Isochorismate pyruvate lyase: A pericyclic reaction mechanism?

Article abstract of DOI:10.1021/ja055871t

Isochorismate pyruvate lyase (IPL) catalyzes the cleavage of isochorismate to give salicylate and pyruvate, a key step in bacterial siderophore biosynthesis. We investigated the enzyme from Pseudomonas aeruginosa using isochorismate selectively deuterated

Full text of DOI:10.1021/ja055871t

Published on Web 10/08/2005
Isochorismate Pyruvate Lyase: A Pericyclic Reaction Mechanism?
†
‡
†
†
,†
Michael S. DeClue, Kim K. Baldridge, Dominik E. K u¨ nzler, Peter Kast, and Donald Hilvert*
Laboratory of Organic Chemistry, Swiss Federal Institute of Technology, ETH H o¨ nggerberg,
CH-8093 Z u¨ rich, Switzerland, and Organisch-Chemisches Institut, UniVersit a¨ t Z u¨ rich,
Winterthurerstrasse 190, CH-8057 Z u¨ rich, Switzerland
Received September 2, 2005; E-mail: hilvert@org.chem.ethz.ch
Despite their broad utility in the laboratory, pericyclic reactions
are rarely exploited in cellular metabolism. The Claisen rearrange-
ment of chorismate to prephenate, the suprafacial [1,5]-sigmatropic
Scheme 1
1
shift of a methyl group in the transformation of precorrin-8x to
2
3
hydrogenobyrinic acid, and several putative Diels-Alder reactions
are the only processes for which actual enzyme catalysts have been
identified. Here we present evidence that isochorismate pyruvate
lyase (IPL) from Pseudomonas aeruginosa,4,5 an enzyme involved
in bacterial siderophore biosynthesis, should be added to this short
list.
IPL catalyzes the elimination of the enolpyruvyl side chain from
isochorismate to give salicylate and pyruvate (Scheme 1a). This
type of aromatization reaction is generally formulated as a base-
initiated process (Scheme 1b),6 but a dissociative mechanism
involving initial cleavage of the C-O bond to give an ion pair
intermediate is conceivable (Scheme 1c). A concerted pericyclic
pathway, in which the hydrogen atom at C2 is transferred to C9 of
the side chain simultaneous with C-O cleavage (Scheme 1d), which
Table 1. Kinetic Parameters for P. aeruginosa Isochorismate
Pyruvate Lyasea
1
substrate
k
cat (s-1)
K
m
(µ
M)
k
cat/K
m
(M- s-1)
5
5
isochorismate
1.01 ( 0.02 1.05 ( 0.08 (9.62 ( 0.73) × 10
2
[2- H]-isochorismate 0.43 ( 0.01 0.79 ( 0.05 (5.49 ( 0.37) × 10
7
8
represents a third possibility, has been proposed as well.
a Enzyme was isolated as described in ref 8. Reactions were performed
We employed isochorismate selectively deuterated at C2 to
distinguish between these mechanistic alternatives. The labeled
in triplicate in 50 mM phosphate buffer (pH 7.5, 30 °C). Kinetic parameters
for the unlabeled substrate are in excellent agreement with previous data
-
1
(
kcat ) 1.06 s , Km ) 0.95 µM).8
substrate was prepared chemoenzymatically from D-mannose.
2
Briefly, the sugar was converted to sodium [6,6- H
2
]shikimate in
in buffer containing dioxane d-8 as an internal standard was
recorded in the absence of enzyme (Figure 1a). After addition of
IPL, the sample was incubated at 30 °C for 30 min. During the
ensuing reaction, the deuterium label was quantitatively transferred
to pyruvate (Figure 1b). The signals for substrate prior to reaction
1
2 steps following a modified procedure for preparing unlabeled
9
shikimic acid. Incubation of labeled shikimate with phospho-
enolpyruvate and cell extracts from an engineered Escherichia coli
strain (KA12/pKAD50/pKS3-02),10 which lacks chorismate mutase
but overproduces the enzymes shikimate kinase, EPSP synthase,
2
and [3- H]pyruvate upon completion of the reaction gave identical
2
chorismate synthase, and isochorismate synthase, afforded [2- H]-
integrations within experimental error (4.66 ( 0.32 mM versus 4.74
isochorismate in ca. 10% yield. The latter was isolated by extraction
and purified by preparative reverse phase HPLC.10
(
0.32 mM, respectively). In contrast to the uncatalyzed reaction
at 60 °C, where significant exchange of the labeled pyruvate with
solvent complicates analysis,10 no exchange was observed over
several hours at the lower temperature of the enzymatic assay; the
small amount of HOD seen at 4.55 ppm in the spectrum of the
reaction mixture (Figure 1b) derives from the added enzyme stock
P. aeruginosa IPL cleanly converts isochorismate to salicylate
1
and pyruvate as verified both by H NMR and HPLC analysis of
typical reaction mixtures. The steady-state kinetic parameters
2
obtained with the unlabeled substrate and [2- H]isochorismate in
5
1
0 mM phosphate buffer (pH 7.5) at 30 °C are reported in Table
. On the basis of these data, the H kinetic isotope effects on kcat
2
solution, which was not prepared from deuterium-depleted H O.
2
The finding that the deuterium originally at C2 of isochorismate
is quantitatively transferred to pyruvate in the IPL-catalyzed
elimination reaction argues strongly against a mechanism requiring
an external base (Scheme 1b). The failure to identify an active site
residue in IPL by mutagenesis and genetic selection that could
and kcat/K are 2.34 ( 0.08 and 1.75 ( 0.18, respectively. The
m
observed effect on kcat establishes that chemistry is significantly
rate determining for the enzyme. Moreover, the magnitude of the
isotope effect is consistent with considerable C-H bond cleavage
in the transition state (Scheme 1b or 1d), but would be unprec-
edentedly large for a â-secondary effect in an ion pair mechanism
8
plausibly serve as such a base further strengthens this conclusion.
2
A significant H kinetic isotope effect and quantitative transfer
(
Scheme 1c).11 The somewhat smaller isotope effect observed on
of the label to pyruvate are both consistent with a pericyclic reaction
mechanism (Scheme 1d). Hybrid density functional theory (HDFT)
calculations13 also support this possibility. A low energy transition
structure for a concerted but asynchronous [1,5]-sigmatropic shift,
in which carbon-oxygen bond cleavage is more advanced than
hydrogen atom transfer from C2 to C9, was located at the
Becke3LYP/DZ+(2d,p) level of theory (Figure 2). This structure,
k
m
cat/K indicates a significant forward commitment to catalysis for
IPL.1
2
The fate of the deuterium atom at C2 was determined directly
2
2
by H NMR spectroscopy. The spectrum of [2- H]isochorismate
†
Swiss Federal Institute of Technology.
Universit a¨ t Z u¨ rich.
‡
15002
9
J. AM. CHEM. SOC. 2005, 127, 15002-15003
10.1021/ja055871t CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Acknowledgment. This work was supported in part by the
Schweizerischer Nationalfonds. We are grateful to Prof. J. Frost
for plasmid pKAD50, Prof. E. Leistner for plasmid pKS3-02, and
2
Prof. B. Jaun for expert assistance with the H NMR experiments.
Supporting Information Available: Coordinates and computational
details for calculation of the theoretical isotope effects, and complete
ref 13b. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Figure 2. Becke3LYP/DZ+(2d,p) structure of the pericyclic transition state
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3
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(
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(
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16
17
salicylate synthase, 4-amino-4-deoxychorismate lyase, and an-
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6
ubiquinones, siderophores, folates, and amino acids, warrants
careful reinvestigation of their mechanism of action.
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