CH5612 Natural Products, Biosynthesis and Enzyme Co-factors

1.

Ethanolamine ammonia lyase is a co-enzyme-B12 dependent enzyme which converts ethanolamine to acetaldehyde and ammonia. In isotope labelling studies, when [1,1- 2H2]-ethanolamine is incubated with the enzyme, then [1,2- 2H2]- acetaldehyde is generated.

(a) Propose a possible mechanism for ethanolamine ammonia lyase. In your answer consider the role of co-enzyme-B12 and rationalise the origin of the isotopically labelled products. [14 marks]

(b) How might you test if the ethanolamine ammonia lyase reaction is stereospecific? [6 marks]

(c) Give an example of another co-enzyme-B12 reaction representing the structures of the substrate and the product. [5 marks]

(d) The scheme below illustrates a bacterial enzymatic chlorination of tryptophan which utilises the co-factor FADH2

Illustrate a mechanism for this reaction, highlighting the role of the cofactor FADH2, the substrate tryptophan, molecular oxygen and chloride ion, for this enzymatic chlorination of tryptophan at the 5-position, to give 5-chlorotrytophan (shown above). [9 marks]

2.

(a) Give an arrow pushing mechanism for the formation of dimethylallylpyrophosphate from acetyl-CoA via the mevalonate pathway. Name all of the enzymes and co-factors involved. [8 marks]

(b) Name the alternative metabolic pathway that also forms the same C5 building block products, but does not utilise acetyl-CoA. [1 mark]

(c) Give an arrow pushing mechanism of a biotransformation that requires the input of energy in the form of ATP, (not a kinase), and explain why ATP is required. [3 marks]

(d) You are given the task of measuring the rate of reaction of an ATP dependent hexose kinase. The by-product ADP is utilised in the coupled assay shown below which involves the reformation of ATP via the reaction of ADP and PEP by the pyruvate kinase (PK) enzyme. Explain how the assay works, including what is monitored to measure the rate of reaction of the hexose kinase. [6 marks]

(e) What kinetic parameters and other factors do you require before you can begin to screen a compound library for inhibitors against this hexose kinase. [3 marks]

(f) Briefly explain how you could quantitatively determine the fatty acid species within olive oil. [3 marks]

(g) Using your own carbohydrate structures, describe the differences in the catalytic function of an endo- and exo– glycosidase, and describe briefly how they can be used to characterise oligosaccharides. [4 marks]

(h) Name two high-energy carbohydrate donors involved in the formation of the N-glycosylation precursor. [2 marks]

(i) Name the enzyme complex and all of the co-factors involved in converting pyruvate into acetyl-CoA within the mitochondria. [3 marks]

3.

(a) Outline a biosynthesis of the piperidine alkaloid A. Your answer should include the starting amino acid and any other precursors required, providing intermediates and, where appropriate, details of any co-factors employed.[11 marks]

(b) Suggest a biosynthetic pathway for polyketide B using 4-hydroxybenzoate-CoA C as a starter unit and explain how [1,2- 13C2]-acetate D would be incorporated.[11 marks]

(c) (i) Sketch the structure of the 8-membered ring containing Type I polyketide that would be produced by a trimodular polyketide synthase (PKS) with the domain structure shown below, and that utilises propionyl-CoA as a starter unit and then malonyl-CoA and 2 xg ´ methylmalonyl-CoA in turn as extender units. [7 marks]

(c) (ii) Macrolide E, a Type I polyketide, has undergone two post-PKS modifications during its biosynthesis. Identify the sites of this post-PKS modification. Your answer should include a clear indication of how specific chain extension units are incorporated into the carbon skeleton. [4 marks]