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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
Actinobacteria
various ecological niches...
... various benefits
• Antibiotics
• Anticancer and other
drugs
• Probiotics
• Hydrolytic enzymes
for bioremediation and
industrial applications
GDS(L) family of
hydrolytic enzymes
GDS(L) enzymes from Streptomyces coelicolor (Sc1L)
and S. rimosus (SrL)
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
66 °C
66 °C
Tm
Multifunctionality, stability, broad
substrate specificity!
Classes of substrates...
p-nitrophenyl
esters
α/β-naphthyl
esters
Activ(ated) in organic solvents
(dioxane, THF, acetone...)
glycerol esters
Fats and oils
Abramić et al, 1999; Bielen et al, 2009
Tween
detergents
GDS(L) family of lipolytic enzymes
GDS(L) esterase from S. scabies
 conserved 3D structure
 low overall sequence homology
Block I
Block II
Block III
Block V
... SEARCHING FOR NOVEL GDS(L) ENZYMES USING TRADITIONAL SEQUENCE SEARCH
METHODS (e.g. Blast) IS NOT SUFFICIENT!
Solution: HMM-based motif scanning techniques
Step 1:
Collect sequences of
experimentally
confirmed family
members
Etc.
Solution: HMM-based motif scanning techniques
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 cutoff discriminates between positive and negative hits
Scores of A. thaliana proteome scanning for GDS(L) motifs
- Viterbi and posterior decoding combined
35
30
25
Score
20
15
10
5
0
1
12
23
34
45
-5
-10
GDS(L) sequences – positive hits
Other sequences – negative hits
56
67
78
89
100
111
122
133
144
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.
2.
3.
4.
genes on plasmids
deviations in codon
usage
incongruency between
GDS(L) gene-phylogeny
and species phylogeny
close homologues in
distant organisms
Actinobacteria
Firmicutes
Cyanobacteria
Proteobacteria
Fungi
Previously unknown variations in motifs
Block III
Block V
Variant
Typical
Block I
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!
Goldstein Pavle
Vujaklija Ivan
Vujaklija Dušica
Paradžik Tina
Bruvo-Mađarić Branka
Pezer Željka