Revealing substrate promiscuity of 1-deoxy-D-xylulose 5-phosphate synthase

Article abstract of DOI:10.1021/ol901961q

A study of DXP synthase has revealed flexibility In the acceptor substrate binding pocket for nonpolar substrates and has uncovered new details of the catalytic mechanism to show that pyruvate can act as both donor and acceptor substrate.

Full text of DOI:10.1021/ol901961q

ORGANIC
LETTERS
2009
Vol. 11, No. 20
4748-4751
Revealing Substrate Promiscuity of
1-Deoxy-D-xylulose 5-Phosphate
Synthase
Leighanne A. Brammer and Caren Freel Meyers*
Department of Pharmacology and Molecular Sciences, Johns Hopkins UniVersity
School of Medicine, Baltimore, Maryland 21205
cmeyers@jhmi.edu
Received August 24, 2009
ABSTRACT
A study of DXP synthase has revealed flexibility in the acceptor substrate binding pocket for nonpolar substrates and has uncovered new
details of the catalytic mechanism to show that pyruvate can act as both donor and acceptor substrate.
Members of the isoprenoid class of natural products are all
biosynthesized from the common precursors isopentenyl py-
rophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).
The mevalonate-independent 2-C-methyl-D-erythritol 4-phos-
phate (MEP) pathway was identified as an alternative to the
mammalian mevalonic acid (MVA) pathway for the production
of these essential isoprenoid precursors.1 This pathway to
isoprenoids is widespread in plants and human pathogens,
including the majority of human bacterial pathogens2 and the
Figure 1. DXP synthase catalyzed first step in the MEP pathway.
malaria parasite Plasmodium falciparum,3 and is under intense
investigation as a potential target for the development of new
anti-infective agents and herbicides.4
3-phosphate (D-GAP). This transformation is reminiscent of
transketolases and decarboxylases, where a two-carbon
intermediate bound to thiamine diphosphate (ThDP) is
condensed with an acceptor substrate to generate a new
carbon-carbon bond. Homologues of DXP synthase include
transketolase and acetolactate synthase which are known to
follow the classical ping-pong kinetic mechanism, where
release of CO2 from the thiamine-bound intermediate pre-
cedes binding of the acceptor substrate. In contrast, DXP
synthase follows an ordered kinetic mechanism,5 where
binding of the second substrate, D-GAP, is required for
release of CO2 and formation of a kinetically competent
The first enzyme in the MEP pathway (Figure 1), 1-deoxy-
D-xylulose 5-phosphate (DXP) synthase, represents a new
class of thiamine-dependent enzymes that catalyzes the
formation of DXP from pyruvate and D-glyceraldehyde
(1) Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H. Biochem.
J. 1993, 295, 517–524.
(2) Testa, C.; Brown, M. Curr. Pharm. Biotechnol. 2003, 4, 248–259.
(3) Jomaa, H.; Wiesner, J.; Sanderbrand, S.; Altincicek, B.; Weidemeyer,
C.; Hintz, M.; Tu¨bachova, I.; Eberl, M.; Zeidler, J.; Lichtenthaler, H.;
Soldati, D.; Beck, E. Science 1999, 285, 1573–1576.
(4) Eoh, H.; Brennan, P.; Crick, D. Tuberculosis (Edinburgh) 2009, 89,
1–11.
10.1021/ol901961q CCC: $40.75
Published on Web 09/24/2009
 2009 American Chemical Society

complex. Interestingly, structural analysis of DXP synthase
indicates the active site is located between domains of the
same monomer of the homodimer, in contrast to its homo-
logues, where the active site is at the dimer interface.6 Taken
together, these studies reveal distinctive characteristics of
DXP synthase, placing this enzyme in the growing list of
potential targets for anti-infective drug development.
We have hypothesized that a study of DXP synthase
substrate specificity will disclose important substrate binding
determinants to help define the substrate binding pockets and
reveal previously undiscovered catalytic activities of this
novel enzyme class. Existing assays used to study DXP
synthase substrate specificity involved laborious TLC resolu-
tion of radioactive products7 or insensitive UV- or refractive
index-based detection of unnatural products.8 These studies
suggested the enzyme processes a small subset of aldose and
aldose phosphate acceptor substrates, closely resembling the
natural acceptor substrate. While the scope of substrates that
could be tested was limited by these assays, the results
nevertheless suggested a potential utility of DXP synthase
for generating 1-deoxysugars.
Figure 2. (a) Derivatization of DXP synthase substrates and product
with 2,4-DNP and (b) HPLC time-course analysis.
Reports describing the incorporation of fluorophores to
enhance sensitivity of detection use harsh and/or labor-
intensive derivatization protocols9 or do not resolve substrates
from products.7 Reports are lacking on tolerance of this
important enzyme class toward structurally diverse substrates.
To more thoroughly explore the scope of DXP synthase
catalytic activity, a sensitive and flexible assay is needed.
Here, we report a versatile, robust HPLC-based assay for
DXP synthase and present results demonstrating its utility
for probing new reactions catalyzed by DXP synthase from
Escherichia coli. Notably, our results indicate that DXP
synthase demonstrates a valuable capacity to process non-
polar aliphatic aldehydes that are structurally distinct from
the natural substrate. Additionally, our assay has uncovered
new details of the catalytic mechanism of DXP synthase.
Here, we provide the first direct evidence to support the
hypothesis formulated by Eubanks and Poulter5 that a second
molecule of pyruvate can bind to DXP synthase. We further
demonstrate that DXP synthase, in fact, utilizes pyruvate as
the second substrate in the generation of acetolactate.
For sensitive detection of DXP synthase activity in this
HPLC-based discontinuous assay, we have exploited the
reactivity of the carbonyl groups in both substrates and
product toward 2,4-dinitrophenylhydrazine (2,4-DNP) to give
the corresponding hydrazones (Figure 2). Derivatization
reactions were performed by treating quenched enzymatic
reaction mixtures with excess 2,4-DNP in sulfuric acid at
room temperature. Hydrazone formation was found to be
complete within 2-3 min (Figure S2b, Supporting Informa-
tion). Acid-catalyzed hydrolysis of the phosphoryl moieties
in D-GAP and DXP was noted in derivatization mixtures after
∼30 min. Thus, derivatization reactions were adjusted to pH
5-7 after 5 min to prevent degradation. Hydrazone mixtures
were then easily resolved and detected using HPLC equipped
with a UV photodiode array detector. For rapid sample
analysis, we have employed the use of a short Rocket column
such that HPLC analysis is complete within 10 min for each
sample.
Confirmation of DXP formation was accomplished fol-
lowing treatment of DXP synthase enzymatic reaction
mixtures with Antarctic phosphatase (AP) and comparison
of the resulting product to authentic 1-deoxy-D-xylulose
hydrazone (Figure S3, Supporting Information). Optimization
of the enzymatic reaction conditions was also possible using
this assay because both substrates and products can be
detected by HPLC. Importantly, we found that pyruvate and
D,L-GAP are unstable in buffers often used in DXP synthase
assays,7,10-13 including Tris and glycylglycine. Enzymatic
reactions carried out at pH 8.0 in HEPES or phosphate buffer
were found to be optimal, as substrates are stable under these
conditions.
Further validation of this assay was accomplished through
Michaelis-Menten kinetic analysis of DXP synthase in the
presence of natural substrates. Kinetic parameters were
measured for pyruvate and D-GAP (Figure S4, Supporting
D-GAP
pyruvate
Information; Km
) 226 ( 32 µM, Km
) 1.1 (
0.3 mM, kcat ) 246 ( 13 min-1) and were found to be in
close agreement with reported values for DXP synthase from
Rhodobacter capsulatus.5
(5) Eubanks, L.; Poulter, C. Biochemistry 2003, 42, 1140–1149.
(6) Xiang, S.; Usunow, G.; Lange, G.; Busch, M.; Tong, L. J. Biol.
Chem. 2007, 282, 2676–2682.
(10) Lee, J.; Oh, D.; Kim, S. J. Biotechnol. 2007, 128, 555–566
(11) Lois, L.; Campos, N.; Putra, S.; Danielsen, K.; Rohmer, M.;
Boronat, A Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2105–2110
(12) Kuzuyama, T.; Takagi, M.; Takahashi, S.; Seto, H. J. Bacteriol.
2000, 182, 891–897
(13) Altincicek, B.; Hintz, M.; Sanderbrand, S.; Wiesner, J.; Beck, E.;
Jomaa, H. FEMS Microbiol. Lett. 2000, 190, 329–333
.
(7) Querol, J.; Grosdemange-Billiard, C.; Rohmer, M.; Boronat, A.;
Imperial, S. Tetrahedron Lett. 2002, 43, 8265–8268.
(8) Schu¨rmann, M.; Schu¨rmann, M.; Sprenger, G. J. Mol. Catal. B:
Enzym. 2002, 19-20, 247–252.
.
.
(9) Han, Y.; Sabbioni, C.; van der Heijden, R.; Verpoorte, R. J. Chro-
matogr., A 2003, 986, 291–296.
.
Org. Lett., Vol. 11, No. 20, 2009
4749

Previous studies have demonstrated that DXP synthase
utilizes glyceraldehyde (GA) as the acceptor substrate,
indicating a tolerance for absence of the phosphoryl moi-
ety.5,12,14,15 We have confirmed this result using our assay.
However, little is known about the effects of removing
substrate hydroxyl groups or lengthening the carbon chain.
This work focuses on investigating enzyme flexibility toward
aliphatic aldehydes as alternative acceptor substrates.
Initial experiments tested acetaldehyde (3) as substrate for
DXP synthase. The enzyme-catalyzed formation of acetoin
(15) was measured by HPLC detection of the corresponding
hydrazone derivative (Figure 3). Acetoin formation was
Table 1. Summary of DXP Synthase Substrate Specificity
toward Aliphatic Aldehydes
Figure 3. Analysis of acetaldehyde (3) as an alternative acceptor
mechanism involving reaction of the thiamine-bound inter-
mediate with a second molecule of pyruvate to form
acetolactate, which then undergoes decarboxylation to form
acetoin (Figure 3b). In a previous kinetic study of DXP
synthase,5 the rate of CO2 release was found to be signifi-
cantly slower in the absence of D-GAP, providing evidence
for an ordered kinetic mechanism. However, in the presence
of high pyruvate concentrations, slow release of CO2 was
observed. The authors speculated that a second molecule of
pyruvate may bind to the active site to promote CO2 release
from the thiamine-bound intermediate leading to a ThDP-
acetaldehyde complex that could dissociate and allow ad-
ditional binding of free ThDP.5 Our results provide the first
direct evidence that pyruvate acts as acceptor substrate to
promote CO2 release and generate a new product, acetolac-
tate.
substrate. (a) HPLC time course showing acetoin (15) formation.
Acetolactate (16) forms in the presence and absence of 3. (b)
Proposed mechanisms to form 15 and 16.
confirmed by comparing the product to authentic acetoin
hydrazone and by mass analysis. An apparent kcat/Km of (7.0
( 0.2) × 102 M-1·min-1 was measured for acetaldehyde that
is comparable to glyceraldehyde (kcat/Km of (21 ( 0.3) ×
102 M-1·min-1, Table 1), revealing relaxed specificity for
simple, nonpolar acceptor substrates.
In addition, a second enzyme-dependent peak was ob-
served (6.7 min, Figure 3a). Mass analysis of the hydrazone
product was consistent with the hydrazone of acetolactate
(16). In principle, acetolactate could be formed in a reaction
of the thiamine-bound intermediate with a second molecule
of pyruvate, similar to the reaction catalyzed by acetolactate
synthase16 (Figure 3b). The acetolactate hydrazone was
observed at 6.7 min, as well as a second peak corresponding
to the acetoin hydrazone. These results are consistent with a
The observation that acetoin is generated from pyruvate,
in the absence of acetaldehyde, led us to seek further proof
that acetaldehyde acts as acceptor substrate to generate
acetoin independently. The rate of acetoin formation from
(14) Bailey, A.; Mahapatra, S.; Brennan, P.; Crick, D. Glycobiology
(16) Crout, D. The Chemistry of Branched Chain Amino Acid Biosyn-
thesis: Stereochemical and Mechanistic Aspects. In Biosynthesis of Branched
Chain Amino Acids; Barak, A., Chipman, J., Eds.; VCH: Weinheim, 1990;
pp 199-242.
2002, 12, 813–820
.
(15) Hahn, F.; Eubanks, L.; Testa, C.; Blagg, B.; Baker, J.; Poulter, C.
J. Bacteriol. 2001, 183, 1–11
.
4750
Org. Lett., Vol. 11, No. 20, 2009

pyruvate is 10-fold lower in the absence of acetaldehyde.
Nevertheless, to rule out any uncertainty about the capacity
of DXP synthase to use acetaldehyde as acceptor substrate
to generate acetoin, the enzyme was incubated with pyruvate
and acetaldehyde-d4. As expected, formation of the corre-
sponding acetoin-d4 hydrazone was observed (Table S1,
Supporting Information).
has imposed limitations in the exploration of substrate
promiscuity. We have developed a robust HPLC-based assay
that permits the detection of both substrates and product and
provides the sensitivity required for detection of compounds
that are not inherently chromophoric. Derivatization of
carbonyl-containing substrates and products is complete
within 2-3 min, and HPLC analysis for the reactions studied
here could be accomplished within 10 min. We have used
this assay to begin exploration of DXP synthase substrate
promiscuity, and to resolve uncertainties about the catalytic
mechanism.
The results obtained in these preliminary experiments
encouraged us to expand the scope of these studies to
examine DXP synthase substrate promiscuity toward other
aldehydes. Notably, DXP synthase exhibits similar reaction
profiles in the presence of several alternative aliphatic
aldehydes (results summarized in Table 1). Propanal (4),
butanal (5), pentanal (6), and hexanal (7) are utilized by the
enzyme (Figures S4-S8, Supporting Information), indicating
DXP synthase is considerably tolerant of long-chain acceptor
substrates. Specificity constants were measured for these
substrates and compared to D-GAP (1), GA (2), and
acetaldehyde (3) (Table 1). A nearly 400-fold decrease in
kcat/Km results from the removal of the phosphoryl moiety
from D-GAP, indicating the contribution of the phosphoryl
moiety in substrate binding. However, removal of either
hydroxyl group of GA or lengthening the carbon chain has
only modest effects on kcat/Km values, demonstrating a
flexibility in this binding pocket for lengthening the carbon
chain and removal of hydroxyl moieties. This finding
indicates that DXP synthase exhibits a relaxed specificity
toward nonpolar aldehyde substrates, rivaling that of TK,17
to generate the corresponding R-hydroxy ketones, an im-
portant compound class in the life-science industry.18,19
Not surprisingly, heptanal (8, Table S1, Supporting
Information) proved to be insoluble under these assay
conditions. Branched aliphatic aldehydes are apparently not
readily accepted by DXP synthase. Although minor product
formation was observed in the presence of ꢀ-branched
aldehyde 9 and cyclopropyl aldehyde 10 (Figures S9 and
S10, Supporting Information), more bulky R-branched al-
dehydes were not processed by the enzyme. Apparent binding
of pentanal to protein was observed, indicating that accurate
determination of specificity constants for longer chain
aldehydes under these conditions will be difficult.
Our findings reveal relaxed specificity of DXP synthase
for nonpolar aliphatic aldehydes, with the exception that
bulky R-branched aldehydes are not well-tolerated by the
enzyme. This relaxed specificity to produce R-hydroxy
ketones points to DXP synthase as a potential biocatalyst
for generating this important compound class and newly
reveals flexibility in the acceptor substrate binding pocket
for nonpolar substrates. The perplexing observation that CO2
release takes place in the absence of D-GAP, albeit at
significantly reduced rates,5 was resolved during the course
of our investigation. The assay reported here enabled the
detection of enzyme-catalyzed acetolactate formation, pro-
viding confirmation that pyruvate acts as the second substrate
to promote CO2 release in the absence of D-GAP. Our
findings raise new questions about opportunities to target
this unique enzyme or to exploit it as a biocatalyst. Studies
are underway to expand the scope of this reaction.
Acknowledgment. We acknowledge Marc Rosen (Johns
Hopkins University School of Medicine) for his assistance
with mass spectrometry and Jason Howard (Johns Hopkins
University School of Medicine) for conducting initial de-
rivatization experiments. We are grateful to Rahul Kohli and
Philip Cole of Johns Hopkins University School of Medicine
and Richard Borch of Purdue University for helpful discus-
sions and careful critiques of this manuscript. This work was
supported by funding from the NIH (T32 GM007445) and
Johns Hopkins Malaria Research Institute Pilot Grant for
L.A.B.
In summary, the lack of an appropriate assay combining
the sensitivity and flexibility needed to study DXP synthase
Supporting Information Available: Assay protocols,
unnatural product characterization, and kinetic analyses. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(17) Hibbert, E.; Senussi, T.; Smith, M.; Costelloe, S.; Ward, J.; Hailes,
H.; Dalby, P. J. Biotechnol. 2008, 134, 240–245.
(18) Mu¨ller, M.; Gocke, D.; Pohl, M. FEBS J. 2009, 276, 2894–2904
.
(19) Marigo, M.; Jørgensen, K. Chem. Commun. 2006, 2001–2011
.
OL901961Q
Org. Lett., Vol. 11, No. 20, 2009
4751