1. Difference in kinetic behaviour of catechol 2,3-dioxygenase variants from a polluted environment *
051217
김혜겸
* Howard et al., 2004

2. Contents
Introduction
Method
Result
Summary
Discussion

3. Introduction
Catechol and its substituted derivatives are common intermediates in aerobic degradation pathways of many natural and xenobiotic aromatic pollutants
Catechol 2,3-dioxygenase(C23O) is predominantly responsible for methyl-catechol related extradiol ring cleavage.

4. Introduction
Single amino acid change or few amino acid differences can bring a great change in catalytic properties of enzyme activity.
Unlike other experiments which are subjected to artificially altered proteins made in laboratory, this study is subjected to naturally found two different C23O.

5. Introduction
Among predominant C23O gene polymorphisms found in BTEX-contaminated environments, Psudomonas stutzeri AN10 identical C23O genes were discovered.
Selected two strains for they have C23O enzymes which differs only one amino acid from one another.

6. I YB VS IYdBTEX2
His 218
Tyr218
Highly contaminated site only
Both in highly and slightly contaminated sites
Benzen, toluene, ethylbenzene
Only benzene
Highest enzyme activity against catechol
Highest enzyme activity against 4-methylcatechol and increased enzyme activity against 3-methylcatechol

7. Here they reports…
Heterologous expression and kinetic characterization of these two C23O enzyme because they differ one amino acid and shows altered enzyme activity.

8. Method Design a pair of primer using conserved region PCR
Ttansformation into E.Coli JM109
Plasmid sequencing
SDS-PAGE
MALDI-TOF/N-terminal sequencing
Enzyme assay

9. Result pC23Ohis218 and pC23Otyr218 : 6 nucleotide difference
: Only one amino acid difference in 218th position

10. Result SDS-PAGE Confirmed as C23O by N-terminal sequencing & MALDI-TOF
C23Otyr C23Ohis218
The level of protein expression is about the same

11. Result

12. Result Alignment of C23O Subfamily
: 218th position usually consists his/phe (rarely with leu)
: Never considered to have any relevant for enzyme function

13. Result
Construction of crystal structure based on C23OMT-2 ( XylE protein )
Points to the tetramer interface at the entrance of a small channel leading from the interface to the active centre

14. Result Functional role of this channel is unclear
His/Tyr218, with a distance of more than 9Å to the active iron and pointing away from it
Unlikely to interfere much with water or oxygen molecules filling the channel

15. Summary
Same enzymes with one amino acid difference were achieved from natural environment
His 218/Tyr218
Enzyme assayed and aligned and built 3D structure

16. His 218
Tyr218
Location
Highly contaminated site only
Both in highly and slightly contaminated sites
Sole carbon and energy source
Benzen, toluene, ethylbenzene
Only benzene
Kinetic parameter
Highest enzyme activity against catechol
Highest enzyme activity against 4-methylcatechol and increased enzyme activity against 3-methylcatechol
Higher turnover rate
Lower km for all substrates

17. Summary
218th amino acid is not involved in the formation of substrate-binding pocket nor interfering role in entrance of substrates
218th amino acid is localized on the side of the second smaller channel leading to the active centre which function is not clear

18. Discussion
Many mutants effect on the enzymic activity of the protein by
Changing its dynamics
Creating small structural change affecting protein kinetics by long range interactions
=> His/Tyr218 variantsare consequences of the different kinetic characteristics of the enzymes or result from other characteristics of the host will need to analysed by site-directed mutagenesis in the same strain