E. coli uses an aerobic degradative pathway for fission of aromatic compounds.

2,3-dihydroxyphenyl propionoic acid is one such compound.

After the benzene ring is broken through oxidative meta-fission by MhpB, the enzyme MhpC hydrolytically cleaves the product. Hydrolytic cleavage of C-C bond is very rare.

MhpC is a member of the α,β-hydrolase family. A conserved catalytic triad is found in these proteins – see ‘The Catalytic Triad’.

Cysteine 270 found very close to triadic histodine in active site. Conserved in all α,β-hydrolase enzymes.

Modification of Cys-270 by certain thiol directed reagents results in inactivation of protein. Function in enzymatic pathway is as yet unknown.

Aim to study function by labelling with thiol directed fluorescent molecule (N-(1-Pyrene) Maleimide), and observe fluorescence changes upon substrate binding. Graph shows change in fluorescence over time for MhpC

labelled with N-(1-Pyrene) Maleimide.

Intensity is very low - see ‘5: Circular Dichroism’.

Time scale is too long to show any changes due to substrate binding.

Initial dip when substrate present probably due to quenching of fluoresced light by the substrate.

More accurate technique required to see fluorescence changes upon substrate binding.

Suggest stopped-flow kinetics. Shows changes over a millisecond timescale.

Acknowledgements and ReferencesSupervised by Prof. Timothy Bugg. Special thanks to Dr. Jian-Jun Li, Dr. Alison Rodger, Chen Li and Rachel Marrington.[1] Catalytic Mechanism of a C-C Hydrolase Enzyme: Evidence for a Gem-Diol Intermediate, Not an Acyl Enzyme, S.M. Fleming, T.A. Robertson, G.J. Langley and T.D.H. Bugg, Biochemistry 2000, 39, 1522-1531.[2] Purification, Characterization, and Stereochemical Analysis of a C-C Hydrolase: 2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic Acid 5,6-Hydrolase, W.Y. Lam and T.D.H. Bugg, Biochemistry 1997, 36, 12242-12251.

Many pollutants contain aromatic carbon chains.

C-C bonds in aromatic compounds are very hard to break.

Micro-organisms have developed pathways to break benzenoid compounds.

Bioremediation is cost effective, but long term effects are unknown.

It is of great interest to understand the enzymatic pathways involved.

Better understanding of metabolic pathways may lead to better usage of micro-organisms in pollution degradation.

Cleavage occurs between C5 and C6.

Results suggest existence of keto-intermediate step. Acts as electron sink.

Insertion of hydrogen at C5 retains regiochemistry.

Two possible mechanisms for attack at C6 – attack by water or active site nucleophile.

Both may involve active site serine – see ‘The Catalytic Triad’.

Lack of proposed acyl enzyme intermediate suggests hydrolytic attack over nucleophilic attack.

Serine as a nucleophile

Previous results suggest the active site serine acts as a nucleophile. This would require the presence of an acyl intermediate step. Studies fail to show this intermediate, which suggests that the serine has some other function.Serine as a baseSerine can also act as a base in hydrolytic attack on the carbon. This would require a gem-diol intermediate step. The exact mechanism is still unknown.

In the α,β-hydrolase family, a triad of amino acids (histodine, aspartate and serine) are conserved. They occur in the active site in close proximity to one another, where they interact to allow Serine to act as a nucleophile. This triad is present in MhpC, but it is unknown whether it has the same function as for the serine proteases. The serine can act as either a nucleophile or as a base.

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Comparison of CD of wildtype and labelled proteins

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Used to check conformational changes upon labelling.

Spectrum suggests predominately β-sheet formation.

Shows 10 times less protein concentration than Bradford assay.

Bradford assay relies on concentration of aromatic amino acids. Bradford reagent may therefore bind heavily to pyrene label. Apparent protein concentration would be much greater than actual concentration.

The subsequent lack of protein in the fluorescence studies would give very low intensity readings.

The addition of the fluorophore may have caused the Bradford assay to overshoot the protein concentration by a large amount. Thus the protein solutions used in the fluorescence studies were much more dilute than originally thought. This would explain the low intensities seen in absorbance and fluorescence studies.

The labelling process seems to have worked, but the addition of the pyrene has affected the protein assay. If the experiment is to be repeated, the protein concentration must be ascertained using a method that will not be affected by the presence of the pyrene label.

By Sam Robson

Oxidative meta-cleavage of benzene ring, followed by hydrolytic cleavage of product by MhpC.

Structure of the amino acid residue cysteine.

Structure of the thiol directed reagent N-(1-Pyrene Maleimide).

Proposed mechanism for C-C bond cleavage by MhpC.

Molecular Organisation and Assembly in Cells