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Scanning for genes encoding GDS(L) hydrolases in Actinobacteria from wide
diversity of ecological niches
Bielen Ana
The 2nd International Symposium “VERA JOHANIDES” Zagreb, May 10-11, 2013
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Actinobacteriavarious ecological niches... ... various benefits
• Antibiotics
• Anticancer and other drugs
• Probiotics
• Hydrolytic enzymes for bioremediation and industrial applications
GDS(L) family of hydrolytic enzymes
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Sc1L SrL
pH activity 8 9.5
pH stability 4 - 9.5 4 - 9.5
Temp. activity 55 °C 55 °C
Temp. stability up to ~ 55 °C up to ~ 55 °C
Tm 66 °C 66 °C
Abramić et al, 1999; Bielen et al, 2009
Activ(ated) in organic solvents (dioxane, THF, acetone...)
p-nitrophenyl esters
α/β-naphthyl esters
Tween detergentsglycerol esters
Fats and oils
GDS(L) enzymes from Streptomyces coelicolor (Sc1L) and S. rimosus (SrL)
Classes of substrates...
Multifunctionality, stability, broad substrate specificity!
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GDS(L) family of lipolytic enzymes- conserved 3D structure- low overall sequence homology
GDS(L) esterase from S. scabies
... SEARCHING FOR NOVEL GDS(L) ENZYMES USING TRADITIONAL SEQUENCE SEARCH METHODS (e.g. Blast) IS NOT SUFFICIENT!
Block I
Block III
Block V
Block II
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Solution: HMM-based motif scanning techniques
Step 1: Collect sequences of experimentally confirmed family members
Etc.
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Step 2: Construct multiple sequence alignment (e.g. seed alignment)
Solution: HMM-based motif scanning techniques
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Step 3: Cut out conserved motifs
Solution: HMM-based motif scanning techniques
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Step 4: Build model representing selected motifs (emission probabilities!)
Step 5: Scan proteomes of selected organisms for desired motifs -> select best possible motifs in every sequence in proteome and assign a score
Solution: HMM-based motif scanning techniques
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Step 6: Combine different motif scanning methods for optimal results -> sharp cut-off discriminates between positive and negative hits
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Sco
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GDS(L) sequences – positive hitsOther sequences – negative hits
Scores of A. thaliana proteome scanning for GDS(L) motifs - Viterbi and posterior decoding combined
Solution: HMM-based motif scanning techniques
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52 actinobacterial proteomes
257 GDS(L) enzymes
cluster into several groups
great diversity,great potential
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Evidence for expansion of genes encoding for GDS(L) hydrolases in Actinobacteria by horizontal gene transfer
1. genes on plasmids2. deviations in codon
usage3. incongruency between
GDS(L) gene-phylogeny and species phylogeny
4. close homologues in distant organisms
ActinobacteriaFirmicutes
CyanobacteriaProteobacteria
Fungi
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Previously unknown variations in motifs
Block I Block VBlock III
Typi
cal
Varia
nt
Novel catalytic properties?
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Conclusions
• Application of proper bioinformatic tools to explore sequence diversity present in the databases (more than blast is out there!)
• Server for motif scanning -> under construction• Use natural diversity of hydrolitic enzymes for
biotechnology
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Thank you for your attention!
Bruvo-Mađarić BrankaVujaklija IvanPezer Željka
Goldstein PavleVujaklija Dušica Paradžik Tina