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J Bennett PhD presentation

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Joe Bennett
Joe Bennett

1) Dr. Joe Bennett studied the enzyme hydroxycinnamoyl CoA hydratase lyase (HCHL) which is able to produce vanillin from ferulic acid. 2) He performed kinetics experiments and x-ray crystallography on wild type and variant forms of HCHL to study its catalytic mechanism. 3) The crystal structures revealed that residue Glu-143 forms hydrogen bonds with the product vanillin, indicating it is essential for catalytic activity, while Ser-123 does not appear to play a major role in catalysis.

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HCHL: A route to Greener cake? Dr Joe Bennett 20th May 2010
INTRODUCTION
Natural vanilla extract • Over 180 compounds  Principle component – vanillin (~85%) • Vanilla orchid – pods and beans Flavour Fragrance Pharmaceuticals INTRODUCTION Vanilla
Natural v Synthetic • 12,000 tons per year  0.5% - natural vanilla  >99% - synthetic $1200 - 4000 kg-1 ~$15 kg-1 1Hocking, M. (1997) J. Chem. Educ. 74, 1055-1059 1 INTRODUCTION Vanillin production
Ferulic acid metabolism • Pseudomonas fluorescens  Ferulic acid – sole carbon source2 • Ligation to coenzyme A  Ligase HCLS Hydroxycinnamoyl CoA Hydratase Lyase – HCHL Hydration / ligation P. fluorescens biofilm 2Narbad, A. et al (1998) Microbiology. 144, 1397-1405 INTRODUCTION HCHL
Additional substrates3 3Mitra, A. et al (1999) Arch. Biochem. Biophys. 365, 10-16 INTRODUCTION HCHL
- Crotonases INTRODUCTION Crotonase superfamily • Structurally related proteins4 ECH PVIAAVNGYALGGGCELAMMCDIIYAGEKAQFGQPEILLGTIPGA 173 HCHL PTIAMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGN 152 *.** ***:.:*** . : **: ..::* ** .** * ** Oxyanion hole conserved ‘Catalytic’ Glu conserved ‘Catalytic’ Glu NOT conserved 4Bennett, J. et al (2007) Biochemistry. 46, 137-144 5Engel, C. et al (1998) J. Mol. Biol. 275, 847-859 • Parent – Enoyl CoA Hydratase (ECH)5
6Leonard, P. et al (2006) Acta. Cryst D. 60, 2343-2345 Tyr239 2.7 INTRODUCTION HCHL model Structure modelling • Native HCHL structure6  Ligand-bound ECH structure  Feruloyl CoA Glu-143: Water activation? Ser-123: Lytic function? Tyr-239: Substrate binding?
RESEARCH
RESEARCH Experiments performed • HCHL variants  Cloning, transformation, expression, purification Project outline Wild Type S123A E143A S123A/E143A Y239F Y239A Michaelis-Menten kinetics X-ray Crystallography Organic synthesis [Substrate] 0 0.02 0.04 0.06 0.08 Rate 0 2 4 Parameter Value Std. Error Enzyme Kinetics Data MECHANISM
7Bennett, J. et al (2008) Biochem. J. 414. 281-289 RESEARCH UV/Vis kinetics Michaelis-Menten kinetics7 • Feruloyl CoA depletion – 400 nm
RESEARCH Structural studies Apo and holo-HCHL • Native wild type HCHL • No ligand • Wild type HCHL  Co-crystallised with feroloyl CoA • Observations  Vanillin  Loop shift on ligand binding  Second Tyr moves ~6 Å
RESEARCH Structural studies Density maps Glu-143: H-bonded to product Tyr-239: H-bonded to product Tyr-75: H-bonded to product
DISCUSSION
8Fraaije, M. et al (1998) Eur. J. Biochem. 253, 712-719 DISCUSSION Examining mechanism Vanillyl Alcohol Oxidase (VAO)8 Green: VAO Blue: HCHL Similar substrate O2+ Tyrosine pincer
VAO mechanism – 4-(methoxymethyl) phenol9 9van den Heuvel, M. et al (2000) J. Biol. Chem. 275, 14799-14808 Quinone methide Intermediate DISCUSSION Vanillyl alcohol oxidase
DISCUSSION Quinone methide mechanism Vanillin Quinone methide enolate
• Substrate specificity: Tyr-75 and Tyr-239 SUMMARY HCHL Glu-143: Essential for activity • Similarities to VAO – not obvious from apo-enzyme • Quinone methide - possible intermediate Ser-123: No major role in catalysis
ACKNOWLEDGEMENTS Gideon Grogan Marek Brzozowski Johan Turkenburg YSBL staff BBSRC

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J Bennett PhD presentation

  • 1. HCHL: A route to Greener cake? Dr Joe Bennett 20th May 2010
  • 3. Natural vanilla extract • Over 180 compounds  Principle component – vanillin (~85%) • Vanilla orchid – pods and beans Flavour Fragrance Pharmaceuticals INTRODUCTION Vanilla
  • 4. Natural v Synthetic • 12,000 tons per year  0.5% - natural vanilla  >99% - synthetic $1200 - 4000 kg-1 ~$15 kg-1 1Hocking, M. (1997) J. Chem. Educ. 74, 1055-1059 1 INTRODUCTION Vanillin production
  • 5. Ferulic acid metabolism • Pseudomonas fluorescens  Ferulic acid – sole carbon source2 • Ligation to coenzyme A  Ligase HCLS Hydroxycinnamoyl CoA Hydratase Lyase – HCHL Hydration / ligation P. fluorescens biofilm 2Narbad, A. et al (1998) Microbiology. 144, 1397-1405 INTRODUCTION HCHL
  • 6. Additional substrates3 3Mitra, A. et al (1999) Arch. Biochem. Biophys. 365, 10-16 INTRODUCTION HCHL
  • 7. - Crotonases INTRODUCTION Crotonase superfamily • Structurally related proteins4 ECH PVIAAVNGYALGGGCELAMMCDIIYAGEKAQFGQPEILLGTIPGA 173 HCHL PTIAMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGN 152 *.** ***:.:*** . : **: ..::* ** .** * ** Oxyanion hole conserved ‘Catalytic’ Glu conserved ‘Catalytic’ Glu NOT conserved 4Bennett, J. et al (2007) Biochemistry. 46, 137-144 5Engel, C. et al (1998) J. Mol. Biol. 275, 847-859 • Parent – Enoyl CoA Hydratase (ECH)5
  • 8. 6Leonard, P. et al (2006) Acta. Cryst D. 60, 2343-2345 Tyr239 2.7 INTRODUCTION HCHL model Structure modelling • Native HCHL structure6  Ligand-bound ECH structure  Feruloyl CoA Glu-143: Water activation? Ser-123: Lytic function? Tyr-239: Substrate binding?
  • 10. RESEARCH Experiments performed • HCHL variants  Cloning, transformation, expression, purification Project outline Wild Type S123A E143A S123A/E143A Y239F Y239A Michaelis-Menten kinetics X-ray Crystallography Organic synthesis [Substrate] 0 0.02 0.04 0.06 0.08 Rate 0 2 4 Parameter Value Std. Error Enzyme Kinetics Data MECHANISM
  • 11. 7Bennett, J. et al (2008) Biochem. J. 414. 281-289 RESEARCH UV/Vis kinetics Michaelis-Menten kinetics7 • Feruloyl CoA depletion – 400 nm
  • 12. RESEARCH Structural studies Apo and holo-HCHL • Native wild type HCHL • No ligand • Wild type HCHL  Co-crystallised with feroloyl CoA • Observations  Vanillin  Loop shift on ligand binding  Second Tyr moves ~6 Ã…
  • 13. RESEARCH Structural studies Density maps Glu-143: H-bonded to product Tyr-239: H-bonded to product Tyr-75: H-bonded to product
  • 15. 8Fraaije, M. et al (1998) Eur. J. Biochem. 253, 712-719 DISCUSSION Examining mechanism Vanillyl Alcohol Oxidase (VAO)8 Green: VAO Blue: HCHL Similar substrate O2+ Tyrosine pincer
  • 16. VAO mechanism – 4-(methoxymethyl) phenol9 9van den Heuvel, M. et al (2000) J. Biol. Chem. 275, 14799-14808 Quinone methide Intermediate DISCUSSION Vanillyl alcohol oxidase
  • 18. • Substrate specificity: Tyr-75 and Tyr-239 SUMMARY HCHL Glu-143: Essential for activity • Similarities to VAO – not obvious from apo-enzyme • Quinone methide - possible intermediate Ser-123: No major role in catalysis