Supporting Online Material

1 SUPPLEMENTARY MATERIAL

2 The glycan alphabet is not universal: a hypothesis 3 4 Jaya Srivastava1*, P. Sunthar2 and Petety V. Balaji1 5 6 1Department of Biosciences and Bioengineering, Indian Institute of 7 Technology Bombay, Powai, Mumbai 400076, India 8 9 2Department of Chemical Engineering, Indian Institute of Technology 10 Bombay, Powai, Mumbai 400076, India 11 12 *Corresponding author 13 Email: [email protected]

14 CONTENTS

Data Description Figure S1 Number of organisms with different number of strains sequenced Figure S2 Biosynthesis pathways Figure S3 Proteome sizes for different number of monosaccharides Figure S4 Prevalence of monosaccharides in species versus that in genomes Figure S5 Bit score distribution plots for hits of various pairs of profiles Table S1 Tools and databases used in this study References References cited in Table S1 Table S2 Comparison of the precursor and nucleotide used for the biosynthesis of two enantiomers of a monosaccharide Flowchart S1 Procedure used to generate HMM profiles Flowchart S2 Precedence rules for assigning annotation to proteins that are hits to two or more profiles and/or BLASTp queries References References to the research articles which describe the pathways (or enzymes of the pathways) of monosaccharide biosynthesis. These formed the basis for generating HMM profiles and choosing BLASTp queries. 15 16 MS-EXCEL file provided separately: Supplementary Data.xlsx 17 Worksheet1 Details of HMM profiles Worksheet2 Details of BLASTp queries Worksheet3 Prevalence of monosaccharides in genomes / species Worksheet4 Abbreviated names of monosaccharides Worksheet5 Enzyme types, enzymes and monosaccharide groups Worksheet6 Precursors of various monosaccharides 18

19 Figure S1 The number of species for which different number of strains are sequenced. 20 Six or fewer strains are sequenced for most of the species. On the other hand, more than 21 50 strains are sequenced for 29 species. Escherichia coli and Salmonella enterica have 22 the highest number of sequenced strains (714 and 602, respectively). Genus and species 23 names are not known for 45 endosymbionts; only their host name is known e.g., 24 Legionella endosymbiont. Each such case is considered as a distinct species.

4 Mutase Glucose-1-phosphate C1 4 (d)TDP- (d)TDP-fucose C1

ThymidylylT (Pyr to Fur) fucofuranose

)

0 6 4

GPE01130 A GPE09510

3 1

(

2 3

- -

: :

4 H a

C 4

(d)TDP-glucose 1 2

–

u E GPE05430 5

4,6-Dehydratase (R) A

e Q

Retention of config @ C5 O

R - 4 4-AminoT 4 (d)TDP-4-keto-6-deoxy-glucose C1 Eq –NH2@C4 3,4-Ketoisomerase 3,4-Ketoisomerase GPE01830 GPE07510 Eq –OH@C4 Ax –OH@C4 GPE01230

(d)TDP-4-amino- e s (d)TDP-3-keto-6-

a (d)TDP-3-keto-6- 4

r 0

0 6-deoxy-glucose C1

3 3

e 4 4 5

4 deoxy-glucose C1 deoxy-galactose C1

2 2

i 0

0 3

p E E 2 1 G

E GPE01910 9 P P 3-AminoT (E) 3-AminoT (E) 2

- GPE01910 - 1 P G G 1 - F E 5 GPE01230 : 1 5 , Eq –NH @C3 Eq –NH @C3 : T o 2 GPE01230 2 3 3 l ( 0 3 D y ) 4 rm 0 8 W t o 1 -a 0 B C e in y (d)TDP-3-amino-6- 4 X c m lT (d)TDP-3-amino-6- C 9 A m i Q n

4 a 0 4 0 4 T - o

C C 3 C deoxy-galactose 1 3 4 )

deoxy-glucose 1 o (

8 2

n 1

(d)TDP-4-keto- 1

0 0

E E 1 m

AcetylT AcetylT N P

L-rhamnose C4 Q6T1W7:1-192 P

A –

- G

Q6TFC6:1-265 G x

(3-amino) Q12KT8:1-152 (3-amino) 4 A 4 (d)TDP-Qui4NAc C1 (d)TDP-Qui4NFo E5 ) 4 O F1 4 E - 4 C ( R 6 6 4 4 1 e 62 : (d)TDP-Qui3NAc C1 (d)TDP-Fuc3NAc C1 e A d 5 1- s 4 x u 6: 31 a C 0 – c 1 8 t 3 O t - @ a 2 (d)TDP-4-amino-6- c 3 H s 7 H @ e 0 u 6 d O 0 C (A 4C e – E 4 ) deoxy-galactose 1 P R q - G 4 E AcetylT Q8FBQ3:1-224 4-Epimerase (4-amino) (d)TDP- (d)TDP-6-deoxy- 1 Q2SYH7:1-363 L-rhamnose C4 L-talose 1C 4 4 (d)TDP-Fuc4NAc C1

D-enantiomer L-enantiomer HMM profile BLASTp query D-enantiomer / pyranose form unless mentioned otherwise 25 26 Figure S2a TDP-/dTDP-linked monosaccharides derived from glucose-1-phosphate. 27 Abbreviated names are used for some of the monosaccharides. Full names of these are 28 given in Supplementary_data.xlsx:Worksheet4.

4 Glucose-1-phosphate C1

GPE00330 CytidylylT

4 CDP-glucose C1

GPE05230 4,6-Dehydratase (R) Retention of config @ C5 GPE40110 C-MeT (E) 3-Epimerase CDP-4-keto-6- CDP-4-keto-3C-methyl- 4C 4C 1 CDP-4-keto-6-deoxy gulose 1 4 GPE02530 deoxy-glucose C1 Eq –Me, Ax-OH@C3 6-deoxy-gulose Ax –OH@C4 4-Reductase GPE06230 4 GPE05630 3-Dehydratase -R e e s E a q d t 4 – u 4 c C O c CDP-6-deoxy gulose C1 u H t CDP-6-deoxy-D3,4-glucocene @ @ a d H GPE06230 s e C e O 4 R – - x P26395:1-330 D3,4-glucocene 4 A Q66DP5:1-329 reductase GPE02530

CDP-3,6-dideoxy-L-glycero- 5-Epimerase glycero 4 CDP-3,6-dideoxy-D- - CDP-cereose C1 CDP-cillose 1C 4 D-glycero-4-hexulose 4 C1 4 D-glycero-4-hexulose C1

GPE06230 4-Reductase 4 e - s R Eq –OH@C4 a E e t 4 q d c C – u u @ O c d H GPE06230 H t 1C e O @ a CDP-L-ascarylose 4 R – C s - x 4 e 4 A

2-Epimerase 4C 4 4C CDP-abequose 1 CDP-paratose C1 CDP-tyvelose 1 P14169:1-338

D-enantiomer / pyranose form unless mentioned otherwise 30 D-enantiomer L-enantiomer HMM profile BLASTp query 31 Figure S2b CDP-linked monosaccharides derived from glucose-1-phosphate. 32 Abbreviated names are used for some of the monosaccharides. Full names of these are 33 given in Supplementary_data.xlsx:Worksheet4.

4 Glucose-1-phosphate C1

GPE00430 UridylylT GPE00530 GPE09510 4,6-Dehydratase (R) 4-Epimerase Mutase UDP-galacto- UDP-4-keto-6- 4 4 UDP-glucose C1 UDP-galactose C1 4 Retention of config @ C5 furanose deoxy-glucose C1 GPE02230 (pyranose GPE05510 to furanose) 2 5 3 3 , - ) 4 5 :1 E - - 8 ( R E 6-Dehydrogenase N p 4 T 4 E e Y o C q d im GPE03130 2 n G – u G i @ e 2 P O c GPE03430 0 m H E H t r A N 0 @ a a 0 A – 5 s s A - 7 C e e 4 q 1 4 4C E 0 ( , Decarboxylase 1 E 4 ) UDP-GlcA C1 UDP-4-keto xylose GPE20030 GPE01830 1 4-Epimerase 4-AminoT (A) UDP-4-amino-6- UDP-L-rhamnose C4 GPE01230 GPE02230 Ax –NH @C4 4C 2 deoxy-glucose 1 4 UDP-xylose C1 4C 4 AcetylT UDP-GalA 1 UDP-L-Ara4N C1 (4-amino) 4-Epimerase FormylT GPE02230 GPE50010 Q5UR11:1-213 (4-amino) 4C 4 UDP-L-arabinose 1 UDP-L-Ara4NFo UDP-Qui4NAc C1 4 C1 D-enantiomer L-enantiomer HMM profile BLASTp query D-enantiomer / pyranose form unless mentioned otherwise 34 35 Figure S2c UDP-linked monosaccharides derived from glucose-1-phosphate. 36 Abbreviated names are used for some of the monosaccharides. Full names of these are 37 given in Supplementary_data.xlsx:Worksheet4.

GPE01330 4 Fructofuranose-6-phosphate Glucosamine-6-phosphate C1 GPE07030 2-AmidoT GPE07130 M lT GPE07230 y u Isomease t 0 G t e 3 P a GPE07330 1 G E c 8 P 0 s A 0 E 9 e Q5SIM4:1-254 E 0 1 P 9 3 G 6 P29954:1-387 3 0 0

1 4C C4 Mannose-6-phosphate 1 GDP-L-galactose GPE09230 4 3,5 Glc2NAc-6-phosphate Glucosamine-1-phosphate C1 -e GPE09330 Mutase 4C R p 1 4N im GPE09630 RR er AcetylT 5: as 1-3 e 4 Mutase GPE08510 50 Mannose-1-phosphate C1 (2-amino) GPE09430 GPE00620 (GlmU-CTD) GPE00720 GuanylylT 4 Glc2NAc-1-phosphate C1 4 6-dehydrogenase 4 GDP-ManA C1 GDP-mannose C1 GPE03210 GPE00831 GPE03430 GPE05020 4,6-dehydratase (R) UridylylT UridylylT Retention of config @ C5 GPE00830 (GlmU-NTD) 4-AminoT (E) 4 4 C1 GDP-Per C1 GDP-4-keto-6-deoxy mannose 4 Eq –NH2@C4 UDP-Glc2NAc C1 AcetylT Q7DBF7:1-221 GPE01430 Eq 4- –OH O85353:1-215 se 3,5-epimerase, re @C (4-amino) ta duc 4 a G tas dr 4-reductase (A) PE0 e 4 y 4- 65 (E) GDP-Per4Ac C1 h 30 re 10 de 56 GPE06030 Ax –OH@C4 du - E0 Ax ct 3 P –O as G H@ e 1C C4 (A 4 GDP-L-fucose 4 ) GDP-rhamnose C1 GDP-4-keto-3,6- 4 4C dideoxy mannose C1 GDP-6-deoxy-talose 1 5-epimerase, 4-reductase (A) GPE06030 Ax –OH@C4

1 GDP-L-colitose C4

D-enantiomer L-enantiomer HMM profile BLASTp query D-enantiomer / pyranose form unless mentioned otherwise 38 39 Figure S2d GDP- and UDP-linked monosaccharides derived from fructofuranose-6- 40 phosphate. Abbreviated names are used for some of the monosaccharides. Full names of 41 these are given in Supplementary_data.xlsx:Worksheet4.

42 43 Figure S2e CMP- and UDP-linked monosaccharides derived from UDP-Glc2NAc (1 of 44 2). Abbreviated names are used for some of the monosaccharides. Full names of these 45 are given in Supplementary_data.xlsx:Worksheet4.

46 47 Figure S2f CMP- and UDP-linked monosaccharides derived from UDP-Glc2NAc (2 of 48 2). CMP-Leg5Ac7Ac may be biosynthesized through GDP-linked or UDP-linked 49 intermediates. Abbreviated names are used for some of the monosaccharides. Full names 50 of these are given in Supplementary_data.xlsx:Worksheet4.

4 Sedoheptulose-7-phosphate C1

P63224:1-192 Isomerase

4 D-glycero-a/b-D-manno-heptose-7-phosphate C1

Kinase Kinase P76658:1-318 Q9AGY8:1-341

D-glycero-a-D-manno- D-glycero-b-D-manno- 4 4C heptose 1,7-bisphosphate C1 heptose 1,7-bisphosphate 1 Q9AGY5:1-179 Phosphatase P63228:1-191 Phosphatase D-glycero-a-D-manno- D-glycero-b-D-manno- 4 4 heptose 1-phosphate C1 heptose 1-phosphate C1

Q9AGY6:1-230 Guanylyltransferase P76658:344-477 AdenylylT

GDP-D-glycero-a-D- ADP-D-glycero-b- 4 4 manno-heptose C1 D-manno-heptose C1

GPE05020 4,6-Dehydratase (R) P76910:1-310 6-Epimerase Retention of config @ C5 ADP-L-glycero-b-D- GDP-4-keto-6-deoxy-a- 3-Epimerase GDP-4-keto-6-deoxy- 4 manno C1 4 -heptose D-arabino-heptose a-D-lyxo-heptose C1 4 GPE02530 C1 4-Reductase (E) GPE06510 4-Reductase (E) GPE06510 Eq –OH@C4 Eq –OH@C4

GDP-6-deoxy-a- GDP-6-deoxy-a-D- 4 4C D-altro-heptose C1 manno-heptose 1

D-enantiomer L-enantiomer HMM profile BLASTp query Pyranose form unless mentioned otherwise 51 52 Figure S2g ADP- and GDP-linked heptoses derived from sedoheptulose-7-phosphate. 53 Abbreviated names are used for some of the monosaccharides. Full names of these are 54 given in Supplementary_data.xlsx:Worksheet4.

55 56 Figure S3 Variations in the proteome size of organisms which encode the same number 57 of monosaccharides. Only the smallest and largest proteome sizes are shown. As can be 58 seen, the number of monosaccharides used by an organism is independent of the 59 proteome size. For instance, Helicobacter pylori PNG84A (proteome size = 1353) uses 60 the same number of monosaccharides (7) as Sorangium cellulosum So0157-2 (proteome 61 size = 10480).

62 A

63 Figure S4 (A) The prevalence of each monosaccharide as percentages of the genomes 64 analyzed in this study (viz., 12939) and the number of species covered by these genomes 65 (viz., 3384; Figure S1(a)). The diagonal line is manually drawn to facilitate visualization 66 of deviations. (B) Zoomed in view of the region near the origin in (A). Data for most of 67 the monosaccharides lie on the diagonal suggesting that the sequencing of a large 68 number of strains for a few species has not biased the outcome, with the exception of 69 TDP-/dTDP-Fuc4NAc and UDP-L-Qui2NAc. TDP-/dTDP-Fuc4NAc (a point below the 70 diagonal line) is present in fewer species but represents a larger fraction of genomes 71 since 679 strains of E. coli contain this monosaccharide. Conversely, presence of UDP-L- 72 Qui2NAc (a point above the diagonal line) is highly strain specific. Abbreviated names 73 are used for some of the monosaccharides. Full names of these are given in 74 Supplementary_data.xlsx:Worksheet4.

76 Figure S5 Setting bit score thresholds for HMM profiles with varying substrate 77 specificities. TrEMBL database was scanned using the profiles shown along the X- and 78 Y-axes in the above scatter plots; for these scans, default values set by HMMer were 79 used for all the parameters. Hits that are common to a pair of profiles (shown along X- 80 and Y-axes) were chosen and bit scores of such hits were plotted against each other. Bit 81 score thresholds (indicated by red lines) were chosen such that a protein is a hit for only 82 one of the two profiles. Threshold was revised for GPE05331 set to exclude PdeG.

83 Table S1 Tools and databases used in this study Tool / Version / URL Reference Database Release

Tools installed and run locally on a Linux platform

BLASTp 2.2.31+ ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.2.31/ (1)

HMMER 3.1b2 http://hmmer.org/download.html (2)

MUSCLE 3.8.31 https://www.ebi.ac.uk/Tools/msa/muscle/ (3)

CD-Hit 4.6 http://weizhongli-lab.org/cd-hit/ (4)

Directly accessed from the website or FTP site

UniProt 2018_07 https://www.uniprot.org/ (5)

Genome 2019_03 https://www.ncbi.nlm.nih.gov/genome/ (6)

Not Pubmed https://www.ncbi.nlm.nih.gov/pubmed/ (6) applicable

CATH-Plus 4.2 http://www.cathdb.info/ (7)

Not PDB https://www.rcsb.org/ (8) applicable

Used through the TrEMBL database

Not UniRule https://www.uniprot.org/help/unirule (5) applicable Not SAAS https://www.uniprot.org/help/saas (5) applicable 84 85

86 References cited in Table S1 87 88 1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment 89 search tool. J Mol Biol. 1990 Oct 5;215(3):403–10.

90 2. Eddy SR. Accelerated Profile HMM Searches. PLoS Comput Biol. 2011 91 Oct;7(10):e1002195.

92 3. Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, et al. The EMBL- 93 EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019 Jul 94 2;47(W1):W636–41.

95 4. Huang Y, Niu B, Gao Y, Fu L, Li W. CD-HIT Suite: a web server for clustering and 96 comparing biological sequences. Bioinformatics. 2010 Mar 1;26(5):680–2.

97 5. UniProt Consortium. UniProt: a worldwide hub of protein knowledge. Nucleic Acids 98 Res. 2019 Jan 8;47(D1):D506–15.

99 6. NCBI Resource Coordinators. Database resources of the National Center for 100 Biotechnology Information. Nucleic Acids Res. 2018 04;46(D1):D8–13.

101 7. Knudsen M, Wiuf C. The CATH database. Human Genomics. 2010;4(3):207.

102 8. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The 103 Protein Data Bank. Nucleic Acids Res. 2000 Jan 1;28(1):235–42.

104

105 Table S2 Monosaccharides whose both enantiomers are considered in the present study: 106 comparison of the nucleotide to which the enantiomer is linked and the precursor for its 107 biosynthesis D enantiomer L enantiomer Monosaccharide Nucleotide Precursor Nucleotide Precursor

Rhamnose GDP Glc-1-P TDP, dTDP, UDP Glc-1-P

6-Deoxytalose GDP Glc-1-P TDP, dTDP Glc-1-P

Galactose UDP Glc-1-P GDP Glc-1-P

Fucose TDP, dTDP Glc-1-P GDP Glc-1-P

Fuc2NAc UDP UDP-Glc2NAc UDP UDP-Glc2NAc

Qui2NAc UDP UDP-Glc2NAc UDP UDP-Glc2NAc 108 109

110 Flowchart S1 Procedure used to generate HMM profiles

1. Generation of Exp dataset and Exp profile, and setting 푇푒푥푝 Step 1a Consider only those enzymes which are characterized by direct enzyme activity assay Step 1b Remove redundancy (80% sequence identity cutoff) and obtain a multiple sequence alignment (MSA) Step 1c Use the MSA as input to generate an HMM profile Step 1d Score Exp dataset sequences against this HMM profile Step 1e Set the bit score of the lowest scoring sequence as the bit score

threshold for Exp dataset, 푇푒푥푝

2. Generation of Extend dataset and Extend profile, and setting 푇푒푥푡푒푛푑 Step 2a Add sequences that meet any of the following criteria to the Exp dataset

(i) SwissProt entries satisfying the threshold 푇푒푥푝

(ii) SwissProt entries scoring < 푇푒푥푝 provided they show conservation of active site residues. Active site residues were collated based on site directed mutagenesis studies or ligand-bound 3D structures (iii) TrEMBL entries for which molecular function has been inferred from experiments other than direct enzyme assays viz., complementation assays, phenotypic studies, etc. (iv) TrEMBL entries with solved 3D structure (v) FunFam members (CATH database) but only in the case of CDP-glucose 4,6-dehydratase (FunFam 20603) and phosphomannoisomerase family 3 (FunFam 54112) Step 2b Remove redundancy (80% sequence identity cutoff) and obtain a multiple sequence alignment (MSA) Step 2c Use the MSA as input to generate an HMM profile Step 2d Score Extend dataset sequences against this HMM profile Step 2e Set the bit score of the lowest scoring sequence as the bit score threshold for Extend dataset, 푇푒푥푡푒푛푑 111

112 Flowchart S2 Precedence rules for assigning annotation to proteins that are hits to two 113 or more profiles and/or BLASTp queries # Case 1 of 14 # specific_aminoTs = [GPE01710, GPE01430, GPE01530] # C3_C4_aminoTs = [GPE01910, GPE01830] # EXPECTED: for a protein which is a hit for one of the specific_aminoTs is expected to be a hit in C3_C4_aminoTs as well as GPE01230

IF (hit for any one of specific_aminoTs) THEN IF (hit for any one of C3_C4_aminoTs) THEN IF (hit for GPE01230) THEN Pass (i.e., this is as expected) ELSE Alert: Hit for one of C3_C4_aminoTs but not GPE01230 ENDIF ELSE Alert: Hit for one of specific_aminoTs but not C3_C4_aminoTs ENDIF ENDIF IF (hit for any one of C3_C4_aminoTs) THEN IF (hit for GPE01230) THEN Pass (as expected) ELSE Alert: Hit for one of C3_C4_aminoTs but not GPE01230 ENDIF ENDIF # Case 2 of 14 IF a protein is a hit for any one of specific_aminoTs, it should be assigned that annotation IF (hit for any one of specific_aminoTs) THEN Assign annotation ENDIF ENDIF # Case 3 of 14 # For a protein which is a hit for one of C3_C4_aminoTs but not any of specific_aminoTs, it should be assigned the former IF (hit for one of C3_C4_aminoTs and not for any of specific_aminoTs) THEN Assign GPE01830/GPE01910 annotation ENDIF

# Case 4 of 14 # For a protein which is a hit for GPE00210 # GPE00210 is used only in combination with GPE00231 # A protein which is a hit for GPE00210 is expected to be a hit for GPE00231 also IF (hit for GPE00210) THEN IF (hit for GPE00231) THEN Assign GPE00210 annotation ELSE Alert: Hit for GPE00210 but not GPE00231 ENDIF ENDIF # Case 5 of 14 # For a protein which is a hit for GPE02430 # GPE02430 is used only in combination with GPE02530 # A protein which is a hit for GPE02430 is expected to be a hit for GPE02530 also IF (hit for GPE02430) THEN IF (hit for GPE002530) THEN Assign GPE02430 annotation ELSE Alert: Hit for GPE02430 but not GPE02530 ENDIF ENDIF # Case 6 of 14 # For a protein that is a hit for GPE03130 # GPE03130 is used only in combination with GPE03430 # A protein which is a hit for GPE03130 is expected to be a hit for GPE03430 also IF (hit for GPE03130) THEN IF (hit for GPE03430) THEN Assign GPE03130 annotation ELSE Alert: Hit for GPE03130 but not GPE03430 ENDIF ENDIF

# Case 7 of 14 # For a protein that is a hit for GPE03210 # GPE03210 is used only in combination with GPE03430 # A protein which is a hit for GPE03210 is expected to be a hit for GPE03430 also IF (hit for GPE03210) THEN IF (hit for GPE03430) THEN Assign GPE03210 annotation ELSE Alert: Hit for GPE03210 but not GPE03430 ENDIF ENDIF # Case 8 of 14 # For a protein that is a hit for GPE09130 # GPE09130 is used only in combination with GPE09630 # A protein which is a hit for GPE09130 is expected to be a hit for GPE09630 also IF (hit for GPE09130) THEN IF (hit for GPE09630) THEN Assign GPE09130 annotation ELSE Alert: Hit for GPE09130 but not GPE09630 ENDIF ENDIF # Case 9 of 14 # For a protein that is a hit for GPE09230 # GPE09230 is used only in combination with GPE09330 and GPE09630 # A protein which is a hit for GPE09230 is expected to be a hit for GPE09630 also IF (hit for GPE09230) THEN IF (hit for GPE09630) THEN Assign GPE09230 annotation ELSE Alert: Hit for GPE09230 but not GPE09630 ENDIF ENDIF

# Case 10 of 14 # For a protein that is a hit for GPE09330 and GPE09630 # GPE09330 is used only in combination with GPE09630 # A protein can be a hit for GPE09330 or GPE09630, but not for both (non- orthologous) IF (hit for GPE09330 AND hit for GPE09630) THEN Alert: Hit for GPE09330 and GPE09630 ENDIF # Case 11 of 14 # For a protein that is a hit for GPE00620 and GPE00720 # GPE00620 is used only in combination with GPE00720 # A protein can be a hit for GPE00620 or GPE00720, but not for both (non- orthologous) IF (hit for GPE00620 AND hit for GPE00720) THEN Alert: Hit for GPE00620 and GPE00720 ENDIF # Case 12 of 14 # Isomerases: GPE07030, GPE07130, GPE07230, and GPE07330 # A protein can be a hit for any one of the above four profiles (non-orthologous) For GPE07030, GPE07130, GPE07230 and GPE07330 IF (hit for more than one) Alert: Hit for (list all profiles which appear as hits from above list)] # Case 13 of 14 # For a protein that is a hit for GPE00430 and GPE00530 # GPE00430 is used only in combination with GPE00530 # A protein can be a hit for GPE00430 or GPE00530, but not for both (non- orthologous) IF (hit for GPE00430 AND hit for GPE00530) THEN Alert: Hit for GPE00430 and GPE00530 ENDIF # Case 14 of 14 # For a protein that is a hit for GPE05332 and Q81A42:1-328 # GPE05332 is used only in combination with Q81A42:1-328 # A protein can be a hit for GPE05332 or Q81A42:1-328, but not for both (non- orthologous) IF (hit for GPE05332 AND hit for Q81A42:1-328) THEN Alert: Hit for GPE05332 and Q81A42:1-328 ENDIF 114

115 Research articles which report the characterization of enzymes involved in the 116 biosynthesis of monosaccharides are listed below. Amino acid sequences of these 117 enzymes were either used to generate HMM profiles or used as BLASTp queries. The 118 PubMed Ids of these research articles are included in the GlycoPathDB 119 (www.bio.iitb.ac.in/glycopathdb/) against respective sequence entry. These PubMed Ids 120 are hyperlinked to the corresponding PubMed webpage.

121 1. Wang-Gillam A, Pastuszak I, Elbein AD. A 17-amino acid insert changes UDP-N- 122 acetylhexosamine pyrophosphorylase specificity from UDP-GalNAc to UDP- 123 GlcNAc. J Biol Chem. 1998 Oct 16;273(42):27055–7.

124 2. Watt G, Leoff C, Harper AD, Bar-Peled M. A bifunctional 3,5-epimerase/4-keto 125 reductase for nucleotide-rhamnose synthesis in Arabidopsis. Plant Physiol. 2004 126 Apr;134(4):1337–46.

127 3. Hinderlich S, Stäsche R, Zeitler R, Reutter W. A bifunctional enzyme catalyzes 128 the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. 129 Purification and characterization of. J Biol Chem. 1997 Sep 26;272(39):24313–8.

130 4. Breazeale SD, Ribeiro AA, McClerren AL, Raetz CRH. A formyltransferase 131 required for polymyxin resistance in Escherichia coli and the modification of lipid 132 A with 4-Amino-4-deoxy-L-arabinose. Identification and function oF UDP-4- 133 deoxy-4-formamido-L-arabinose. J Biol Chem. 2005 Apr 8;280(14):14154–67.

134 5. Yoo H-G, Kwon S-Y, Karki S, Kwon H-J. A new route to dTDP-6-deoxy-l-talose 135 and dTDP-L-rhamnose: dTDP-L-rhamnose. Bioorg Med Chem Lett. 2011 Jul 136 1;21(13):3914–7.

137 6. Yoshida Y, Nakano Y, Nezu T, Yamashita Y, Koga T. A novel NDP-6- 138 deoxyhexosyl-4-ulose reductase in the pathway for the synthesis of thymidine 139 diphosphate-D-fucose. J Biol Chem. 1999 Jun 11;274(24):16933–9.

140 7. Swan MK, Hansen T, Schönheit P, Davies C. A novel phosphoglucose isomerase 141 (PGI)/phosphomannose isomerase from the crenarchaeon Pyrobaculum 142 aerophilum is a member of the PGI superfamily: structural evidence at 1.16-A 143 resolution. J Biol Chem. 2004 Sep 17;279(38):39838–45.

144 8. Jiang H, Wang S, Dang L, Wang S, Chen H, Wu Y, et al. A novel short-root gene 145 encodes a glucosamine-6-phosphate acetyltransferase required for maintaining 146 normal root cell shape in rice. Plant Physiol. 2005 May;138(1):232–42.

147 9. DeHaven JE, Robinson KA, Nelson BA, Buse MG. A novel variant of glutamine: 148 fructose-6-phosphate amidotransferase-1 (GFAT1) mRNA is selectively 149 expressed in striated muscle. Diabetes. 2001 Nov;50(11):2419–24.

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