We study structure-function of enzymes involved in
Our laboratory is interested in aldolases and phosphotriesterases involved in the degradation of aromatics (eg polychlorinated biphenyls) and organophosphorous pollutants (pesticides and chemical warfare agents), respectively. We are performing structure-function studies to understand their catalytic mechanisms and the molecular basis for their unique substrate specificities.
This will form the basis for our long term goal to engineer
these enzymes to enhance their bioremediation capabilities or to
Crystals of Enzymes used for structure determination by X-ray crystallography
(2) Disruption of quorum sensing
Many pathogenic bacteria regulate the expression of virulence factors in a population dependent manner (eg in formation of biofilms or production of toxins). Bacteria sense population density by production and secretion of chemical signals, such as N-acylhomoserine lactones. We are working on enzymes that degrade these signals, thereby interfering with quorum sensing. These enzymes could have potential medical or industrial applications to control bacterial infections or prevent biofouling.
(3) Biosynthesis of siderophores
Knockout of a specific pyoverdine synthesis gene in P. aeruginosa resulting in the loss of green fluorescence under UV.
Pseudomonas
aeruginosa is an opportunistic pathogen that infects
immuno-compromised and cystic fibrosis patients. Successful colonization by this bacteria depends on their
ability to competitively acquire iron, an essential growth element, from tightly
bound iron stores within their hosts. Acquisition
of iron is facilitated by the synthesis and secretion of specialized molecules,
termed siderophores, that bind and transport iron from the host into the
bacteria cell. While the structure
of pyoverdine, the major siderophore produced by P. aeruginosa, was
elucidated more than twenty years ago, the mechanism of how pyoverdine is
synthesized and transported across the bacterial cell wall is still poorly
understood. It has however been
recognized that disruption of this mechanism of iron acquisition would be
therapeutically useful against Pseudomonas infections.
Structure
of the pyoverdine chromophore
Our laboratory is actively involved in
elucidating the biosynthetic pathway of pyoverdine using a combination of in-vivo and in-vitro approaches.
Research techniques used
The techniques we use in our research are
multidisciplinary and include:
(1) Molecular Genetics - PCR, Gene
cloning, Gene knockout, Directed/Random mutagenesis
(2) Protein Purification by FPLC
(3) Enzymology - Steady-state and Pre-steady-state kinetics
(4) Spectroscopy – UV-Visible, Fluorescence, Circular Dichroism, Nuclear Magnetic Resonance (NMR)
(5) Analytical Chemistry –HPLC, Mass
spectrometry
(6) X-ray crystallography
(7) Computer based molecular modelling
Selected
Research Publications
1. Carere, J., Baker, P. and Seah, S.Y.K. (2011) Investigating the molecular determinants for substrate channeling in BphI-BphJ, an aldolase-dehydrogenase complex from the polychlorinated biphenyls degradation pathway. Biochemistry
2. Baker, P., Carere, J. and Seah, S.Y.K. (2011) Probing the molecular basis of substrate specificity, stereospecificity, and catalysis in the class II pyruvate aldolase, BphI. Biochemistry 50:3559-3569.
3. Ng, F.S.W., Wright, D. and Seah, S.Y.K. (2011) Characterization of a phosphotriesterase-like lactonase from Sulfolobus solfataricus and its immobilization for quorum quenching. Appl Environ Microbiol 77:1181-1186
4. Wang, W., Mazurkewich, S., Kimber, M.S., and Seah, S.Y.K. (2010) Structural and kinetic characterization of 4-hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase: a protocatechuate degradation enzyme evolutionarily convergent with the HpaI and DmpG pyruvate aldolases. J. Biol. Chem. 285:36608-36615
5. Wang, W., Baker, P. and Seah, S.Y.K. (2010) Comparison of two metal-dependent pyruvate aldolases related by convergent evolution: substrate specificity, kinetic mechanism, and substrate channeling. Biochemistry 49:3774-3782.
6. Baker P., Pan, D., Carere, J., Rossi, A., Wang, W. and Seah, S.Y.K. (2009) Characterization of an aldolase/dehydrogenase complex that exhibits substrate channeling in the polychlorinated biphenyls degradation pathway. Biochemistry 48:6551-6558.
7. Wang, W. and Seah, S.Y.K. (2008) The role of a conserved histidine residue in a pyruvate specific Class II aldolase. FEBS Lett. 582:3385-3388.
8. Horsman, G.P., Bhowmik, S., Seah, S.Y.K., Kumar, P., Bolin, J.T., Eltis, L.D. (2007) The tautomeric half-reaction of BphD, A C-C bond hydrolase: Kinetic and structural evidence supporting a key role for histidine 265 of the catalytic triad. J. Biol. Chem.282:19894-19904
9.. Seah, S.Y.K. Ke, J., Denis, G., Horsman, G.P., Fortin, P.D., Whiting, C.J. and Eltis, L.D. (2007) Characterization of a C-C bond hydrolase from Sphingomonas wittichii RW1 with novel specificity towards PCB metabolites. J. Bacteriol. 189:4038-4045
10. Ge, L. and Seah,
S.Y.K. (2006) Heterologous expression,
purification and characterization of an L-ornithine N5-hydroxylase
involved in pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa. J.
Bacteriol. 188:7205-7210..
11. Horsman, G.P, Ke, J., Dai, S., Seah, S.Y.K., Bolin, J.T. and Eltis, L.D. (2006) Kinetic and structural insight into the mechanism of BphD, a C-C bond hydrolase in the biphenyl degradation pathway. Biochemistry 45:11071-11086.
12. Wang,
W. and Seah, S.Y.K. (2005) Purification and Biochemical characterization of a
pyruvate-specific Class II aldolase, HpaI. Biochemistry 44:9447-9455.
13. Wang, P. and Seah,
S.Y.K. (2005) Substrate and metal cofactor specificity of a hydratase involved in the
degradation pathway of biphenyl/chlorobiphenyl. FEBS
Journal 272:966-974
14. Vandenende, CS, Vlasschaert, M. and Seah,
S.Y.K. (2004). Functional
characterization of an aminotransferase involved in pyoverdine siderophore
biosynthesis. J.
Bacteriol. 186:5596-5602