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

Contents Introduction Method Result Summary Discussion

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.

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.

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.

I YB2 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

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

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

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

Result SDS-PAGE Confirmed as C23O by N-terminal sequencing & MALDI-TOF C23Otyr218 C23Ohis218 The level of protein expression is about the same

Result

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

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

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

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

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

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

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