACAAGAAGATTGCTTAC-3’ Phe315Ala-R 5’-GTTTTAATCCTATTTTGCGTAAGGGTTTTGCTACAAGAAGATTGCTTAC-3’ Arg318Ala-F 5’-GCGTAAGGGTTTTTTTACAAGAGCATTGCTTACAGAATCTGTATTTCAAG-3’ Arg318Ala-R 5’-CTTGAAATACAGATTCTGTAAGCAATGCTCTTGTAAAAAAACCCTTACGC-3’ Leu320Ala-F 5’-CGTAAGGGTTTTTTTACAAGAAGATTGGCTACAGAATCTGTATTTCAAGC-3’ Leu320Ala-R 5’-GCTTGAAATACAGATTCTGTAGCCAATCTTCTTGTAAAAAAACCCTTACG-3’ COH1Gtf3-BamHI 5’-GATCA 3’ GGATCCSK36Gtf3-

TCACATGCTATTTAATGCAC-3’ SalI 5’-GATCA 5’ GTCGAC

COH1Gtf3-ATGAAAGTTAATATTACC-3’

BamHI 5’-GATCA 5’ GGATCCCOH1Gtf3-

GTGAGGACATATATTACAAACTTG-3’ XhoI 5’-GATCA 3’ CTCGAG

SK36Gtf3-TCACATGCTATTTAATGCAC-3’

BamHI 5’-GATCA 5’ GGATCCSK36Gtf3-

ATGAAAGTTAATATTACC-3’ XhoI 5’-GATCA 3’ CTCGAG

SK36Gtf3-CTAAGATAAGACTTCCATC-3’

BamHI 5’-GATCA 3’ GGATCCTIGR4Gtf3-

CTAAGATAAGACTTCCATC-3’ SalI 5’-GATCA 5’ GTCGAC

TIGR4Gtf3-ATGAAACTACATTTAACAAATTTATAC-3’

BamHI 5’-GATCA 5’ GGATCCTIGR4Gtf3-

ATGAAACTACATTTAACAAATTTATAC-3’ XhoI 5’-GATCA 3’ CTCGAG

Gtf3-TTAATCAATTCCCAAGTGATAG-3’

SalI 5’-GATCA 5’ GTCGACGtf3-

ATGCGTGTATATATCACAAAT-3’ KpnI 5’-CATCA 3’ GGTACC

COH1Gtf3-ATCACATATAGCTTGAAATAC-3’

SalI 5’-GATCA 5’ GTCGACCOH1Gtf3-

ATGAGGACATATATTACAAACTTG-3’ BamHI 5’-GATCA 3’ GGATCC

SK36Gtf3- TCACATGCTATTTAATGCAC-3’

SalI 5’-GATCA 5’ GTCGACATGAAAGTTAATATTACC-3’

Supplemental Table 3: List of structures most similar to Gtf3 (molecule A) as determined by the DALI server. Protein PDB id

code Fold CAZY

Family Mechanism Oligomer DALI Z-score RMSD

(Å) Alignment length

Seq. ID (%)

PimA(5) 2GEJ/ 2GEK

GT-B GT-4 Retaining Monomer 22.5 (Chain A)/ 22.3 (Chain A)

3.6 300/298 10

MshA (6)

3C4V/ 3C4Q

GT-B GT-4 Retaining Dimer 21.3 (Chain A,B)/ 21.8 (Chain A,B)

3.3 300 11

BshA (7) 2JJM/ 3MBO

GT-B GT-4 Retaining Tetramer 21.4 (Chain A-L)/ 21.2 (Chain A-H)

3.6 295/296 12/13

PimB(8) 3OKA/ 3OKC

GT-B GT-4 Retaining Dimer 20.8 3.3/3.4 306/300 10

GLGA glycogen synthase (9)

3FRO/ 2BIS/ 3L01

GT-B GT-5 Retaining Trimer/ Monomer

20.2 (Chain A,C)/ 20.1 (Chain A,B,C)/ 20.0 (Chain A,B)

3.3 291/290/289

13

α-trehalose phosphate synthase (OtsA) (10)

1GZ5 GT-B GT-20 Retaining Monomer/Dimer/ Tetramer

19.6 (Chain A-D)

3.4 287 10

α,α-trehalose phosphate synthase(11)

2WTX GT-B GT-20 Retaining Dimer 19.5 (Chain A,B) 3.3 286 12

Results and discussion of DALI search for structurally similar proteins: A DALI search using only molecule A from Gtf3 revealed numerous hits for structurally similar proteins, mainly glycosyltransferases, as well as other sugar-converting enzymes (Table S1). The most similar protein to Gtf3 was phosphatidylinositol mannosyltransferase (PimA) from Mycobacterium smegmatis (PDB id code 2gej and 2gek; Z-score 22.5-22.3, rmsd 3.6 Å, 10% sequence identity), which catalyzes the initial mannosylation of phosphatidylinositol and is important for the biosynthesis of the mycobacterial cell envelope(5). The second most similar structure to Gtf3 was MshA from Cornybacterium glutamicum (PDB id codes 3c4v and 3c4q; Z-score 21.8-21.3, rmsd 3.3 Å, 11% sequence identity), which mediates the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-L-myo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis(6) . The third most similar structure was the ORF BA1558 (BshA) from Bacillus anthracis (PDB id codes 2jjm and 3mbo; Z-score 21.4-21.2, rmsd 3.6 Å, 12/13% sequence identity), which transfers GlcNAc from UDP-GlcNAc to malate and is essential for biogenesis of bacillithiol (Cys-GlcN-malate, BSH) (7). The fourth most similar structure to Gtf3 was another member of the phosphatidylinositol mannoside (PIM) biosynthetic pathway; PimB. PimB catalyzes the reaction that attaches the second mannosyl residue at the 6-hydroxyl to yield mono-acetylated phosphatidylinositol mannosides. It is interesting to note that the four most similar structures to Gtf3 are all members of the GT-4 family of retaining glycosyltransferases, however they share a very low sequence identity (<12%) to Gtf3.

A surprising result from the DALI search was the Pyrococcus abyssi glycogen synthase in both open and closed conformations (PDB id codes 2bis and 3fro). This enzyme belongs to GT-5 family of the glycosyltransferases(9) and, as all other structures similar to Gtf3, displays the canonical GT-B type fold. Gtf3 (molecule A) and the P. abyssi glycogen synthase structures superimpose with an rmsd of 3.3Å with a Z-score of 20.2-20.0 (13% sequence identity). Intriguingly, this enzyme catalyzes the transfer of

SK36Gtf3-BamHI 5’-GATCA 3’ GGATCC CTAAGATAAGACTTCCATC-3’ TIGR4Gtf3-SalI 5’-GATCA 5’ GTCGACTIGR4Gtf3-

ATGAAACTACATTTAACAAATTTATAC-3’ BamHI 5’-GATCA 3’ GGATCCTTAATCAATTCCCAAGTGATAG-3’

glucosyl residues from ADP- or UDP-glucose to the non-reducing end of a growing α-1,4-glucan chain. We have shown that Gtf3 also transfers glucosyl residue from UDP-Glucose to GlcNAc modified Fap1 substrate, the structural similarity may account for the same (or similar) sugar transfer activity. In addition, the OtsA α-trehalose-phosphate synthase (PDB id code 1GZ5) is next on the list of structurally similar proteins to Gtf3, with an rmsd of 3.4Å and a Z-score of 19.6 for 287 residues (10% sequence identity). OtsA a-trehalose-phosphate synthase engages the transfer of glucose from a UDP-glucose donor to glucose-6-phosphate to form alpha, alpha-1,1-trehalose-6-phosphate. OtsA belongs to GT-20 family of glycosyltransferases based upon amino acid sequence similarity analysis(12) (see http://afmb.cnrs-mrs.fr/CAZY/).

References

1. Cole, R. M., Calandra, G. B., Huff, E., and Nugent, K. M. (1976) J Dent Res 55, A142-153 2. Zhou, M., Zhu, F., Dong, S., Pritchard, D. G., and Wu, H. (2010) J Biol Chem 285, 12140-

12148 3. Wu, H., Mintz, K. P., Ladha, M., and Fives-Taylor, P. M. (1998) Mol Microbiol 28, 487-

500 4. Zhou, M., Fives-Taylor, P., and Wu, H. (2008) J Microbiol Methods 72, 249-256 5. Guerin, M. E., Kordulakova, J., Schaeffer, F., Svetlikova, Z., Buschiazzo, A., Giganti, D.,

Gicquel, B., Mikusova, K., Jackson, M., and Alzari, P. M. (2007) J Biol Chem 282, 20705-20714

6. Vetting, M. W., Frantom, P. A., and Blanchard, J. S. (2008) J Biol Chem 283, 15834-15844

7. Ruane, K. M., Davies, G. J., and Martinez-Fleites, C. (2008) Proteins 73, 784-787 8. Batt, S. M., Jabeen, T., Mishra, A. K., Veerapen, N., Krumbach, K., Eggeling, L., Besra, G.

S., and Futterer, K. (2010) J Biol Chem 285, 37741-37752 9. Horcajada, C., Guinovart, J. J., Fita, I., and Ferrer, J. C. (2006) J Biol Chem 281, 2923-

2931 10. Gibson, R. P., Turkenburg, J. P., Charnock, S. J., Lloyd, R., and Davies, G. J. (2002)

Chemistry & Biology 9, 1337-1346 11. Errey, J. C., Lee, S. S., Gibson, R. P., Martinez Fleites, C., Barry, C. S., Jung, P. M.,

O'Sullivan, A. C., Davis, B. G., and Davies, G. J. (2010) Angew Chem Int Ed Engl 49, 1234-1237

12. Gibson, R. P., Tarling, C. A., Roberts, S., Withers, S. G., and Davies, G. J. (2004) J Biol Chem 279, 1950-1955

Figure S1: Ramachandran plot for all four molecules in the Gtf3 tetramer.

Figure S2: Ramachandran plot for the two molecules in the Se-Met Gtf3 strucuture.

Figure S3: Structural superimpositions of Gtf3 (molecule A) with similar proteins (molecule A) as found by the DALI search.