1. Protein engineering of toluene ortho-
monooxygenase of Burkholderia cepacia G4 for
regio specific hydroxylation of indole to form
various indigoid compounds
BURAK YENER
GENETIC AND BIOENGINEERING
50091127

2. What is Indigo?
Indigo dye is an organic compound with a distinctive
blue. Historically, indigo was a natural dye extracted
from plants, and this process was important
economically because blue dyes were once rare.
Nearly all indigo dye produced today — several
thousand tons each year — is synthetic. It is the blue
of blue jeans.
http://en.wikipedia.org/wiki/Indigo_dye

3. Indigo
Nature indigo plant extract
sample
The production of indigo is primarily
by chemical syntheses, such as the
Adolf von Baeyer chemical synthesis of
1890 (Gillam et al. 2000)
which resulted in the fifth Noble prize in
chemistry.
http://upload.wikimedia.org/wikipedia/commons/1/18/Indigo_plant_
extract_sample.jpg

4. Chemical Structure of Indigo
http://upload.wikimedia.org/wikipedia/co
mmons/c/c7/Indigo.svg

6. C-3 oxidation and forming indigo

7. C-2 oxidation and forming isoindigo

8. Aims Of This Study
 The aims of this study were to create different color
producing TOM variants via DNA shuffling, to discern
the important residues that cause the formation of these
various colored products.
 Explore the altered patterns of indole hydroxylation by E.
coli TG1 expressing TOM with amino acid variations at
positions V106 and A113 of the hydrolase α-subunit
(TomA3)
 Explanation at the molecular level for why indole was
oxidized in three possible ways (hydroxylation of indole
at C-3, C-2, and at both C-2 and C-3 )

9. Methods
 E. Coli TG1 strain and pBS(kan)TOM plasmid used.
 DNA shuffling of TOM
 Saturation mutagenesis and DNA sequencing
 Thin-layer chromatography (TLC)
 High performance liquid chromatography (HPLC)
 Liquid Chromatography - Mass Spectroscopy ( LC-
MS)

10. DNA shuffling
http://www.nature.com/nrmicro/journa
l/v2/n7/box/nrmicro925_BX2.html

11. Saturation mutagenesis is a form of site-directed mutagenesis, in which one
tries to generate all possible (or as close to as possible) mutations at a specific site,
or narrow region of a gene.
Thin layer chromatography (TLC) is a chromatography technique used to
separate mixtures. Thin layer chromatography can be used to monitor the
progress of a reaction, identify compounds present in a given mixture, and
determine the purity of a substance.
HPLC, is a chromatographic technique used to separate a mixture of
compounds in analytical chemistry and biochemistry with the purpose of
identifying, quantifying and purifying the individual components of the
mixture.
Liquid chromatography–mass spectrometry
Generally its application is oriented towards the general detection and potential
identification of chemicals in the presence of other chemicals (in a complex
mixture)

12. Results

13. Results
Fig. 2 a,b Colored compoundsproduced by TOM variants. a) Colored
chloroform culture extracts of TOM variants with mutations at position
TomA3 A113, along with standards (indigo, indirubin, isatin, isoindigo).
b) Cell color extracts of TomA3 A113G mutants

14. Discussion
 Results showed that random mutagenesis of TOM
led to the identification of the key sites V106 and
A113 that are responsible for the different indole
hydroxylation patterns.
 Saturation mutagenesis at these key sites resulted in
the discovery of more diversified indole
hydroxylation which allows a single enzyme for the
first time to make a diverse range of indigoid
compounds.

15. Discussion
 Single or double amino acid change can create catalytically
distinct enzyme variants that hydroxylate indole in different
regiospecific positions on the pyrrole and benzene rings.
 TOM may partly, if not completely, control the oxidative
coupling of indole derivatives to form dimeric conjugates,
although indoxyl is normally believed to form indigo by non-
enzymatic condensation and oxidation.
These dramatic effects on indole hydroxylation are caused by
A113 and/or V106 substitution.
Although there is no direct evidence, it seems that the identity of
residues V106 and A113 not only determines the binding
orientation of the substrate and its regiospecific hydroxylation
but also influences the subsequent dimerization.

16. Discussion
 Along with the diversified products distributions in
Table 1 that are not easily explained by different
hydroxyindole formation rates, it appears the
enzyme may be controlling both dimerization and
the position of hydroxylation of the benzene or
pyrrole ring.

17. Thank You