J Bennett PhD presentation
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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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J Bennett PhD presentation
- 1. HCHL: A route to Greener cake? Dr Joe Bennett 20th May 2010
- 2. INTRODUCTION
- 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?
- 9. RESEARCH
- 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 Å
- 13. RESEARCH Structural studies Density maps Glu-143: H-bonded to product Tyr-239: H-bonded to product Tyr-75: H-bonded to product
- 14. DISCUSSION
- 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
- 19. ACKNOWLEDGEMENTS Gideon Grogan Marek Brzozowski Johan Turkenburg YSBL staff BBSRC