bielen ana
DESCRIPTION
Scanning for genes encoding GDS(L) hydrolases in Actinobacteria from wide diversity of ecological niches. Bielen Ana. The 2 nd International Symposium “VERA JOHANIDES” Zagreb, May 10-11, 2013. Actinobacteria. various ecological niches. ... various benefits. Antibiotics - PowerPoint PPT PresentationTRANSCRIPT
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
Actinobacteriavarious ecological niches... ... various benefits
• Antibiotics
• Anticancer and other drugs
• Probiotics
• Hydrolytic enzymes for bioremediation and industrial applications
GDS(L) family of hydrolytic enzymes
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!
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
Solution: HMM-based motif scanning techniques
Step 1: Collect sequences of experimentally confirmed family members
Etc.
Step 2: Construct multiple sequence alignment (e.g. seed alignment)
Solution: HMM-based motif scanning techniques
Step 3: Cut out conserved motifs
Solution: HMM-based motif scanning techniques
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
Step 6: Combine different motif scanning methods for optimal results -> sharp cut-off discriminates between positive and negative hits
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-10
-5
0
5
10
15
20
25
30
35
Sco
re
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
52 actinobacterial proteomes
257 GDS(L) enzymes
cluster into several groups
great diversity,great potential
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
Previously unknown variations in motifs
Block I Block VBlock III
Typi
cal
Varia
nt
Novel catalytic properties?
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
Thank you for your attention!
Bruvo-Mađarić BrankaVujaklija IvanPezer Željka
Goldstein PavleVujaklija Dušica Paradžik Tina