Stereochemistry of the reaction catalysed by 2-hydroxy-6-keto-6-phenyl-hexa-2,4-dienoic acid 5,6-hydrolase (BphD).

Article abstract of DOI:10.1039/b415491f

The stereochemical course of the reaction catalysed by C-C hydrolase BphD from Burkholderia xenovorans LB400 occurs with replacement of a benzoyl group by hydrogen with overall retention of stereochemistry.

Full text of DOI:10.1039/b415491f

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Stereochemistry of the reaction catalysed by 2-hydroxy-6-keto-6-
phenyl-hexa-2,4-dienoic acid 5,6-hydrolase (BphD)
Jian-Jun Li and Timothy D. H. Bugg*
Received (in Cambridge, UK) 6th October 2004, Accepted 20th October 2004
First published as an Advance Article on the web 26th November 2004
DOI: 10.1039/b415491f
9
3-bromo-2-acetoxy-propenoate. C-C hydrolase from Burkholderia
The stereochemical course of the reaction catalysed by C-C
hydrolase BphD from Burkholderia xenovorans LB400 occurs
with replacement of a benzoyl group by hydrogen with overall
retention of stereochemistry.
10,11
xenovorans LB400
was expressed as an N-terminal His
6
fusion protein, and purified to near homogeneity (specific activity
1
1.8 u/mg). Steady-state kinetic parameters for (His) -BphD were
6
21
determined to be K
M
2.0 mM and kcat 6.5 s , similar to reported
11
C-C hydrolase enzymes catalyse the hydrolytic cleavage of a
carbon–carbon bond adjacent to a ketone, yielding a carboxylic
1
acid product. A class of C-C hydrolases are found on bacterial
values for native BphD. Enzymatic conversion in 50 mM
potassium phosphate buffer, pH 8.0, followed by extraction of the
12
unstable 2-hydroxypentadienoic acid product (t 5–10 min ),
1/2
1
gave the H NMR spectrum shown in Fig. 2A, identical to that of
meta-cleavage pathways responsible for the degradation of
2
aromatic compounds in soil. Amino acid sequence alignments
3
of these enzymes, and structure determination of hydrolase
4
BphD from Rhodococcus RHA1, have revealed that they are
6
the MhpC enzymatic product. The separation of the H-5E
hydrogen (5.42 ppm) and H-5Z hydrogen (5.18 ppm) allows the
1
determination of stereochemistry by H NMR spectroscopy.
2
2
members of the ab-hydrolase family, containing a Ser-His-Asp
catalytic triad.
Attempted enzymatic conversions of substrate in H O resulted in
2
very rapid H exchange in the substrate dienol, obscuring the
Mechanistic studies of C-C hydrolase MhpC, on the phenyl-
propionic acid catabolic pathway of Escherichia coli, have
established that the reaction occurs with enzymatic insertion
of the H-5E hydrogen of the product 2-hydroxypentadienoic
stereochemical determination. Therefore, the deuteriated substrate
was prepared by exchange of H-5 and H-3 of the dienol substrate,
2
by incubation of substrate in H
2
O. Conversion of 10 mg of the
1
deuteriated substrate in H
O by 8 units of BphD, and direct
1
monitoring of the reaction by H NMR spectroscopy, gave the
2
acid, and replacement of
a succinyl group with overall
5,6
1
retention of stereochemistry. Pre-steady state kinetic analysis
7
of MhpC has provided evidence for a keto-intermediate,
spectrum shown in Fig. 2B. Integration of the H signals for H-5
(89%) and H-5 (31%, see Table 1) shows that the H-5 hydrogen
is inserted by BphD. There is 31% exchange of the H-5 hydrogen,
E
Z
E
which is attacked by water via a general base-catalysed mechan-
8
ism. We have recently established a synthetic route to the aryl
Z
6
similar to that observed for MhpC, which is due to partial
release of the ketonised reaction intermediate, followed by non-
specific re-enolisation in solution. Therefore, the stereochemistry of
meta-ring fission intermediates on the biphenyl catabolic
pathway, responsible for the degradation of polychlorinated
9
biphenyls. The availability of synthetic intermediates on this
pathway enables mechanistic studies of C-C hydrolase BphD,
which bears 50% amino acid sequence identity to E. coli
MhpC. Here we report the elucidation of the stereochemistry
of the BphD-catalysed reaction (Fig. 1), and pre-steady state
kinetic analysis.
2-Hydroxy-6-keto-6-phenyl-hexa-2,4-dienoic acid was synthe-
sised by Heck coupling of 1-phenyl-prop-2-en-1-ol and ethyl
Fig. 1 Reaction catalysed by C-C hydrolase BphD. H-3 and H-5
1
2
2
exchanged with H prior to reaction in H O.
1
1
1
2
Fig. 2 Part of H NMR spectra for (A) conversion of H-RFP in H O,
2
1
*T.D.Bugg@warwick.ac.uk
2
(B) conversion of H-RFP in H O.
1
30 | Chem. Commun., 2005, 130–132
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Table 1 Integration for H-4, H-3, H-5 , H-5
Z
pentadienoic acid from conversion of H-labelled substrate in H O by
2
BphD vs. MhpC
E
of product 2-hydroxy-
to propose a more detailed catalytic mechanism for the
BphD-catalysed reaction, shown in Fig. 3. The substrate is
predicted to bind with the C-1 carboxylate interacting with
Arg-188 at the bottom of the active site, and with the C-6
carbonyl positioned between (and beneath) the sidechains of
His-263 and Ser-110. Our previous observation that BphD is
able to process a reduced substrate containing a secondary
2
1
H-4
H-3
H-5
Z
H-5
E
BphD
MhpC
1.00
1.00
0.15
0.02
0.31
0.30
0.89
1.00
6
13
alcohol at C-6, together with mechanistic studies on C-C
6–8
hydrolase MhpC,
implies that C–C cleavage proceeds via a
the BphD-catalysed reaction occurs with replacement of the
benzoyl substituent by hydrogen with overall retention of
stereochemistry.
general base mechanism, not a nucleophilic mechanism. His-263
appears to be responsible for both keto–enol tautomerisation
and deprotonation of the catalytic water molecule, since
there are no other acid–base residues in the vicinity of the
In order to examine the kinetic mechanism for the BphD-
catalysed reaction, pre-steady state kinetic analysis of the enzyme-
catalysed reaction was carried out at 30.3 mM substrate and
4
active site.
There are two stereochemical mechanisms that could give rise to
30.3 mM BphD in 50 mM potassium phosphate buffer pH 8.0.
insertion of the H-5 hydrogen: either protonation at the C-5 proS
E
Observation at 430 nm gave a single exponential curve for
hydrogen, followed by C–C cleavage onto the re face of the 3,4-
double bond; or protonation at the C-5 proR hydrogen,
21
substrate consumption (k 5 9.4 s ). Observation of product
5
followed by C–C cleavage onto the si face. The orientation of
appearance at 270 nm also gave a single exponential curve
2
1
(
k 5 10.2 s ). Therefore, only a single step kinetic mechanism is
the bound substrate, in relation to Ser-110 and His-263, requires
that the scissile C5–C6 bond must rotate towards Ser-110, and
therefore that C–C cleavage occurs onto the re face. Twisting of
the dienol substrate towards Ser-110 would facilitate protonation
at C-5 at the proS hydrogen by His-263, hence the mechanism
shown in Fig. 3 would result in the observed labelling of the H-5E
position.
observed for BphD, whereas a two-step kinetic mechanism is
observed for MhpC, comprising a fast initial keto–enol tautomer-
7
isation, followed by rate-limiting C–C cleavage. This result implies
that the initial ketonisation step is much slower, and rate-limiting,
in the BphD catalytic cycle.
In order to probe further the relative energy barriers in
the BphD reaction, the solvent kinetic isotope was measured
2
in 100% H O. A value of 1.76 ¡ 0.02 was measured on
In summary, we have found that the stereochemistry of the
reaction catalysed by C-C hydrolase BphD occurs with insertion of
2
v
max, higher than the value of 1.42 measured previously for
the H-5 hydrogen, and overall replacement of a benzoyl group by
E
6
MhpC. The higher solvent kinetic isotope is consistent with
hydrogen with retention of stereochemistry. This is the same
the initial tautomerisation step being rate-limiting, since keto–
enol tautomerisation involves proton transfer with an active
site base. The slower tautomerisation by BphD may reflect
the greater resonance stabilisation of the aryl substrate for
BphD, or a lesser degree of substrate destabilisation than in
stereochemical course as C-C hydrolase MhpC from E. coli, but
the two enzymes show different kinetic behaviour under single
turnover conditions, implying that keto–enol tautomerisation is
rate-limiting in this enzyme. The availability of a synthetic route to
the BphD substrate will allow a more detailed examination of the
catalytic roles of His-263 and Ser-110 in this enzyme, which will be
reported in due course.
7
MhpC.
The availability of a crystal structure for BphD from
4
Rhodococcus, together with these mechanistic data, allows us
This work was supported by BBSRC (grant B20467). We
thank Dr Sharon Mendel and Chen Li (University of
Warwick) for assistance with molecular biology and stopped flow
kinetics.
Jian-Jun Li and Timothy D. H. Bugg*
Department of Chemistry, University of Warwick, Coventry, CV4 7AL,
UK. E-mail: T.D.Bugg@warwick.ac.uk; Fax: 02476-524112;
Tel: 02476-573018
Notes and references
1
2
3
4
5
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7
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Fig. 3 Proposed catalytic mechanism for C-C hydrolase BphD,
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illustrating the reaction stereochemistry.
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32 | Chem. Commun., 2005, 130–132
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