Mechanistic insight with HBCH2CoA as a probe to polyhydroxybutyrate (PHB) synthases

Article abstract of DOI:10.1021/cb5002735

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1-2 MDa. A substrate analogue HBCH₂CoA, in which the S in HBCoA is replaced with a CH₂ group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with K_is of 40 and 14 μM, respectively. To probe the elongation steps of the polymerization, HBCH₂CoA was incubated with a synthase acylated with a [ ³H]-saturated trimer-CoA ([³H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [³H]-sTCoA ([³H]-sT-CH₂-CoA), saturated dimer-([³H]-sD- CO₂H), and trimer-acid ([³H]-sT-CO₂H), distinct from the expected methylene analogue of [³H]-saturated tetramer-CoA ([³H]-sTet-CH₂-CoA). Detection of [³H]-sT- CH₂-CoA and its slow rate of formation suggest that HBCH ₂CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.

Full text of DOI:10.1021/cb5002735

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Article
2
Mechanistic Insight with HBCHCoA as A Probe
to Polyhydroxybutyrate (PHB) Synthases
Wei Zhang, Ruben Shrestha, Rachael M Buckley, Jamie
Jewell, Stefan H. Bossmann, JoAnne Stubbe, and Ping Li
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb5002735 • Publication Date (Web): 04 Jun 2014
Downloaded from http://pubs.acs.org on June 15, 2014
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Mechanistic Insight with HBCH CoA as A Probe to
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Polyhydroxybutyrate (PHB) Synthases
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Wei Zhang, Ruben Shrestha, Rachael M. Buckley, Jamie Jewell, Stefan H. Bossmann, JoAnne
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Stubbe, * and Ping Li *
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Department of Chemistry, Kansas State University, Manhattan, KS 66506; Department of Chemistry,
≠
§
Ohio Dominican University, Columbus, OH 43219; and Department of Chemistry and Biology,
Massachusetts Institute of Technology, Cambridge, MA 02139
pli@ksu.edu; stubbe@mit.edu
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
ABSTRACT: Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3ꢀRꢀ
hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1 to 2 MDa. A substrate analog
HBCH CoA, in which the S in HBCoA is replaced with a CH group, was synthesized in 13 steps using
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a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor
of a class I and a class III PHB synthases, with K of 40 and 14 ꢀM, respectively. To probe the
is
3
elongation steps of the polymerization, HBCH CoA was incubated with a synthase acylated with a [ H]ꢀ
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saturated trimerꢀCoA ([ H]ꢀsTCoA). The products of the reaction were shown to be the methylene
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analog of [ H]ꢀsTCoA ([ H]ꢀsTꢀCH ꢀCoA), saturated dimerꢀ([ H]ꢀsDꢀCO H) and trimerꢀacid ([ H]ꢀsTꢀ
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3
3
CO H), distinct from the expected methylene analog of [ H]ꢀsaturated tetramerꢀCoA ([ H]ꢀsTetꢀCH ꢀ
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CoA). Detection of [ H]ꢀsTꢀCH ꢀCoA and its slow rate of formation suggest that HBCH CoA may be
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reporting on the termination and reꢀpriming process of the synthases, rather than elongation.
Polyhydroxyalkanoates (PHAs) are carbon and energy storage polymers synthesized by a variety of
bacteria when they find themselves limited in an essential nutrient such as nitrogen or phosphorous but
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with an abundant carbon source available. Under conditions of maximum accumulation, PHAs can
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constitute up to 90% of the cell dry weight. When the limiting nutrient is restored, the bacteria degrade
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, 4
the PHAs to release energy and monomers for other biological processes.
PHAs are of general
interest as they can provide a biodegradable alternative to environmentallyꢀunfriendly, petroleumꢀbased
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, 6
plastics.
PHA synthases (PhaCs) catalyze the polymerization of 3ꢀRꢀhydroxyalkanoate coenzyme A (CoA) to
form PHA. They have been divided into four classes based on their substrate specificity, subunit
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composition, and molecular weight. Class I and III PhaCs utilize shortꢀchainꢀlength monomers such as
3
ꢀRꢀhydroxybutyrate (HB) coenzyme A (HBCoA) to generate polyhydroxybutyrate (PHB) polymers.
The synthase from Ralstonia eutropha (PhaC ) is the prototypical class I enzyme and is a dimer,
Re
8
composed of two 65 kDa subunits. The synthase from Allochromatium vinosum (PhaC ) is the
Av
prototypical class III synthase and is a tetramer of two ~40 kDa subunits: PhaC (the synthase) and PhaE
9
, 10
(a protein of unknown function that is essential for activity).
Studying the mechanism of
polymerization (the priming and initiation, elongation, and termination) has been challenging because
8, 11
the elongation process is much faster than the initiation process.
Since PhaCs are largely responsible
for determining the molecular weight and polydispersity of the PHA, understanding the PhaC
mechanism can contribute to the ultimate goal of producing PHA products in an economically
competitive fashion.
Two models have been proposed for the polymerization catalyzed by PhaCs and involve the covalent
catalysis by a CysꢀHis dyad and general base catalysis of an Asp to activate the 3ꢀhydroxyl of a second
1
, 12
HBCoA for ester bond formation.
The model shown in Scheme 1A is based on analogy to fatty acid
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synthases and predicts that the growing PHB chain is always covalently attached to the protein and
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, 14–16
alternates between the two PhaC monomers.
This model requires the active site sits on the
interface of two monomers in order to facilitate chain transfer, which is contradictory to the results from
17
structural study that indicated the active site should be deeply buried. Thus, an alternative model based
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8
on the type III polyketide synthases is shown in Scheme 1B. It predicts that addition of the second
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, 12
HBCoA will generate a nonꢀcovalently bound (HB) CoA that then rapidly reacylate the Cys.
2
Detection of nonꢀcovalent intermediates will provide an effective way to distinguish between the two
models.
Our recent investigation with a mutant of PhaEC in which the active site cysteine was replaced with
Av
a serine revealed nonꢀcovalent intermediates (dimers and trimers) providing evidence in support of the
model in Scheme 1B. However, the turnover of this mutant was 5 × 10 −4 the rate of the wildꢀtype
11
(wt) PhaEC . The slow rate suggests that additional mechanistic links to the wt enzyme need to be
Av
established. To obtain further evidence for the model shown in Scheme 1B, HBCH CoA 1 (Scheme 2),
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an analog of HBCoA in which the S of the thioester is replaced with a CH group, was designed. Our
2
hypothesis was 1 could be incorporated into the growing PHB chain reporting on the chain elongation
process and resulting in chain termination.
RESULTS AND DISCUSSION
Chemoenzymatic Synthesis of HBCH CoA. Preparation of HBCH CoA 1 using a chemoenzymatic
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2
19
approach is shown in Scheme 2. Synthesis of the acid 5 has been previously reported. Although the
method is convenient, traces of byproduct tertꢀbutyldimethylsilyl (TBDMS) alcohol lowered the yield
of the next coupling reaction. Therefore, a different procedure of protecting carboxylic and hydroxyl
groups, respectively, with benzyl (Bn) and TBDMS followed by hydrogenation was developed for acid
2
0
5. Formation of the Weinreb amide 6 was achieved using a standard coupling condition. The terminal
alcohol 7 was obtained through the addition of Grignard reagent generated in situ from trimethylene
2
1–23
chlorohydrin.
Treatment of 7 with methansulfonyl chloride (MsCl) followed by sodium azide
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resulted in an azide 9. Various methods have been reported to reduce the organic azides to amines, in
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which the Staudinger reduction is the most widely used one.
produced in high yield based on TLC were not successful, presumably because during concentration of
0, it underwent intramolecular cyclization to form a cyclic imine. Therefore, we decided to perform the
reduction without purification and concentration using Pd/Cꢀcatalyzed hydrogenation in methylene
However, efforts to purify the amine
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chloride. After filtration of the catalyst, the mixture was directly reacted with protected pantothenic
acid 11 to give HBꢀcarba(dethio)pantetheine 12. The precursor 13 for enzymatic transformation was
obtained after treating 12 with 4% HF in CH CN for 15 min at 0 °C. It has to be pointed out that the
3
yield from 12 to 13 was very low if the diol in 12 was protected with benzylidene. Compound 13 was
obtained in 10 steps in a total 15% yield from 2.
Preparation of HBCH CoA 1 was achieved by use of three enzymes involved in CoA biosynthesis: a
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pantothenate kinase from Staphylococcus aureus (SaPanK), a phosphopantetheine adenylyltransferase
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0
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(EcCoaD), and a dephosphoꢀCoA kinase (EcCoaE) from Escherichia coli. Although our knowledge
of the substrate specificity of these enzymes remains incomplete, previous work has established that
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2–36
they can accept a wide spectrum of pantothenateꢀbased substrates.
It has been demonstrated that the
key regulatory enzyme in CoA biosynthesis from E. coli is the pantothenate kinase (EcPanK) due to its
37
feedback inhibition by CoA and/or its thioesters. This feedback inhibition will limit the conversion of
pantothenate analogs to CoA analogs and thus cause low yields. However, in contrast to all known
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pantothenate kinases, SaPanK is not feedback regulated by CoA and/or its thioesters, which will allow
the accumulation of CoA. Therefore, in order to maximize the conversion yield of enzymatic precursor
1
3 to HBCH CoA 1, SaPanK was employed for the first phosphorylation step in our reaction. An HPLC
2
method was developed to monitor the progress of enzymatic conversions as depicted in Supplementary
Figure S1. The retention times for enzymatic precursor 13, 4′ꢀphospho derivative 14, 3′ꢀdephosphoꢀCoA
derivative 15, and HBCH CoA 1 are at 48, 33, 39, and 32 min, respectively. Their identities have been
2
confirmed by mass spectroscopy (MS). Compound 1 was obtained in a 50% yield from 13 after
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isolation by semiꢀpreparative HPLC. Its identity was confirmed by NMR and HRMS (see Supporting
Information).
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Inhibition Study of HBCH CoA with Class I and III Synthases. HBCH CoA was evaluated as an
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inhibitor to wtꢀPhaC and wtꢀPhaEC . The kinetics of recombinant PhaC and PhaEC , monitoring
Re
Av
Re
Av
CoA release via a discontinuous 5,5'ꢀdithiobis(2ꢀnitrobenzoic acid) (DTNB) assay, are distinct from
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each other and are multiphasic. PhaC exhibits a lag phase followed by a linear phase and PhaEC
Re
Av
8,39
exhibits a fast linear phase followed by a slower phase. In the former case the lag phase was ignored
and in the latter case only the fast phase was examined. The data (Supplementary Figures S4 and S5)
were fit to the MichaelisꢀMenten equation and the results in both cases showed competitive inhibition as
summarized in Table 1.
4
0–42
Although the K of HBCoA with PhaC has been reported to be 103 to 381 ꢀM,
the assays were
M
Re
carried out in high concentrations of fructose or detergents. Therefore, the kinetic parameters of PhaCRe
were determined in the presence of 0.05% Hecameg (Supplementary Figure S6), providing a k similar
cat
−1 41
to the previously reported value (188 s ), but a K that is 10ꢀfold higher. Neither synthase was found
M
to have any timeꢀdependent component to the observed inhibition that might, for example, be associated
with hemithioketal formation between the active site cysteine of PhaC and the carbonyl group in
HBCH CoA (data not shown). The tight binding of 1 relative to the K for HBCoA suggests that it
2
M
might be a reasonable probe of the elongation process.
Predicted Products from Reactions between Acylated Synthase and HBCH CoA. Differentiation
2
of two mechanistic models in Scheme 1 has been difficult as with both class I and III synthases, the rate
8, 43
of chain elongation is much faster than that of chain initiation.
Thus, even at very low substrate to
enzyme ratios, a small amount of protein is modified with high molecular weight PHB polymer, while
43
most of the protein remains unmodified. In order to overcome this problem, we developed a method
3
8
to uniformly load PhaC, so that elongation process could be examined. Specifically, an artificial
3
3
primer, [ H]ꢀsaturated trimericꢀCoA ([ H]ꢀsTCoA, 16 in Scheme 3) is used to acylate the enzyme,
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which can then be incubated with 1. Our hypothesis is illustrated in Scheme 3. If the model in Scheme
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1
A operates, 1 cannot acylate the active site cysteine. However, if the model in Scheme 1B is operative,
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HBCH CoA would extend the sT chain to generate a methylene analog of [ H]ꢀsaturated tetramericꢀ
2
3
CoA ([ H]ꢀsTetꢀCH ꢀCoA, 17, Scheme 3), and further elongation would not be possible. Moreover, it
2
was expected that the rate of elongation would be fast, given the K for 1 and rate of elongation with
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is
HBCoA.
Products from Reactions between Acylated Synthase and HBCH CoA. Although both the PhaC
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Re
3
19
and PhaECAv can be labeled with [ H]ꢀsTCoA with similar stoichiometry (0.5 labels/monomer), the
3
stability of the covalent linkage in PhaC is much higher. Thus PhaC was acylated with [ H]ꢀsTCoA,
Re
Re
purified by Sephadex Gꢀ50 chromatography and then reacted with HBCH CoA. Aliquots of the reaction
2
mixture were removed as a function of time, the reaction stopped in 10% trichloroacetic acid, and the
protein separated from the small molecules. The supernatant was neutralized and analyzed by reverse
phase HPLC by A260 nm and the fractions monitored by scintillation counting. A typical elution profile in
which radioactivity and A260 nm are monitored is shown in Figure 1. In the experiment fractions 19−22
(sample A), 26–30 (sample B), and 31–34 min (sample C) were pooled based on radioactivity,
concentrated and analyzed by either ESIꢀMS (samples A and C) or MALDIꢀTOF MS (sample B). The
results are shown in Figure 2 and Supplementary Figures S7 and S8. The material in sample A and C
3
3
correspond to [ H]ꢀsDꢀCO H 18 and [ H]ꢀsTꢀCO H 19, respectively (Scheme 4). No absorption at 260
2
2
nm is apparent in either sample (Figure 1). Sample B, on the other hand has absorbance at 260 nm,
associated with CoA that accounts for 90% of the sample. The MALDIꢀTOF MS of this sample is
3
consistent with [ H]ꢀsTꢀCH ꢀCoA 20 (Figure 2). Finally, the retention times of species 18, 19, and 20
2
are identical to the standards prepared synthetically. The kinetics of formation of these species is shown
in Supplementary Figure S9 and the rate constants from this data are summarized in Table 2. A control
experiment in the absence of HBCH CoA was also carried out and the results are shown in Figure 1 as
2
black solid line. The rate constants are again summarized in Table 2. The rate of hydrolysis to form
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[
H]ꢀsTꢀCO H 19 is 5ꢀfold faster than that of [ H]ꢀsDꢀCO H 18. When HBCH CoA is present,
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hydrolysis is suppressed. However, the rate of formation of [ H]ꢀsTꢀCH ꢀCoA 20 is slow and only 3ꢀ
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fold faster than the total rate of formation of [ H]ꢀsDꢀCO H 18 and [ H]ꢀsTꢀCO H 19.
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2
Mechanistic Implications. Several unexpected results were obtained from this study. First was the
3
3
identification of [ H]ꢀsTꢀCH ꢀCoA 20 (Scheme 4) rather than [ H]ꢀsTetꢀCH ꢀCoA 17 (Scheme 3) as the
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major product. Second was the very slow rate constant for its formation relative to k . Our
cat
interpretation of these results is that 1 is not reporting on chain elongation as we intended, and as an
alternative model we suggest that it may be reporting on chain termination and PhaC reꢀpriming. We
1
know that the size of the PHB polymers are 1 to 2 MDa and the rate constant for polymerization is 196
s− (this study). Thus chain termination occurs every 1−2 × 10 turnovers, proceeding with a rate
1
4
−
1
3
constant of 0.6 to 1.2 min . This number is very similar to the rate constant of [ H]ꢀsTꢀCH ꢀCoA
2
−1
formation of 0.8 min . Thus, we propose that 1 binds and dissociates from the active site of PhaC many
3
times before ester formation occurs. Detection of [ H]ꢀsTꢀCH ꢀCoA suggests that the chain termination
2
occurs by reaction at the penultimate HB unit (Scheme 4) leaving an acylatedꢀHB unit. We suggest that
this HBꢀPhaC (Scheme 4) may function as a primer of the polymerization process. Although a number
44–48
of studies have reported on chain termination events,
the actual chain terminator and termination
site in the biological systems remain unknown. The proposed formation of HBꢀPhaC is in accord with
our previous study of mutant class III D302AꢀPhaEC , in which proteins modified with (HB) (n =
Av
n
48
3
−10) were identified. Furthermore, a recent study on class I Aeromonas caviae PhaC has suggested
4
9
that HBCoA causes conversion of the monomeric form of the synthase into its active, dimeric form.
We have also shown previously that acylation of PhaC with a (HB) CoA (n = 2 or 3) decreases the lag
Re
n
38
phase of the reaction, increases the amount of dimeric PhaC and increases the activity of the enzyme.
The similarity in structure between HBCH CoA and HBCoA, suggest that HBCoA itself could function
2
3
3
as a chain terminator in vivo. The observation of [ H]ꢀsDꢀCO H 18 and [ H]ꢀsTꢀCO H 19 (Scheme 4) is
2
2
3
also interesting as water may function as a chain terminator as well. The observed ratio of [ H]ꢀsTꢀ
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CO H/[ H]ꢀsDꢀCO H of 5 (calculated from the rate constants), rather than 100 based on the differences
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9
1
1
1
1
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1
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6
5
0
in chemical reactivity of an oxo vs thioester, suggests again that there is something special about the
penultimate HB unit. Thus these results may have unexpectedly given us new insight about the
termination and reꢀpriming processes and are in line with other published results described above.
Additional studies with this analog are in progress and will be reported in a due course.
0
1
2
3
4
5
6
7
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9
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1
2
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Conclusion. A methylene analog, HBCH CoA 1 was prepared through a chemoenzymatic approach.
2
Inhibition studies with class I and III synthases reveals that 1 is a competitive inhibitor of HBCoA with
3
tight binding relative to K for HBCoA. The unexpected product ([ H]ꢀsTꢀCH ꢀCoA) identified when
M
2
3
HBCH CoA was incubated with the [ H]ꢀsTꢀacylated PhaC and its slow rate of formation suggest that
2
HBCH CoA may be reporting on chain termination process rather than elongation based on our original
2
3
3
designed. The detection of [ H]ꢀsTꢀCH ꢀCoA and [ H]ꢀsDꢀCO H not only highlight the importance of
2
2
penultimate HB unit in PHB chain, but may also shed light on the possible termination site and chain
terminator in vivo. Further investigations are needed in order to better understand the challenging
reactions catalyzed by PHA/PHB synthases.
METHODS
Materials and Enzymes. All commercial chemicals were purchased at the highest purity grade. All
solvents were anhydrous. All reactions were performed under argon atmosphere unless otherwise
3
19
specified. Radiolabeled [ H]ꢀsTCoA was synthesized according to our previously developed method.
Thin layer chromatography (TLC) was performed using 60 mesh silica gel plates and visualization was
performed using short wavelength UV light (254 nm) and/or basic KMnO staining.
4
Hisꢀtagged pantothenate kinase from Staphylococcus aureus (SaCoaA), phosphopantetheine
adenylyltransferase (EcCoaD) and dephosphoꢀCoA kinase (EcCoaE) from E. coli were purified
29, 34
according to the published methods.
The activity of each enzyme was measured following the
−
1
−1
reported procedure. The specific activities (SA) were measured at 45, 27, and 20 µmol min mg at 25
°
C for SaCoaA, EcCoaD, and EcCoaE, respectively. Synthases of wtꢀPhaCRe and wtꢀPhaECAv were
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1
0, 39
purified following the published procedures.
The SA were measured at 50 (25 °C) and 120 µmol
1
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9
1
1
1
1
1
1
1
1
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5
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5
5
5
5
6
−
1
−1
min mg (37 °C) for PhaC and PhaEC , respectively.
Re
Av
Enzymatic Synthesis of HBCH CoA 1. A 2ꢀmL reaction mixture consisted of 13 (20.0 mM), ATP
2
(
50.0 mM), MgCl (10.0 mM), SaCoaA (80.0 ꢀg), EcCoaD (80.0 ꢀg) and EcCoaE (80.0 ꢀg) in 100 mM
2
0
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0
TrisꢀHCl (pH 7.60). The reaction was initiated by addition of the enzymes and incubated at 37 °C for 3
h. The reaction was stopped by heating the reaction mixture in a 95 °C water bath for 5 min, and the
precipitated protein was removed by centrifugation (14,000 rpm × 5 min). The supernatant was loaded
onto a semiꢀpreparative HPLC column (Luna C18ꢀ2, 5 ꢀm, 10 mm × 250 mm) that was eluted at 3.00
mL/min using a linear gradient from 0 to 30% methanol in 10.0 mM ammonium acetate (pH 5) over 60
min. The fractions containing the product were pooled, concentrated in vacuo, and lyophilized to give a
white powder (15.0 mg, 50% yield, HPLC: t = 33 min). The final product was desalted by HPLC using
methanol and water as the eluents.
Inhibition Studies. The reaction was carried out at 25 and 30 °C for wtꢀPhaCRe and wtꢀPhaECAv,
respectively, in a final volume of 300 ꢀL consisting of buffer (for wtꢀPhaC : 150 mM KPi, pH 7.20 and
Re
0
2
.05% Hecameg; for wtꢀPhaEC : 20 mM TrisꢀHCl, pH 7.80 and 50 mM NaCl), wt synthase (140 and
Av
5 nM for PhaC and PhaEC , respectively), HBCoA, and HBCH CoA. The reaction was initiated by
Re
Av
2
the addition of enzyme. At defined time points, 20 ꢀL aliquots were removed from the reaction mixture
and quenched with 20 ꢀL of 10% TCA. Each sample was centrifuged to remove the precipitated
protein, and then 38 ꢀL of the quenched reaction was added to 382 ꢀL of 0.25 mM DTNB in 500 mM
KPi (pH 7.80), and A412 was measured. The rates of the reaction were determined by the slope of the
initial fast phase in the case of PhaECAv and ignoring lag phase in the case of PhaC . Each point was
Re
done in triplicate. The data were fit to MichaelisꢀMenten equation for the best inhibition mode
(
competitive, uncompetitive, and noncompetitive) using SigmaPlot.
Reaction of *sT-PhaC and HBCH CoA. In a final volume of 50 ꢀL, 500 ꢀM wtꢀPhaC and 5.00
Re
2
Re
mM *sTꢀCoA (3120 cpm/nmol) in buffer A consisting of 150 mM KPi (pH 7.20) and 0.05% Hecameg
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were incubated at 25 °C for 1 min before loading the mixture onto a Sephadex Gꢀ50 column (5 mm ×
1
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7
8
9
1
1
1
1
1
1
1
1
1
1
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2
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3
150 mm) preꢀequilibrated with buffer A at 4 °C. The acylated protein [ H]ꢀsTꢀPhaC was isolated in
Re
3
9
7% yield and labeled as 0.5 equiv. [ H] per monomer. The protein was then divided equally into two
portions.
0
1
2
3
4
5
6
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9
0
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One portion was reacted with HBCH CoA. The reaction was carried out at 25 °C in a final volume of
2
3
5
00 ꢀL containing 20 ꢀM [ H]ꢀsTꢀPhaC and 15.0 mM HBCH CoA. At defined time points, 100 ꢀL
Re
2
aliquots were removed from the reaction mixture. The reaction was then stopped by addition of 50 µL
0% TCA followed by centrifugation at 14,000 rpm for 15 min at 4 °C. The supernatant was removed
1
and the protein precipitate was washed three times with 100 ꢀL ddH O. The supernatant and washes
2
were combined and the pH was adjusted to 7 using 1 M NaOH. The mixture was then analyzed by
HPLC and scintillation counting. HPLC was performed using a reverseꢀphase C18 column (10 ꢀm, 250
×
4.6 mm, Alltech) eluting with 50 mM KPi (pH 6.72) (A) and methanol (B) and at a flow rate of 1
mL/min. The gradient (B) was from 5% to 40% from 0 to 20 min; 40% to 70% from 20 to 55 min; and
0% to 95% from 55 to 75 min and to 95% from 75 to 85 min. One min fractions were collected and
7
analyzed by scintillation counting. The protein precipitate from the each reaction was dissolved in 10%
SDS and analyzed by scintillation counting. The inventory of radioactivity is summarized in
Supplementary Table S3. A control experiment (hydrolysis) was performed in parallel with the other
3
portion of [ H]ꢀsTꢀPhaC , in which HBCH CoA was replaced with buffer A. The inventory of
Re
2
radioactivity is summarized in Supplementary Table S4.
The rates of product formation were obtained by fitting the kinetic data to the equation y = a ×
−
bx
(
1−e ) using SigmaPlot.
Sample Preparation for MS Analysis. For the above reaction, fractions eluted at 19–22 (sample A),
2
6–30 (sample B), and 31–34 min (sample C) were combined and concentrated to dryness using a
speedvac. Each sample was then dissolved in 80 µL ddH O and subjected to A 260nm measurement.
2
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Quantitation by A 260nm and radioactivity showed that sample B contained more than 90% CoA
analog. Samples A/C and B were analyzed by ESIꢀMS and MALDIꢀTOF MS, respectively.
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ACKNOWLEDGMENT This work was supported by funds from Kansas State University and
Johnson Cancer Research Center to P. L. and National Institute of Health grant to J.S. (GM49171). J. J.
was supported by the ASSURE program of the Department of Defense in partnership with the National
Science Foundation REU Site program under grant number 1004991.
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Supporting Information Available. Experimental details and spectral data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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2. Zhang, S. M., Yasuo, T., Lenz, R. W., and Goodwin, S. (2000) Kinetic and mechanistic
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characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha. Biomacromolecules 1,
244–251.
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3. Tian, J. M., Sinskey, A. J., and Stubbe, J. (2005) Class III polyhydroxybutyrate synthase:
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Involvement in chain termination and reinitiation. Biochemistry 44, 8369–8377.
44. Lawrence, A. G., Choi, J., Rha, C., Stubbe, J., and Sinskey, A. J. (2005) In vitro analysis of the
chain termination reaction in the synthesis of polyꢀ(R)ꢀbetaꢀhydroxybutyrate by the class III synthase
from Allochromatium vinosum. Biomacromolecules 6, 2113–2119.
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5. Madden, L. A., Anderson, A. J., Shah, D. T., and Asrar, J. (1999) Chain termination in
polyhydroxyalkanoate synthesis: involvement of exogenous hydroxyꢀcompounds as chain transfer
agents. Int. J. Biol. Macromol. 25, 43–53.
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6. Shi, F. Y., Ashby, R., and Gross, R. A. (1996) Use of poly(ethylene glycol)s to regulate poly(3ꢀ
hydroxybutyrate) molecular weight during Alcaligenes eutrophus cultivations. Macromolecules 29,
753–7758.
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7. Shi, F. Y., Gross, R. A., and Rutherford, D. R. (1996) Microbial polyester synthesis: Effects of
poly(ethylene glycol) on product composition, repeat unit sequence, and end group structure.
Macromolecules 29, 10–17.
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8. Tian, J., Sinskey, A. J., and Stubbe, J. (2005) Detection of intermediates from the polymerization
reaction catalyzed by a D302A mutant of class III polyhydroxyalkanoate (PHA) synthase. Biochemistry
4, 1495–1503.
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9. Numata, K., Motoda, Y., Watanabe, S., Tochio, N., Kigawa, T., and Doi, Y. (2012) Active
Intermediates of Polyhydroxyalkanoate Synthase from Aeromonas caviae in Polymerization Reaction.
Biomacromolecules 13, 3450–3455.
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0. Jencks, W. P., Cordes, S., and Carriuolo, J. (1960) The free energy of thiol ester hydrolysis. J.
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Biol. Chem. 235, 3608–3614.
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Scheme 1. Two mechanistic models for chain elongation catalyzed by PhaCs
His
HN
His
HN
A
His
HN
Asp
O
Asp
Asp
O
O
O
N
NH
S
NH
S
O
O
OH
O
CoAS
SCoA
CoAS
Cys
0
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0
Cys
Cys SH
Cys SH
(HBCoA)
O
OH
OH
O
OH
O
O
OH
HN
SCoA
Cys
HN
S
N
O
O
Asp
Cys SH
HN
His
NH
His
O
O
Asp
N
O
O
Asp
His
His
HN
His
HN
Cys SH
Asp
His
HN
Asp
O
O
O
NH
Asp
O
N
SCoA
O
NH
S
OH
O
Cys
S
O
Cys
O
O
OH
O
O
OH
Cys
HN
S
O
O
OH
O 2 OH
HBCoA
Cys SH
HN
O
Cys
HN
S
NH
His
HO
O
NH
O
N
O
Asp
O
O
Asp
Asp
His
His
B
Asp
His
His
HN
O
His
HN
Asp
O
Asp
O
CoAS
O
O
OH
O
HN
OH
NH
O
N
NH
OH
O
O
SCoA
OH
O
HBCoA
SCoA
S
Cys
HS Cys
S
Cys
His
His
Asp
Asp
OH
O
O
HN
S
NH
Cys
O
HN
OH
NH
O
OH
O
O
O
O
O
SCoA
S
Cys
SCoA
(
HB) CoA
2
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Scheme 2. Chemoenzymatic synthesis of methylene analog HBCH CoA 1
2
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Scheme 3. Differentiation of two models using HBCH CoA 1
2
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Scheme 4. Model of product formation
3H
O
O
O
3
1
8: n = 0, [ H]-sD-CO H
2
3
O
O
OH 19: n = 1, [ H]-sT-CO
H
2
n
hydrolysis
H
O
H
O
3H
O
O
0
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O
O
S Cys
OH
O
PHA chain
termination
CoA
1
3H
O
O
O
O
O
CoA
+
HO
S Cys
O
3
HB-PhaC, primed synthase
2
0: [ H]-sT-CH -CoA
2
3
Figure 1. Typical HPLC and radioactivity profiles of the reaction between [ H]ꢀsTꢀPhaC (20.0 µM)
Re
and HBCH CoA and a control reaction. The experiment in the presence of HBCH CoA at 15.0 mM is
2
2
shown in blue solid and dashed lines for A260 nm (left Yꢀaxis) and radioactivity (right Yꢀaxis),
respectively. The control in the absence of HBCH CoA is shown in black solid line for radioactivity.
2
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3
Figure 2. Negative mode of MALDIꢀTOF MS spectrum of sample B that is assigned as [ H]ꢀsTꢀCH ꢀ
2
−
CoA 20 [(M−H) : calc. 990.2669; found 989.9526].
Table 1. Summary of kinetic constants.
a
PhaCRe
PhaECAv
−1
b
k (s )
196
65.0
cat
b
K (µM)
M
330 × 10
130
K (µM)
is
14.1
40.1
a
b
lag phase ignored. from ref. 39.
Table2. Rate constants of product formation.
Product
Hydrolysis (control) (min )
18
0.09 0.44
0.025 0.26 0.78
19
20
−
1
−1
With HBCH CoA (min )
2
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