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Supplementary Materials
Mutational analysis of conserved regions harbouring catalytic triad residues of the levansucrase protein encoded by lsc-3 (lsc3) gene of Pseudomonas syringae pv. tomato DC3000
Karin Mardo, Triinu Visnapuu, Heiki Vija, Triin Elmi and Tiina Alamäe
Supplementary tables S1, S2
Supplementary figures S1, S2
Supplementary references
Table S1. Oligonucleotides* for site-specific mutation of lsc3 gene.
Oligonucleotidename / Codon exchange / Oligonucleotide sequence
W61A Rev / TGG → GCG / 5’-ATGGTGTCCGCGATGAATACCG-3’
W61N Rev / TGG → AAC / 5’-ATGGTGTCGTTGATGAATACCG-3’
D62A Rev / GAC → GCC / 5’-CATGGTGGCCCAGATGAAT-3’
T63A Rev / ACC → GCC / 5’-AGCGGCATGGCGTCCCAGATG-3’
L66A Rev / CTG → GCG / 5’-TCTCGCGCCGGCATGGTG-3’
D219A Rev / GAC → GCC / 5’-ACTGGGGGCGCGAAAGTTC-3’
P220A Rev / CCC → GCC / 5’-ATGAACGGACTGGCGTCGCG-3’
D225A Rev / GAT → GCC / 5’-CATCATTAGGGGCAATGAACGGAC-3’
D225N Rev / GAT → AAT / 5’-CATCATTAGGATTAATGAACGGAC-3’
Q301A Fw / CAG → GCG / 5’-TGAATGATGCGACCGAGCGC-3’
Q301E Fw / CAG → GAG / 5’-TGAATGATGAGACCGAGCGC-3’
T302M Fw / ACC → ATG / 5’-AATGATCAGATGGAGCGCCC-3’
E303A Fw / GAG → GCG / 5’-TGATCAGACCGCGCGCC-3’
R304A Fw / CGC → GCC / 5’-TCAGACCGAGGCCCCGCACTATG-3’
R304C Fw / CGC → TGC / 5’-TCAGACCGAGTGCCCGCACTATG-3’
H306A Fw / CAC → GCC / 5’-AGCGCCCGGCCTATGTTTTTCAAG-3’
*Name and sequence of oligonucleotides and concomitant codon exchange are presented. Mismatched nucleotides with regard to wild-type lsc3 are underlined and mutated codon is highlighted by gray background
Plasmid / ReferencepURI3 / [1-2]
pURI3-lsc3 / [3]
pURI3-lsc3W61A / This study
pURI3-lsc3W61N / This study
pURI3-lsc3D62A / This study
pURI3-lsc3T63A / This study
pURI3-lsc3L66A / This study
pURI3-lsc3D219A / This study
pURI3-lsc3P220A / This study
pURI3-lsc3D225A / This study
pURI3-lsc3D225N / This study
pURI3-lsc3D300N / [3]
pURI3-lsc3Q301A / This study
pURI3-lsc3Q301E / This study
pURI3-lsc3T302P / [3]
pURI3-lsc3T302M / This study
pURI3-lsc3E303A / This study
pURI3-lsc3R304A / This study
pURI3-lsc3R304C / This study
pURI3-lsc3H306A / This study
Table S2. Plasmids used in this study.
Fig. S1. Three conserved regions of bacterial levansucrases with catalytic triad amino acids marked by asterisks (*). Numbering of amino acid positions for each region is shown according to Lsc3. Protein sequences of 25 bacterial and archaeal levansucrases retrieved from CAZy [4] and BRENDA [5] databases were aligned using MUSCLE program [6] and visualized by Jalview software [7]. The background shading shows identity value between the residues at homologous positions. Conservation scores for each position calculated in ConSurf server [8-10] are indicated above the alignment. Most thoroughly studied levansucrases are marked with dark grey background and red frame. Levansucrases aligned and their accession numbers in protein/genomic sequence databases are as follows: Pseudomonas syringae pv. tomato, Lsc3 (Q88BN6; ENA accession No HE985190); Burkholderia indica (182678986); Burkholdera phymatum (B2JVY2); Burkholderia pseudomallei (Q3JGQ0); Arthrobacter sp. K-1 (Q8VW87); Actinomyces viscosus (304364376); Streptomyces viridochromogenes (302555562); Cellulomonas flavigena (296131231); Pseudomonas chlororaphis subsp. aurantiaca, LscA (Q93FU9); Gluconacetobacter diazotrophicus, LsdA (Q43998); Erwinia amylovora (Q46654); Rahnella aquatilis (O54435); Pseudomonas fluorescens (C3K8D6); Serratia odorifera (D1RZ84); Pseudomonas syringae pv. phaseolicola (Q48JY3); Pseudomonas syringae pv. phaseolicola (Q48BC9); Zymomonas mobilis (Q60114); Gluconacetobacter oxydans (58039338); Clostridium acetobutylicum (15895050); Halorubrum lacusprofundi (B9LT89); Haloalkalicoccus jeotgali (300710312); Halomicrobium mukohataei (C7P4M9); Haloarcula marismortui (Q5V249); Bacillus megaterium, SacB (D5DC07); Bacillus subtilis, SacB (P05655).
Fig. S2. TLC analysis of reaction products synthesized by levansucrase Lsc3 and its mutants from 1200 mM (410.8 g/L) sucrose during 20 h. Reactions and TLC analysis were performed as in [3] and [11]. For the reactions 2.7 U of levansucrase was used. 100 µg of protein was reacted in case of mutants with very low activity W61A, W61N, D62A, D219A, E303A, R304C.
Reference sugars R1: fructose (DP 1; 30 mM), 1-kestose (DP 3; 8 mM), levan (DP >7; 0.7%); R2: sucrose (DP 2; 30 mM), nystose (DP 4; 8 mM), (DP >7; 0.7%). C – control sample without the protein which was handled identically to reaction samples. All reaction mixtures were diluted 4 times in distilled water and 0.5 µl of the dilution was applied onto the TLC plate.
Supplementary references
[1] Rivas, B. de Las, Curiel, J.A., Mancheño, J.M., Muñoz, R. (2007) Expression vectors for enzyme restriction- and ligation-independent cloning for producing recombinant His-fusion proteins. Biotechnol. Prog. 23, 680-686.
[2] Curiel, J.A., de Las Rivas, B., Mancheño, J.M., Muñoz, R. (2011) The pURI family of expression vectors: a versatile set of ligation independent cloning plasmids for producing recombinant His-fusion proteins. Protein Expr. Purif. 76, 44-53.
[3] Visnapuu, T., Mardo, K., Mosoarca, C., Zamfir, A.D., Vigants, A., Alamäe T. (2011) Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: Substrate specificity, polymerizing properties and usage of different acceptors for fructosylation. J. Biotechnol. 155, 338-349.
[4] Carbohydrate-Active Enzymes Database: http://www.cazy.org
[5] Brenda.The Comprehensive Enzyme Information System: http://www.brenda-enzymes.org
[6] Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797.
[7] Waterhouse, A.M., Procter, J.B., Martin, D.M.A, Clamp, M., Barton, G.J. (2009) Jalview Version 2 – a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189-1191.
[8] Glaser, F., Pupko, T., Paz, I., Bell, R.E., Bechor, D., Martz, E., Ben-Tal, N. (2003) ConSurf: identification of functional regions in proteins by surface-mapping of phylogenetic information. Bioinformatics 19, 163-164.
[9] Landau, M., Mayrose, I., Rosenberg, Y., Glaser, F., Martz, E., Pupko, T., Ben-Tal, N. (2005) ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33, 299-302.
[10] Ashkenazy, H., Erez, E., Martz, E., Pupko, T., Ben-Tal, N. (2010) ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, 529-533.
[11] Visnapuu, T., Zamfir, A.D., Mosoarca, C., Stanescu, M.D., Alamäe, T. (2009) Fully automated chip-based negative mode nanoelectrospray mass spectrometry of fructooligosaccharides produced by heterologously expressed levansucrase from Pseudomonas syringae pv. tomato DC3000. Rapid Commun. Mass Spectrom. 23, 1337-1346.