The results show that the epimerase reac-tion follows a nonstereospecific retroaldol⁄ aldol mech-anism with the same reaction intermediate as that of the aldolase reaction and that SaDHNA
Trang 1NMR, equilibrium and transient kinetic studies of the
Staphylococcus aureus and Escherichia coli enzymes
Yi Wang, Yue Li, Yan Wu and Honggao Yan
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA
Dihydroneopterin aldolase (DHNA, EC 4.1.2.25)
catalyzes the conversion of 7,8-dihydro-d-neopterin
(DHNP) into 6-hydroxymethyl-7,8-dihydropterin (HP)
in the folate biosynthetic pathway, one of the principal
targets for developing antimicrobial agents [1] Folate
cofactors are essential for life [2] Most
micro-organ-isms must synthesize folates de novo In contrast,
mam-mals cannot synthesize folates because of the lack of
three enzymes in the middle of the folate pathway, and
they therefore obtain folates from the diet DHNA is
the first of the three enzymes that are absent in
mam-mals and therefore an attractive target for developing
antimicrobial agents [3]
DHNA is a unique aldolase in two respects First, it requires neither the formation of a Schiff’s base between the substrate and enzyme nor metal ions for catalysis [4] Aldolases can be divided into two classes based on their catalytic mechanisms [5,6] Class I aldo-lases require the formation of a Schiff’s base between
an amino group of the enzyme and the carbonyl of the substrate, whereas class II aldolases require a Zn2+ion
at their active sites for catalysis The proposed catalytic mechanism for DHNA [4,7,8] is similar to that of class I aldolases, but the Schiff’s base is embedded in the substrate (Fig 1) Secondly, in addition to the aldo-lase reaction, DHNA also catalyzes the epimerization
Keywords
dihydroneopterin aldolase; Escherichia coli;
folate biosynthesis; mechanism;
Staphylococcus aureus
Correspondence
H Yan, Department of Biochemistry and
Molecular Biology, Michigan State
University, East Lansing, MI 48824, USA
Fax: +1 517 353 9334
Tel: +1 517 353 5282
E-mail: yanh@msu.edu
Website: http://www.bch.msu.edu/faculty/
yan.htm
*These authors have contributed equally to
this work
(Received 13 January 2007, revised 14
February 2007, accepted 28 February 2007)
doi:10.1111/j.1742-4658.2007.05761.x
Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-d-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydro-pterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-l-monapterin (DHMP) Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotona-tion and reprotonadeprotona-tion of C2¢ of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent No deuteration of C2¢ was observed for the newly formed DHMP This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction In contrast with an earlier observation, the DHNA-catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol⁄ aldol mechanism for the epimerization reaction The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) were determined by equilib-rium binding and transient kinetic studies A complete set of kinetic con-stants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them
Abbreviations
DHMP, 7,8-dihydro-L-monapterin; DHNA, dihydroneopterin aldolase; DHNP, 7,8-dihydro- D -neopterin; EcDHNA, E coli dihydroneopterin aldolase; GA, glycolaldehyde; HP, 6-hydroxymethyl-7,8-dihydropterin; HPO, 6-hydroxymethylpterin; MP, L -monapterin; NP, D -neopterin; SaDHNA, S aureus dihydroneopterin aldolase.
Trang 2at C2¢ of DHNP to generate 7,8-dihydro-l-monapterin
(DHMP) [7], but the biological function of the
epi-merase reaction is not known at present The aldolase
and epimerase reactions are believed to involve a
com-mon intermediate as shown in Fig 1 [4,7,8] Both
reac-tions involve the retroaldol cleavage of the C–C bond
between C1¢ and C2¢ Epimerization results from the
re-formation of the C–C bond after the reorientation
of glycolaldehyde, which exposes the opposite face of
the aldehyde The mechanism of the epimerization
reaction is very similar to that catalyzed by
l-ribulose-5-phosphate 4-epimerase [9], which also follows aldol
chemistry [10], but the two enzymes are different in
structure and have no apparent sequence identity
l-Ribulose-5-phosphate 4-epimerase has 26% identity
with the class II l-fuculose-1-phosphate aldolase and
requires a Zn2+ion for catalysis [9] DHNA is unique
because it catalyzes both aldolase and epimerase
reac-tions, whereas l-ribulose-5-phosphate 4-epimerase and
l-fuculose-1-phosphate aldolase catalyze only one type
of reaction
Interestingly, DHNAs from Gram-positive and
Gram-negative bacteria have some unique sequence
motifs Figure 2 shows the amino-acid sequence
align-ment of DHNAs from 11 bacteria The first five enzymes
are from Gram-positive bacteria, and the rest are from
Gram-negative bacteria The identities between enzymes
from Gram-positive bacteria range from 39% to 45%
and those between Gram-negative bacteria are 49–91%,
but the identities between positive and
Gram-negative bacterial enzymes are < 30% Many
differ-ences between enzymes from positive and Gram-negative bacteria are at or near their active centers [8] DHNA was first identified in Escherichia coli (EcDHNA) by Mathis and Brown in 1970 [4] There were few studies on DHNA until 1998, when Hennig and coworkers determined the crystal structures of DHNA from Staphylococcus aureus (SaDHNA) and its complex with the product HP [8] In the same year, Haussmann and coworkers demonstrated that the enzyme has both aldolase and epimerase activities and determined the steady-state kinetic parameters for both reactions [7] In 2000, the Wu¨thrich group pub-lished the total sequential resonance assignment of the 110-kDa homo-octomeric SaDHNA [11], which was
a model system for the development of TROSY (transverse relaxation optimized spectroscopy) NMR [12–14] Also in 2000, Deng and coworkers measured the pKa of N5 of SaDHNA-bound 7,8-dihydrobio-pterin by Raman spectroscopy [15] In 2002, Illarionova and coworkers showed that the protonation of the reac-tion intermediate prefers the pro-S posireac-tion [16]
We are interested in understanding the catalytic mechanism of DHNA and the biochemical conse-quences of the significant sequence differences des-cribed above Most recently, we studied the dynamic properties of apo-SaDHNA and the product complex SaDHNA–HP by molecular dynamics simulations [17] and began to investigate the functional roles of the act-ive-site residues by site-directed mutagenesis [18] In this paper, we address the issue of whether the epim-erase reaction follows a nonstereospecific retroaldol⁄
Fig 1 Proposed catalytic mechanism for
the DHNA-catalyzed reactions Both aldolase
and epimerase reactions follow the same
reaction intermediate generated by the
clea-vage of the bond between 1¢ and 2¢ carbons
of the substrate The epimerization product
is generated by the re-formation of the C–C
bond after the reorientation of GA, which
exposes the opposite face of the aldehyde.
Trang 3aldol mechanism [7] or an alternative mechanism via
the deprotonation and re-protonation of C2¢ and
report a comprehensive equilibrium and kinetic study
of SaDHNA and EcDHNA, which represent DHNAs
from Gram-positive and Gram-negative bacteria,
respectively The results show that the epimerase
reac-tion follows a nonstereospecific retroaldol⁄ aldol
mech-anism with the same reaction intermediate as that
of the aldolase reaction and that SaDHNA and
EcDHNA have significantly different equilibrium and
kinetic constants, which form the basis for elucidating
the catalytic mechanism of DHNA and developing
antimicrobial agents specifically against Gram-positive
or Gram-negative bacteria
Results
NMR analysis
Although it is reasonable that the epimerase reaction
follows the same reaction intermediate as that of the
aldolase reaction, as described above (Fig 1), it is also possible that it follows an alternative mechanism, i.e the deprotonation and reprotonation of C2¢ The alter-native reaction can be initiated by deprotonation of C1¢ and protonation of N5 to form an enol intermedi-ate, which can turn into a keto intermediate by tau-tomerization for the subsequent deprotonation and reprotonation of C2¢ Whether the epimerase reaction follows the same reaction intermediate as that of the aldolase reaction or the mechanism of deprotonation and reprotonation of C2¢ can be tested by NMR The key difference between the two reaction mechanisms is that H2¢ is always attached to C2¢ if the epimerase reaction follows the same reaction intermediate as that
of the aldolase reaction (Fig 1), whereas it has to be extracted by a base if the epimerase reaction follows the mechanism of deprotonation and reprotonation of C2¢ Therefore, when the reaction is run in D2O, the H2¢ occupancy will change if the epimerase reaction involves the deprotonation and reprotonation of C2¢, but will not change if it follows the same reaction
Fig 2 Amino-acid sequence alignment of DHNAs The top five DHNAs are from Gram-positive bacteria: Staphylococcus aureus (SA), Bacillus subtilis (BS), Strepto-coccus pyogenes (SP), Listeria innocua (LI), and Streptomyces coelicolor (SC) The bot-tom six DHNAs are from Gram-negative bacteria: Escherichia coli (EC), Yersinia pes-tis (YP), Vibrio cholerae (VC), Haemophilus influenzae (HI), Pseudomonas aeruginosa (PA), and Shewanella oneidensis (SO) The highly conserved residues among all DHNAs are shaded in black Residues that are char-acteristic of Gram-positive or Gram-negative bacteria are highlighted in gray Residues that comprise the active centers are indica-ted by horizontal bars The residue number-ing at the top of the alignment is that of SaDHNA.
Trang 4intermediate as that of the aldolase reaction The
pro-ton occupancy can be quantified by NMR The result
of such an experiment is shown in Fig 3 The NMR
signals were assigned on the basis of their multiplicity
patterns, decoupling experiments, and comparison with
the NMR spectrum of authentic DHMP (the top
spec-trum in Fig 3) As shown in Fig 3, the NMR signals
of all 2¢ and 3¢ protons of DHNP and DHMP are well
separated, except those of the 3¢Hb protons of the two
compounds, which are overlapping The proton
occu-pancy at the 2¢ position of the newly formed DHMP
could be quantified by comparing the integrals of the
2¢H and 3¢Ha NMR signals of DHMP, because 3¢
pro-tons do not participate in the chemical reaction in
either mechanism and cannot be replaced with
deuter-ons The result showed that the intensities of the 2¢H
and 3¢Ha NMR signals were the same throughout the
time course of the reaction (18, 35, and 70 min) The
1 : 1 intensities of the 2¢H and 3¢Ha NMR signals indi-cated a 100% proton occupancy at the 2¢ position, strongly suggesting that there is no deprotonation and reprotonation at C2¢ and the epimerase reaction fol-lows the aldol chemistry
Is the DHNA-catalyzed reaction reversible? Although aldolase-catalyzed reactions are generally reversible, the DHNA-catalyzed reaction was shown previously to be irreversible [4] However, it was noticed that the E coli enzyme preparation used in the experiment had a low activity and furthermore, the glycoaldehyde (GA) concentration (150 lm) was rather low, especially considering that it exists in various forms in solution and only a small fraction is in the correct form for the reaction [19,20] To further investigate the issue of the reversibility of the DHNA-catalyzed reaction, we ran the reverse reaction with our recombinant enzymes and high concentrations of
GA One such result obtained with SaDHNA is shown
in Fig 4 Clearly, the SaDHNA-catalyzed reaction was reversible Furthermore, the reverse reaction was rather rapid in the presence of SaDHNA The appar-ent Kmfor GA obtained by varying GA at a fixed HP
Fig 3 NMR analysis of the SaDHNA-catalyzed reactions in D2O.
The bottom spectrum was obtained before the addition of the
enzyme, and the middle three spectra were obtained 18, 35, and
70 min after the addition of the enzyme The top spectrum is that
of DHMP for comparison Only the NMR signals of the 2¢ and 3¢
protons of DHNP and DHMP are shown The chemical structures
of DHNP and DHMP are also shown at the top, with atom
number-ing labeled for DHNP For clarity, the NMR signals of the aldolase
reaction products HP and GA are not shown.
Fig 4 HPLC analysis of the reverse reaction catalyzed by SaDHNA The initial reaction mixture in 100 m M Tris ⁄ HCl, pH 8.3, contained 100 l M HP and 20 m M GA The reaction was initiated with 10 l M SaDHNA at 25 C and quenched with 1 M HCl The reverse reaction generated both DHNP and DHMP HP, DHNP, and DHMP were oxidized to HPO, NP, and MP, respectively, before the HPLC analysis as described in Experimental procedures.
Trang 5concentration (100 lm) was 10 mm Both DHNP
and DHMP were generated in the reverse reaction,
which also lent support to a nonstereospecific
retroal-dol⁄ aldol mechanism for the epimerization reaction
Equilibrium binding studies
As the epimerase reaction uses the same reaction
inter-mediate as that of the aldolase reaction and the
aldo-lase reaction is reversible, we can draw a unified
kinetic scheme for the DHNA-catalyzed reactions as
shown in Scheme 1, where A, B, I, P, and Q represent
DHNP, DHMP, the reaction intermediate, HP, and
glycolaldehyde, respectively
The major goal of this work was to determine the
rate constants of the individual steps of the reactions
Our strategy to achieve this goal was a comprehensive
one, involving the measurements of both equilibrium
and kinetic constants of the physical steps by
equilib-rium and stopped-flow fluorimetric analysis and the
determination of the rate constants of the chemical
steps by quench-flow analysis of both forward and
reverse reactions We first measured the dissociation
constants by fluorimetry A typical fluorimetric
titra-tion curve is shown in Fig 5 The results are
summar-ized in Table 1 To facilitate the purification of
SaDHNA, we engineered a His-tag at the N-terminus
of the enzyme The binding properties of the
His-tagged and unHis-tagged enzymes were essentially the same
(data not shown), and the binding data for SaDHNA
in Table 1 are those of the His-tagged enzyme
d-Neopterin (NP), l-monapterin (MP), and
6-hydroxy-methylpterin (HPO) are the oxidized forms of DHNP,
DHMP, and HP, respectively The only difference
between the two sets of pterin compounds is that the
link between C7 and N8 is a single bond in the
reduced pterins but a double bond in the oxidized
pterins Consequently, there is a hydrogen atom
attached to N8 in the reduced pterins and the NH
group can serve as a hydrogen-bond donor, whereas in
the oxidized pterins, there is no hydrogen attached to
N8 and it can only serve as a hydrogen-bond acceptor
NP, MP, and HPO are all DHNA inhibitors The binding of the inhibitors to the enzymes cause a decrease in their fluorescence intensities The increasing fluorescence intensities in Fig 5A were obtained by subtracting the control titration data in the absence of the enzymes from the titration data in the presence of the enzymes The results of the equilibrium binding studies showed that, in comparison with EcDHNA, SaDHNA has significantly higher Kd values for the measured pterin compounds, particularly HPO, whose the Kd value for SaDHNA was 240 times that for
EPQ
E + P + Q
k6
k-6
k1
k-1
k2
k-2
k5
k-5
k3
k-4 k4
k-3
Scheme 1 Kinetic mechanism of the DHNA-catalyzed reactions.
A
B
Fig 5 Fluorimetric titration of SaDHNA with NP (A) and of HPO with SaDHNA (B) (A) A 2-mL solution containing 15 l M SaDHNA in
100 m M Tris ⁄ HCl, pH 8.3, was titrated with NP by adding aliquots
of a 1.94 m M NP stock solution in the same buffer at 24 C The final enzyme concentration was 14 l M The top axis indicates the
NP concentrations during the titration A set of control data was obtained in the absence of the enzyme and was subtracted from the corresponding data set obtained in the presence of the enzyme (B) A 2-mL solution containing 1 l M HPO in 100 m M Tris ⁄ HCl, pH 8.3, was titrated with SaDHNA by adding aliquots of a 1.55 m M SaDHNA stock solution in the same buffer at 24 C The final HPO concentration was 0.93 l M The top axis indicates the SaDHNA concentrations during the titration A set of control data was obtained in the absence of HPO and was subtracted from the corresponding data set obtained in the presence of the enzyme The solid lines were obtained by nonlinear least-squares regression
as previously described [25].
Trang 6EcDHNA Furthermore, whereas the Kd values of
SaDHNA for the reduced and oxidized pterin
com-pounds (HP and HPO, respectively) were the same, the
Kd value of EcDHNA for the reduced pterin
com-pound (the product HP) was higher than that for the
oxidized pterin compound (the oxidized product
HPO) Finally, the Kd value of SaDHNA for NP was
slightly higher than that for MP, whereas the Kdvalue
of EcDHNA for NP was lower than that for MP
Stopped-flow analysis
We then measured the rate constants of the physical
steps of the reaction by stopped-flow fluorimetric
ana-lysis Because GA has a very low affinity for the
enzymes (data not shown) and exists in solution in
multiple forms, of which the correct form for the
reac-tion is a minor one [19,20], we focused our analysis of
product binding and dissociation on HP Because
DHNP and DHMP undergo chemical reactions in the
presence of DHNA, we measured the binding and
dis-sociation of the structurally related DHNA inhibitors
NP and MP To assess the differences in the rate
con-stants of the reduced and oxidized pterins, we also
measured the association and dissociation rate
con-stants of HPO and compared them with those of HP
A representative set of the stopped-flow analysis data
is shown in Fig 6 The rate constants measured by the
stopped-flow experiments are summarized in Table 2,
where k1 and k)1 are the association and dissociation
rate constants, respectively The Kd values calculated
as k)1⁄ k1 were in excellent agreement with those
meas-ured by equilibrium binding studies (Table 1) The
results show that the association rate constants for NP
and MP are very similar and slightly lower than those
for HP and HPO, which are very similar This
phe-nomenon is presumably related to the sizes of the
molecules NP and MP are the same size and are slightly larger than HP and HPO Furthermore, the results also show that, for SaDHNA, the association and dissociation rate constants of the reduced pterin
HP are the same as those of the oxidized pterin
Fig 6 Stopped-flow analysis of the binding of HPO to SaDHNA The concentration of SaDHNA was 2 l M , and the concentrations of HPO were 10, 20, 30, and 40 l M for traces 1, 2, 3, and 4, respect-ively All concentrations were those immediately after the mixing of the two syringe solutions Both SaDHNA and HPO were dissolved
in 100 m M Tris ⁄ HCl, pH 8.3 The fluorescent signals were rescaled
so that they could be fitted into the figure with clarity The solid lines were obtained by nonlinear regression as described in Experi-mental procedures The inset is a replot of the apparent rate con-stants versus the HPO concentrations The solid line in the inset was obtained by linear regression.
Table 2 Association and dissociation rate constants of S aureus and E coli DHNAs measured by stopped-flow experiments SaDHNA has a His-tag (MHHHHHH) at the N-terminus The Kd val-ues were calculated as k)1⁄ k 1
k1 (l M )1Æs)1)
k)1 (s)1)
Kd (l M )
k1 (l M )1Æs)1)
k)1 (s)1)
Kd (l M )
NP 0.24 ± 0.01 4.5 ± 0.1 19 0.32 ± 0.02 0.29 ± 0.03 0.88
MP 0.29 ± 0.02 4.2 ± 0.2 15 0.26 ± 0.01 0.58 ± 0.03 2.3
HP 0.47 ± 0.04 13 ± 1 28 0.65 ± 0.08 0.26 ± 0.02 0.4 HPO 0.45 ± 0.02 10 ± 1 24 0.55 ± 0.04 0.062 ± 0.006 0.11
Table 1 Dissociation constants (l M ) of S aureus and E coli
DHNAs measured by equilibrium binding experiments.
a The chemical structures of the measured compounds are as
fol-lows:
N
N
HN
N
H 2 N
O
OH OH
N HN N
H 2 N
O
OH OH
OH
MP
N
N HN N
H 2 N
O
OH
b SaDHNA has a His-tag at the N-terminus.
Trang 7(HPO), in accordance with the same Kd value for the
two pterin compounds On the other hand, for
EcDHNA, the association rate constants for HP and
HPO are essentially the same, but the dissociation
con-stant of HP is larger than that of HPO, in agreement
with a larger Kd value for HP Finally, the higher Kd
values are all mainly due to the higher dissociation
rate constants
Quench-flow analysis
The rate constants of the chemical steps were
meas-ured by quench-flow experiments We ran the forward
reaction (the formation of HP) using both DHNP and
DHMP as the substrates and the reverse reaction (the
formation of DHNP and DHMP) with HP and GA
For the forward reaction, three concentrations each
for DHNP and DHMP were used For the reverse
reaction, the concentration of HP was fixed, and eight
concentrations of GA were used for the
SaDHNA-cat-alyzed reaction and six concentrations of GA for the
EcDHNA-catalyzed reaction Each reaction generated
three curves, one each for DHNP, DHMP, and HP
This multitude of quench-flow data was then fitted
glo-bally to Scheme 1 by nonlinear least-squares regression
using the program dynafit [21] The enzyme-bound
intermediate (EI) was assumed to isomerize to the
aldol product HP during the acid quench and therefore
treated as HP in the global fitting analysis The initial
values for the physical steps were derived from the
stopped-flow analysis described in the previous section
The rate constants for the chemical steps were
estima-ted by global fitting with fixed rate constants for the
physical steps Then the dissociation rate constants
were allowed to vary by 20% to obtain the best fit of
the data via an iterative process For SaDHNA, both
the association and dissociation rate constants of the
oxidized pterin HPO (0.45 lm)1Æs)1 and 10 s)1,
respectively) were virtually the same as those of the
reduced pterin HP (0.47 lm)1Æs)1 and 13 s)1,
respect-ively), suggesting that HPO is an excellent analogue
for HP and, by analogy, NP and MP are excellent
analogues of DHNP and DHMP for the kinetic study
of the physical steps (association and dissociation)
Therefore, the rate constants for the binding of DHNP
and DHMP were fixed at the values measured for the
corresponding oxidized pterins NP and MP during the
initial global fitting analysis For EcDHNA, the
associ-ation rate constant of HPO (0.55 lm)1Æs)1) was very
similar to that of HP (0.65 lm)1Æs)1), but the
dissoci-ation rate constant of HPO (0.062 s)1) was about a
quarter of that of HP (0.26 s)1), suggesting that the
oxidation does not have significant effects on the
association rate constant but increases the dissociation rate constant by a factor of 4 Therefore, during the initial global fitting of the EcDHNA quench-flow data, the association constants for the binding of DHNP and DHMP were fixed at the values measured for the corresponding oxidized pterins NP and MP, and the dissociation rate constants were fixed at four times the values measured for the corresponding oxidized pterins With these constraints, the rate constants for the chemical steps were well determined with standard error less than 15% for both SaDHNA-catalyzed and EcDHNA-catalyzed reactions, except the rate con-stants for the interconversion of the enzyme-bound intermediate (Sa.I in Fig 8) and enzyme-bound prod-ucts (Sa.HP.GA in Fig 8) in the SaDHNA-catalyzed reaction The rate constants for the interconversion of Sa.I and Sa.HP.GA are considered to be approximate low limits, because they were sensitive to lower values but not to higher values This is probably due to their high values relative to those of the rate constants for other steps and the fact that the reaction rate is insen-sitive to this step when its rate constants increase beyond certain high values Typical results of the for-ward reaction are shown in Fig 7 for the SaDHNA-catalyzed reaction The results of the quench-flow analysis are summarized in Fig 8 For SaDHNA, the epimerase activity is insignificant in comparison with its aldolase activity, the rate-limiting step in the forma-tion of HP is the generaforma-tion of the reacforma-tion intermedi-ate, and the interconversion of Sa.I and Sa.HP.GA
is very fast in comparison with other steps For EcDHNA, in contrast, the epimerase activity is highly significant (comparable to the aldolase activity), the rate-limiting step in the formation of HP is the prod-uct release, and the interconversion of the enzyme-bound intermediate (Ec.I in Fig 8) and enzyme-enzyme-bound products (Ec.HP.GA) is much slower than in the SaDHNA-catalyzed reaction
Discussion
DHNA catalyzes the cleavage of the bond between C1¢ and C2¢ of DHNP to form HP (an aldolase reaction) and also the formation of DHMP (an epimerase reac-tion) [7] A nonstereospecific retroaldol⁄ aldol mechan-ism has been proposed for the epimerization reaction (Fig 1) [7], but no experimental evidence in support of such a mechanism has been reported, and one cannot exclude a priori an alternative mechanism of deproto-nation and reprotodeproto-nation of C2¢ for the epimerization reaction In this work, we considered these two alter-native mechanisms for the epimerization reaction Our NMR analysis of DHMP generated in the reaction in
Trang 8D2O clearly indicates that there is no deuteration of
C2¢ of the epimerase product The lack of deuteration
of the C2¢ of DHMP is not due to the lack of
deute-rons, because it has been shown previously that the
6-hydroxymethyl group of the aldolase reaction
prod-uct, HP, can be significantly deuterated (at least half
of the –CH2– protons of the hydroxymethyl group) if
the reaction occurs in D2O [16] Another possibility is
that deprotonation and reprotonation occur without
the proton exchanging with bulk water However,
de-protonation and rede-protonation in the aldolase reaction
involve the proton exchanging with bulk water [16]
The residues that function as the general acid and base
in the aldolase reaction are probably the same as those
in the epimerase reaction Therefore, it is unlikely that
deprotonation and reprotonation in the epimerase
reaction occur without the proton exchanging with
bulk water The NMR data strongly support the hypo-thesis that the epimerase reaction follows a nonstereo-specific retroaldol⁄ aldol mechanism as depicted in Fig 1 without deprotonation and reprotonation of C2¢ In further support of this mechanism, we demon-strated that both epimers (DHNP and DHMP) can be generated from the aldolase products (HP and GA)
We also observed that, in the transient kinetic experi-ments, the epimerization product (DHMP from DHNP
or DHNP from DHMP) accumulated more extensively
in the early part of the reaction course and decreased
in the late part of the reaction course (data not shown) It suggests that the aldolase and epimerase reactions follow the same reaction intermediate The product distribution is determined by kinetics in the early part of the reaction course and by thermodynam-ics in the late part of the reaction course, and therefore the epimerization product increases early and decreases
as the reaction progresses to the equilibrium
Because DHNA catalyzes both aldol and epimeriza-tion reacepimeriza-tions and the epimerizaepimeriza-tion product, DHMP, can also be converted into the aldol reaction product,
HP, it is particularly important to determine the rate constants for elementary steps if one intends to deter-mine how the enzyme catalyzes both reactions Fur-thermore, steady-state kinetic analysis is insufficient for DHNA, because the steady-state kinetic parameters cannot adequately describe the two reactions catalyzed
by the enzyme and the formation of DHMP will be underestimated because of its conversion into HP Haussmann and coworkers previously determined the steady-state kinetic constants for EcDHNA [7] According to the steady-state kinetic data, the epime-rase activity is one-sixth of the aldolase activity, which significantly underestimates the epimerase activity of EcDHNA (see Fig 8, lower panel) Furthermore, the
kcatvalues for the aldolase and epimerase activities are significantly lower than the rate constants of the chem-ical steps
A critical issue in the kinetic analysis is whether the reaction is reversible or not Although aldolase-cata-lyzed reactions are in general readily reversible, it has been shown previously that DHNA is an exception and the DHNA-catalyzed reaction is apparently irre-versible [4] The apparent irreversibility is probably due to the low activity of the enzyme preparation used
in the experiment, the low concentration of GA, and the low reaction rate of the EcDHNA-catalyzed reverse reaction With pure recombinant enzymes and high concentrations of GA, it is clear that the DHNA-catalyzed reaction is reversible In fact, for SaDHNA, the reverse reaction is much faster than the forward reaction
Fig 7 Global analysis of the quench-flow data of the
SaDHNA-cata-lyzed reaction Data 1, 2, 3, 7, 8, and 11 were obtained with DHNP
as the substrate Because the commercial DHNP contained a
min-ute amount of DHMP, the initial reaction mixtures contained both
DHNP and DHMP The initial DHNP and DHMP concentrations for
these data were 29.7 and 0.3, 19.8 and 0.2, 9.9 and 0.1 l M ,
respectively Data 4, 5, 6, 9, 10, and 12 were obtained with DHMP
as the substrate The initial DHMP concentrations for these data
were 10, 20, and 30 l M , respectively The enzyme concentration
was 20 l M for all reactions All concentrations were those
immedi-ately after the mixing of the two syringe solutions The buffer
con-tained 100 m M Tris ⁄ HCl, pH 8.3, and 5 m M dithiothreitol Data 1–6
are the concentrations of the aldolase product, HP, and data 7–12
are the concentrations of the epimerase product, MP or NP The
solid lines were obtained by global nonlinear least-squares
regres-sion using the program DYNAFIT [21] For clarity, the changes in the
substrate concentrations were not plotted The data for the reverse
reactions, i.e with HP and GA as the substrates, were not plotted.
Trang 9The rate constants of individual steps, as
summar-ized in Fig 8, were determined by a comprehensive
strategy using a combination of stopped-flow and
quench-flow analyses The philosophy behind the
strat-egy is to isolate the different steps of the reaction
whenever possible and design experiments to determine
rate constants for the specific steps We began the
comprehensive kinetic analyses by measuring the rate
constants of the physical steps (i.e the binding steps)
by stopped-flow fluorimetry To avoid the chemical
reactions, we substituted NP and MP (see Table 1 for
their chemical structures) for DHNP and DHMP,
respectively, and measured the binding of HP in the
absence of GA NP and MP are the oxidized forms of
the pterins, with a double bond between C7 and N8
instead of a single bond as in the reduced pterins
(DHNP and DHMP) To assess the differences in the
binding rate constants between the closely related pairs
of oxidized and reduced pterins, we also measured the
rate constants for the binding of HPO, the oxidized
form of HP These measured rate constants are reliable
and accurate, because of (a) the high quality of the
stopped-flow data as illustrated in Figs 7 and 8 and (b)
the consistency between the Kd values calculated from
the association and dissociation rate constants
(Table 2) and those measured by equilibrium titration
experiments (Fig 5 and Table 1) These measured rate
constants are also reasonable in that the association
rate constants are similar between NP and MP and
between HP and HPO in accordance with the similar shapes and sizes between NP and MP and between HP and HPO The different Kd values are proportional to the different values of the dissociation rate constants,
as expected The results also show that for SaDHNA,
HP and HPO have essentially the same rate constants,
in accordance with the crystal structure of the complex
of SaDHNA with HP, which reveals that NH at posi-tion 8 of HP has no hydrogen bond with the protein [8] and suggest that the rate constants for the binding
of the corresponding reduced and oxidized pterins to SaDHNA may be essentially the same For EcDHNA,
HP and HPO have very similar association rate con-stants, but their dissociation rate constants are signifi-cantly different The dissociation rate constant of HP
is about four times that of HPO, suggesting that the corresponding reduced and oxidized pterins may have significantly different dissociation rate constants for binding to EcDHNA
The rate constants of the chemical steps were deter-mined by quench-flow experiments Because the reac-tion is reversible, we were able to run the reacreac-tion in all three directions with DHNP, DHMP, or HP and
GA as the substrate(s) so that both forward and reverse rate constants could be defined Because the three pterin components of the reaction mixtures could
be resolved by HPLC (Fig 4), each set of the quench-flow experiments generated three sets of data The rate constants of the chemical steps were evaluated by the
Fig 8 Summary of the kinetic constants for the SaDHNA-catalyzed (top panel) and EcDHNA-catalyzed (lower panel) reactions.
Sa and Ec represent SaDHNA and EcDHNA, respectively I represents the reaction inter-mediate as shown in Fig 1 The rate con-stants for the interconversion of Sa.I and Sa.HP.GA are considered to be approximate low limits, and the standard errors for other rate constants are within 15%.
Trang 10global fitting of the multitude of the quench-flow data
(total nine sets) using the widely used program for
kin-etic analysis dynafit [21], which uses the numerical
integration of simultaneous first-order ordinary
differ-ential equations to calculate the time-course of the
chemical reaction and the Levenberg–Marquardt
algo-rithm for nonlinear regression fitting Such transient
kinetic analysis has been the standard method for the
determination of individual rate constants of enzymatic
reactions [22–24] and has been successfully used in our
transient kinetic analysis of E coli
6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase [25] and yeast
cytosine deaminase [26]
Under the reaction conditions, the quench flow data
are sensitive to both the physical and chemical steps of
the enzymatic reaction and are insufficient for the
deter-mination of the rate constants for both the physical and
chemical steps However, when the rate constants of the
physical steps are available, the quench-flow data can
be used to determine the rate constants of the chemical
steps The rate constants for the physical steps can be
estimated from the stopped-flow measurements of the
pterin analogues As the rate constants for the binding
of the pair of the reduced and oxidized pterins to
SaDHNA are essentially the same, the rate constants
for the physical steps of the SaDHNA-catalyzed
reac-tion (the first step in each direcreac-tion) are well defined
For the EcDHNA-catalyzed reaction, the association
rate constants for the physical steps were assumed to be
the same as those for the binding of the oxidized pterins
(NP and MP), because the oxidation has no significant
effects on the association rate constants, and the
stereo-chemistry of the trihydroxypropyl tail has no significant
effects either The dissociation rate constants for
DHNP and DHMP were estimated from those for NP
and MP and the difference between HP and HPO and
finalized by iterative fittings as described in the Results
section When the rate constants for the physical steps
were fixed, the rate constants for the chemical steps
were well defined in the sense that > 15% variations in
the rate constants, except those for the conversion of
the reaction intermediate into the aldolase products
(HP and GA) in the SaDHNA-catalyzed reaction,
would have significant detrimental effects on the
fittings The rate constants for the conversion of the
reaction intermediate into the aldolase products in
the SaDHNA-catalyzed reaction must be considered to
be the low limits, because decreasing the values of these
rate constants had significant detrimental effects but
increasing the values of these rate constants had
insigni-ficant effects on the fittings
Our equilibrium and kinetic data also show that
SaDHNA and EcDHNA have significantly different
binding and catalytic properties, in accordance with the significant sequence differences between the two enzymes EcDHNA is biochemically different from SaDHNA in several aspects (a) EcDHNA has much higher affinities for the substrate, products, and inhibi-tors as measured in this work, particularly for HPO (b) EcDHNA has a much higher epimerase activity than SaDHNA (c) The rate-limiting step in the for-ward reaction (the formation of HP) is the product release for EcDHNA but is the formation of the reaction intermediate for SaDHNA (d) The intercon-version of the enzyme-bound intermediate and enzyme-bound aldolase products is much slower in the EcDHNA-catalyzed reaction than in the SaDHNA-catalyzed reaction The marked differences in the lig-and-binding properties of SaDHNA and EcDHNA, which must stem from the significant differences in the structures of their active sites, suggest that it may be possible to develop antimicrobial agents specifically against DHNA from S aureus or E coli Because many DHNAs from Gram-positive and Gram-negative bacteria are highly homologous within their own groups but significantly different between the two groups, it may be possible to develop antimicrobial agents specifically against positive or Gram-negative bacteria by targeting respective DHNAs
Experimental procedures
Materials HPO, HP, DHNP, DHMP, NP, and MP were purchased from Schircks Laboratories (Jona, Switzerland) Restriction enzymes and T4 ligase were purchased from New England Biolabs (Ipswich, MA, USA) Pfu DNA polymerase and the pET-17b vector were purchased from Stratagene (La Jolla, CA, USA) and Novagen (Madison, WI, USA), respectively Other chemicals were from Sigma-Aldrich (St Louis, MO, USA)
Cloning The SaDHNA gene was cloned into the prokaryotic expression vector pET-17b and a home-made derivative (pET17H) by PCR from S aureus genomic DNA The pET17H vector was used for the production of a His-tagged SaDHNA The primers for the PCR were 5¢-GG AATTCCATATGCAAGACACAATCTTTCTTAAAG-3¢ (forward primer with a Nde I site) and 5¢-CGGGATCCT CATTTATTCTCCCTCACTATTTC-3¢ (reverse primer with a BamHI site) The EcDHNA gene was cloned into the prokaryotic expression vector pET-17b by PCR from E coli genomic DNA The primers for the PCR were