Molecular
Organisation and
Assembly in Cells
By Sam Robson
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.
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.
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 base
Serine 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.
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,βhydrolase family. A conserved catalytic
triad is found in these proteins – see ‘The
Catalytic Triad’.
25
Results suggest existence of ketointermediate 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.
Aim to study function by labelling with
thiol directed fluorescent molecule (N(1-Pyrene) Maleimide), and observe
fluorescence changes upon substrate
binding.
Structure of the thiol directed
reagent N-(1-Pyrene Maleimide).
Wildtype
5
0
190
Labelled
200
210
220
230
240
250
260
-5
Wavelength (nm)
Used to check conformational changes upon labelling.
Spectrum suggests predominately β-sheet formation.
Shows 10 times less protein concentration than
Bradford assay.
Lack of proposed acyl enzyme
intermediate suggests hydrolytic attack
over nucleophilic attack.
Proposed mechanism for
C-C bond cleavage by MhpC.
3
2.5
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.
2
LABELLED PROTEIN
PROTEIN + SUBSTRATE
1.5
SUBSTRATE CONTROL
BUFFER CONTROL
1
0.5
0
0
10
20
30
40
50
60
70
80
90
100
Time (secs)
Graph shows change in fluorescence over time for MhpC
labelled with N-(1-Pyrene) Maleimide.
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.
Structure of the amino
acid residue cysteine.
10
-15
Intensity is very low - see ‘5: Circular Dichroism’.
Oxidative meta-cleavage of benzene ring, followed by
hydrolytic cleavage of product by MhpC.
15
-10
Both may involve active site serine –
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.
20
Ellipticity (millidegrees)
C-C bonds in aromatic compounds are very hard
to break.
E. coli uses an aerobic degradative
pathway for fission of aromatic
compounds.
Cleavage occurs between C5 and C6.
Intensity
Many pollutants contain aromatic carbon chains.
Comparison of CD of wildtype and labelled proteins
Suggest stopped-flow kinetics. Shows changes
over a millisecond timescale.
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.
Acknowledgements and References
Supervised 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 5,6Hydrolase, W.Y. Lam and T.D.H. Bugg, Biochemistry 1997, 36, 12242-12251.