Mechanism-based inactivation of benzoylformate decarboxylase, a thiamin diphosphate-dependent enzyme

Article abstract of DOI:10.1021/ja068636z

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is a thiamin diphosphate-dependent enzyme that catalyzes the non-oxidative decarboxylation of benzoylformate. Here we report the discovery of a mechanism-based inhibitor of BFD that is unusual in

Full text of DOI:10.1021/ja068636z

Published on Web 03/17/2007
Mechanism-Based Inactivation of Benzoylformate Decarboxylase, A Thiamin
Diphosphate-Dependent Enzyme
Asim K. Bera,† Lena S. Polovnikova,† Juliatek Roestamadji,§ Theodore S. Widlanski,§
George L. Kenyon,‡ Michael J. McLeish,*,‡ and Miriam S. Hasson†,||
Department of Biological Sciences, Purdue UniVersity, West Lafayette, Indiana 47907, Department of Chemistry,
Indiana UniVersity, Bloomington, Indiana 47405, and College of Pharmacy, UniVersity of Michigan,
Ann Arbor, Michigan 48109
Received December 1, 2006; E-mail: mcleish@umich.edu
Scheme 1
Benzoylformate decarboxylase from Pseudomonas putida (BFD;
EC 4.1.1.7) belongs to a family of thiamin diphosphate (ThDP,
vitamin B1)-dependent enzymes that catalyze the non-oxidative
decarboxylation of 2-keto acids (Scheme 1).1 Mechanism-based
Scheme 2
inhibitors of such thiamin-dependent decarboxylases are rare.2 In
this communication, we report the discovery of a mechanism-based
inhibitor of benzoylformate decarboxylase that covalently modifies
the enzyme via active site phosphorylation, a type of mechanism-
based inactivation that is virtually unprecedented.
There are several instances where phosphonate analogues of
2-keto acids have proved to be effective inhibitors of non-oxidative
decarboxylation.3 In particular, acetylphosphonate (1) and its
monoester, methyl acetylphosphonate (2), have been shown to
inhibit enzymes catalyzing the decarboxylation of pyruvate via the
reversible addition of the cofactor to these inhibitors to form
structures such as 3.3a,b
importantly, it was also observed that prolonged incubation of BFD
with 4 resulted in permanent inactivation of the enzyme. Further
investigation showed that the inactivation of BFD was both time-
and concentration-dependent (Figure 1A). The data could most
simply be described by the scheme in which 4 initially binds
reversibly to the benzoylformate binding site, and in a subsequent
slower reaction, BFD is inactivated:
We hypothesized that benzoylphosphonate (4), the phenyl
analogue of 1, might function as a mechanism-based, irreversible
inactivator of BFD. It was thought that, as shown in Scheme 2, 4
would undergo nucleophilic attack by the ylid of ThDP to provide
the adduct, 5. However, unlike 3, 5 has an anion-stabilizing phenyl
group. This gives 5 the potential for a more facile elimination of
metaphosphate which might lead to enzyme phosphorylation and,
concomitantly, irreversible inhibition. Two possible pathways for
this process are shown in Scheme 2.
kinact
E + I y
\
Kzi EI
8 E - I
Kitz and Wilson5 (Figure 1B) analysis of the inactivation data,
using eq 1, provided an inactivation rate constant (kinact) of 0.0185
min-1, that is, a half-life of ca. 38 min at 30 °C.
To explore this possibility, 4 was prepared using a modification
of the method of Karaman et al.4 and was found to act as a
competitive inhibitor of BFD. The Ki value of 0.36 ( 0.03 mM
indicated that it was likely to be acting as a ground-state analogue
of benzoylformate, which has a Km value around 0.4 mM.1e More
k
inact[I]
kobs
)
(1)
Ki + [I]
To identify the residue(s) affected by reaction with 4, a sample
of the inactivated BFD was crystallized at pH 7.0 using the hanging
drop vapor diffusion method. The inactivated enzyme crystallized
in space group I222, the same form as that of the WT BFD crystal
used to determine the original BFD structure (PDB entry 1BFD).6
† Purdue University.
§ Indiana University.
‡ University of Michigan.
| Died 1/16/2006.
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J. AM. CHEM. SOC. 2007, 129, 4120-4121
10.1021/ja068636z CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
that the phenyl analogue of 1, benzoylphosphonate (4), displays a
distinctly different pattern of inhibition with BFD. It seems likely
that two important factors govern this difference in reactivity. First,
the conjugate base of hydroxybenzyl thiamin diphosphate (HBTh-
DP, 6) is more stable than that of its pyruvate-derived analogue
(the pKa of HBThDP is ca. 2 units lower than that of HEThDP, F.
Jordan, personal communication). Therefore, 6 could serve as a
sufficiently good leaving group to enable direct phosphoryl transfer
to an active site nucleophile (Scheme 2). Second, the presence of
a nucleophilic serine prepositioned to attack the phosphoryl group
may be a requirement for the phosphoryl transfer process. Such
nucleophilic precoordination to phosphorus is known to be a
requirement for the mechanistically similar Conant-Swan frag-
mentation.11
Figure 1. (A) Inactivation of BFD showing saturation at increasing
concentrations of benzoylphosphonate (4). (B) Reciprocal (Kitz and Wilson5)
plot of 1/observed kinact versus 1/[benzoylphosphonate]. The solid line
represents the fit of the data to eq 1 and provides a Ki of 0.71 mM and a
kinact of 0.0185 min-1
.
In summary, we suggest that 4 acts as a mechanism-based
inhibitor of BFD, with inactivation resulting from the phosphoryl-
ation of Ser26.
Acknowledgment. Dedicated to the memory of Miriam S.
Hasson. We thank Prof. Janet Smith (University of Michigan) for
critical reading of the manuscript. We also appreciate discussions
with Dr. John W. Burgner, II (Purdue University), and the assistance
of Ryan P. Tyler and Dr. Alejandra Yep (University of Michigan)
with collection of some of the kinetic data. This work was
supported, in part, by the NSF at Purdue (97333552-MCB) and at
Michigan (EF-0425719). Use of the Advanced Photon Source was
supported by the U.S. Department of Energy. Use of BioCARS
14-BMC was supported by NIH-NCRR. Assistance of the Bio-
CARS staff is gratefully acknowledged.
Figure 2. (A) View of the active site region near Ser26 in 4-modified
BFD. The unbiased Fo - Fc (green, 3.5 σ) electron density at 1.39 Å
resolution is based on phases from the BFD model before addition of the
phosphate group. (B) 2Fo - Fc (blue) electron density map contoured at
1.5 σ.
The crystals of the modified enzyme diffracted to 1.39 Å.
Significant electron density was found near Ser26 (Figure 2A) with
the 2Fo - Fc electron density map clearly showing that Ser26 had
been phosphorylated, with the phosphorus atom being covalently
attached to the γ-oxygen of Ser26 (Figure 2B). The cofactor was
found to be intact, and there was no evidence for benzaldehyde
remaining in the active site. Coordinates and structure factors for
the modified enzyme are deposited as Protein Data Bank entry
2FWN.
Both site-directed mutagenesis and X-ray crystallography have
shown that Ser26 is important in the catalytic mechanism of BFD.1e
Based on the structure of BFD in complex with the substrate
analogue, R-mandelate, the Ser26 side chain was predicted to
interact with the carboxyl group of the substrate benzoylformate.
Further, the BFD S26A variant showed more than 20-fold increase
in Km value for benzoylformate as well as a 50-fold decrease in
the value of kcat.1e Presumably, phosphorylation of Ser26 would
render the active site unavailable for productive binding of
benzoylformate, resulting in irreversible inhibition. This suggestion
would also lead to the prediction, subsequently proven to be correct,
that the BFD S26A variant would not be irreversibly inactivated
by 4 (data not shown).
Phosphonates have been used extensively as inhibitors of various
enzyme types, including serine proteases,7 protein kinases,8 and
phosphatases.9 Generally, the inhibitory properties of the phospho-
nates are based on their ability to mimic the tetrahedral intermediates
of the enzymatic reactions while maintaining chemical stability.10
By contrast, it has been shown that 1 reversibly inhibits the
decarboxylase activity of both pyruvate oxidase and pyruvate
dehydrogenase simply by acting as an analogue of pyruvate. Given
the mechanistic similarities between PDC and BFD, it is notable
Supporting Information Available: Experimental details and
crystallographic statistics. This material is available free of charge via
the Internet at http://pubs.acs.org.
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