bielen ana

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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-5

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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