Oefin isomerization regiochemistries during tandem action of BacA and BacB on prephenate in bacilysin biosynthesis

Article abstract of DOI:10.1021/bi300254u

BacA and BacB, the first two enzymes of the bacilysin pathway, convert prephenate to an exocylic regioisomer of dihydrohydroxyphenylpyruvate (ex-H ₂HPP) on the way to the epoxycyclohexanone warhead in the dipeptide antibiotic, bacilysin. BacA decarboxylates prephenate without aromatization, converting the 1,4-diene in prephenate to the endocyclic 1,3-diene in Δ⁴,Δ⁸-dihydrohydroxyphenylpyruvate (en-H ₂HPP). BacB then performs an allylic isomerization to bring the diene into conjugation with the 2-ketone in the product Δ³, Δ⁵-dihydrohydroxyphenylpyruvate (ex-H₂HPP). To prove that BacA acts regiospecifically on one of the two prochiral olefins in prephenate, we generated 1,5,8-[¹³C]-chorismate from bacterial fermentation of 5-[¹³C]-glucose and in turn produced 2,4,6-[ ¹³C]-prephenate via chorismate mutase. Tandem action of BacA and BacB gave 2,4,8-[¹³C]-7R-ex-H₂HPP, showing that BacA isomerizes only the pro-R double bond in prephenate. Nonenzymatic isomerization of the BacA product into conjugation gives only the Δ³E- geometric isomer of Δ³,Δ⁵-ex-H₂HPP. On the other hand, acceleration of the allylic isomerization by BacB gives a mixture of the E- and Z-geometric isomers of the 7R- product, indicating some rerouting of the flux, likely through dienolate geometric isomers.

Full text of DOI:10.1021/bi300254u

Article
pubs.acs.org/biochemistry
Olefin Isomerization Regiochemistries during Tandem Action
of BacA and BacB on Prephenate in Bacilysin Biosynthesis
Jared B. Parker and Christopher T. Walsh*
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave, Boston,
Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: BacA and BacB, the first two enzymes of the
bacilysin pathway, convert prephenate to an exocylic regio-
isomer of dihydrohydroxyphenylpyruvate (ex-H2HPP) on the
way to the epoxycyclohexanone warhead in the dipeptide
antibiotic, bacilysin. BacA decarboxylates prephenate without
aromatization, converting the 1,4-diene in prephenate to the
endocyclic 1,3-diene in Δ4,Δ8-dihydrohydroxyphenylpyruvate
(en-H2HPP). BacB then performs an allylic isomerization to
bring the diene into conjugation with the 2-ketone in the
product Δ3,Δ5-dihydrohydroxyphenylpyruvate (ex-H2HPP).
To prove that BacA acts regiospecifically on one of the two
prochiral olefins in prephenate, we generated 1,5,8-[13C]-
chorismate from bacterial fermentation of 5-[13C]-glucose and
in turn produced 2,4,6-[13C]-prephenate via chorismate mutase. Tandem action of BacA and BacB gave 2,4,8-[13C]-7R-ex-
H2HPP, showing that BacA isomerizes only the pro-R double bond in prephenate. Nonenzymatic isomerization of the BacA
product into conjugation gives only the Δ3 E-geometric isomer of Δ3,Δ5-ex-H2HPP. On the other hand, acceleration of the allylic
isomerization by BacB gives a mixture of the E- and Z-geometric isomers of the 7R- product, indicating some rerouting of the
flux, likely through dienolate geometric isomers.
acilysin, a dipeptide antibiotic1−3 produced by Bacillus
subtilis strains, contains L-Ala and the nonproteinogenic
previously unassigned.14 Most notably, the first two enzymes in
the pathway, BacA and BacB, act to divert prephenate away
from typical aromatization fates to the exocyclic diene Δ3,Δ5-
dihydrohydroxyphenylpyruvate (ex-H2HPP) (Figure 1A).
The BacA enzyme and homologues we have subsequently
characterized in other bacterial pathways to nonproteinogenic
dihydro- and tetrahydroamino acid antimetabolites15,16 catalyze
a novel decarboxylative transformation on prephenate. When
canonical prephenate decarboxylases liberate CO2 from
prephenate, the electrons that flow into the 1,4-cyclohexadiene
ring lead to aromatization as the C7-OH is ejected and phenyl-
pyruvate is formed.17,18 By contrast, the BacA subfamily enzymes,
during the comparable decarboxylation, capture the electrons by
protonation at one end of the starting 1,4-diene (Figure 1B). This
leaves the C7-OH group intact, resulting in formation of a
methylene group at C8 and a net isomerization of the starting 1,4-
diene of prephenate into the 1,3 endocyclic diene of the product
Δ4,Δ8-dihydrohydroxyphenylpyruvate (en-H2HPP). Instead of
aromatization of the cyclohexadiene, the ring remains at the
dihydroaromatic oxidation state.14
B
anticapsin, bearing the antibiotic warhead. Anticapsin (Figure 1A)
has an expoxycyclohexanone moiety attached to C3 of alanine.
Once taken up by the dipeptide permeases of susceptible bacterial
or fungal cells,4−6 bacilysin is subjected to peptidase action to
release the free anticapsin as an analogue of glutamine.7,8
Anticapsin can then bind to glutaminase domain active sites.
The epoxyketone acts as an electrophile to capture the nucleo-
philic cysteine thiolate in the glutaminase domain of glucos-
amine-6-phosphate synthetase. Inactivation of both fungal and
bacterial glucosamine-6-phosphate synthetases thereby blocks
supply of this key amino sugar for cell wall peptidoglycan
assembly and leads to the cell lysis that explains the name
bacilysin.8,9
The epoxycyclohexanone ring in the anticapsin amino acid
moiety of bacilysin is an unusual modification. It was presumed
to arise by shunting some of the flux from the chorismate
pathway3,10 that would normally yield the aromatic amino acids
Phe and Tyr.11,12 Genetic analysis of bacilysin-producing strains
of B. subtilis identified a bac gene cluster,13 which was originally
identified as five contiguous genes but has since been expanded
by biochemical studies to the seven genes bacA-E and ywf GH.
In previous work we have shown that the purified BacAB and
YwfGH enzymes can convert prephenate to a 2S-tetrahydrotyro-
sine diastereomer (Figure 1A); there are three stereogenic
centers in H4Tyr, with the C4 and C7 stereochemistries
BacB acts next and accelerates isomerization to the Δ3,Δ5-ex-
H2HPP product. The Δ4 double bond has migrated from being
Received: February 24, 2012
Revised: March 23, 2012
Published: April 6, 2012
© 2012 American Chemical Society
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Figure 1. (A) Proposed pathway for the formation of bacilysin from prephenate. Important stereochemical questions are highlighted. (B) Possible
enantiomers of en-H2HPP formed from the enzymatic decarboxylation of prephenate by BacA.
Figure 2. Stereoisomers of ex-H2HPP that could possibly be obtained through the tandem action of BacA decarboxylation of prephenate followed by
nonenzymatic or BacB enzymatic isomerization of the internal diene.
endocyclic to the exocyclic Δ3 position (the other double bond
has not moved but the numbering priority has changed (see
Figure 1A)). This is a thermodynamically favored allylic
isomerization (Δ4 to Δ3) bringing the diene into conjugation
with the 2-ketone (and generating a yellow chromophore14).
The rate of isomerization is accelerated about 103-fold by
BacB.19 Subsequent conjugate addition of a hydride equivalent
from NADH at C4 by YwfH and transamination by YwfG yields
tetrahydrotyrosine, presumably two steps (C7-alcohol to ketone
oxidation and epoxidation of the remaining double bond) away
from anticapsin.
To assist in understanding the novel mechanism by which
the BacA subfamily separates decarboxylation from aromatiza-
tion, we have undertaken examination of its stereochemical
specificity. The 1,4-diene system in prephenate is symmetric
and prochiral, containing pro-R and pro-S olefins. As shown in
path 1 of Figure 1B, decarboxylation and isomerization of the
5,6-olefin (pro-R) with protonative quenching at C6 would
yield the 7R-OH product (carbon numbering schemes can be
viewed in Figure S4 of the Supporting Information). On the
other hand, isomerization of the pro-S olefin (path 2) would
yield the 7S-OH product. This stereochemistry may have
consequences for the action of subsequent enzymes in the
pathway. Analogously, as the allylic isomerization occurs from
en-H2HPP to ex-H2HPP, moving the double bond from the
endocyclic Δ4 to the exocyclic Δ3 position, two possible olefin
isomers, the Z- or E-geometric isomers (Figure 2), could arise
for either the 7R or 7S alcohol generated by BacA action (3Z-
7R, 3E-7R, 3Z-7S, and 3E-7S: Figure 2). In turn, the configura-
tion at those carbon centers could affect recognition and pro-
cessing by downstream enzymes in this and related pathways
which generate distinct chemical outcomes, such as the bio-
synthesis of the antimetabolite 2,5-dihydrophenylalanine16 or
2-carboxy-6-hydroxyoctahydroindole (Choi) in aeruginosin
assembly.20
This paper describes how we have addressed these two
stereochemical questions in the bacilysin pathway. With BacA
we desired a source of asymmetrically labeled [13C]-prephenate
to assess if the BacAB ex-H2HPP product was 13C-enriched at
an sp2 carbon (C6) (7S-alcohol) or sp3 carbon (C8) (7R-alcohol)
(Figure 3). In turn, because of the problematic symmetry of
prephenate, we decided to make a labeled [13C]-chorismate as
precursor to asymmetric prephenate, convertible via chorismate
mutase action. For determination of the E-/Z-geometric isomer
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Figure 3. Strategy of positional trace of 13C-label from 5-[13C]-glucose to ex-H2HPP to reveal which en-H2HPP (and thereby ex-H2HPP)
enantiomer(s) is being formed via BacA action on prephenate.
content of the ex-H2HPP from BacB action, we were able to
separate by HPLC and assign by NMR spectroscopy the two
geometric isomers of the Δ3,Δ5-exocyclic diene product.
protein expression. The cloning of BacA and BacB and their
transformation into BL21(DE3) E. coli cells have been previously
described.14 For CM, BacA, and BacB expression, transformed cells
were grown at 37 °C in Luria broth media supplemented with
50 μg/mL kanamycin until the OD600 nm was ∼0.2. The tem-
perature was then reduced to 15 °C for 30 min, and protein
expression was induced via addition of 1 mM IPTG. Protein
was allowed to express overnight (∼16 h) at 15 °C before cells
were harvested by centrifugation (4000g for 20 min). Cells/
protein were then kept at 4 °C or on ice for all remaining
purification steps. Pelleted cells were resuspended in cold lysis
buffer (50 mM potassium phosphate pH 8, 500 mM NaCl,
5 mM β-mercaptoethanol, 15 mM imidazole, 5% glycerol) and
lysed by two passes of exposure to 5000−15000 psi in an
Avestin EmulsiFlex-C5 homogenizer. The cell lysate was
clarified via centrifugation (50000g for 35 min), and the
supernatant was filtered through a 0.45 μm PES syringe filter
before being loaded onto a 5 mL His-Trap HP column at
1.5 mL/min via an AKTA FPLC. Bound protein was eluted
with a linearly increasing gradient of lysis buffer containing
500 mM imidazole. Fractions were analyzed via SDS-PAGE
with visualization by Coomassie blue staining. Proteins were
then dialyzed into S-75 buffer (50 mM potassium phosphate
pH 8.0, 150 mM NaCl, 1 mM dithiothreitol, and 5% glycerol)
using a dialysis membrane. CM aliquots were flash frozen at
this point and stored at −80 °C. CM concentration was deter-
mined using an extinction coefficient of 8480 M−1 cm−1 at
280 nm. BacA and BacB were subjected to further purification
by gel filtration on a Sephadex 75 26/60 HiLoad column.
Fractions were analyzed as described above, and BacA aliquots
were flash frozen at this point and stored at −80 °C. BacA
concentration was determined using an extinction coefficient of
17 420 M−1 cm−1 at 280 nm.
MATERIALS AND METHODS
■
Materials and Instrumentation. Prephenic acid barium
salt and unlabeled chorismic acid were purchased from Sigma-
Aldrich. DNA primers were purchased from Integrated DNA
Technologies. NMR solvent (D2O) and [13C]-glucoses were
purchased from Cambridge Isotope Laboratories. B. subtilis sp.
168 genomic DNA was purchased from ATCC. Herculase II
DNA polymerase was purchased from Agilent. Restriction
enzymes and T4 DNA ligase were purchased from New England
Biolabs. Competent cells were purchased from Invitrogen. Vector
(pET-28a) was purchased from Novagen. DNA purification was
performed with kits purchased from Qiagen. DNA sequencing was
performed by Genewiz. 5 mL His-Trap columns were purchased
from GE Healthcare. Any kD SDS-PAGE gels were purchased
from Bio-Rad. Protein was dialyzed using 10 000 MWCO
SnakeSkin Pleated Dialysis tubing from Thermo Scientific, and
protein was concentrated using Amicon Ultra 10 000 MWCO
centrifugal filters from Millipore. Restriction-grade thrombin was
purchased from EMD Biosciences. Hypercarb 5 μm columns were
purchased from Thermo Scientific.
1H and 2D NMR spectra were collected on a Varian VNMRS
600 MHz spectrometer equipped with a triple-resonance probe.
13C NMR spectra were recorded on a Varian MR 400 MHz
spectrometer (100.497 MHz for 13C) equipped with a OneNMR
probe. NMR data were analyzed with ACD/Laboratories software.
High-resolution LC/MS data were collected on an Agilent
Technologies 6520 Accurate-Mass Q-TOF LC/MS and analyzed
with its accompanying software. HPLC was performed on a
Beckman Coulter System Gold instrument. Protein purification
was performed on an Amersham Pharmacia Biotech AKTA FPLC.
Cloning, Expression, and Purification of Chorismate
Mutase and BacAB. The chorismate mutase (CM) gene
(aroH)21 was amplified from B. subtilis sp. 168 genomic DNA
via PCR using primers encoded with NdeI and BamHI restric-
tion sites (5′-GGCAGCCATATGATGATTCGCGGAATTC-
GCGGAG-3′; 5′-GAATTCGGATCCTTACAATTCAGTAT-
TTTTTGTCAATGATAAATCGGGCCTC-3′, respectively).
The amplified gene was ligated into vector pET-28a and trans-
formed into chemically competent TOP10 E. coli cells (Invitrogen).
Proper gene insertion was confirmed by DNA sequencing of the
purified plasmid DNA. The sequence-confirmed plasmid was then
transformed into chemically competent BL21(DE3) E. coli cells for
The N-terminal hexahistidine tag of BacB was then removed
via thrombin treatment at 16 °C. (The His6-tag was removed to
prevent any interactions between the hexahistidine tag and the
bound divalent metals of BacB.19) Thrombin cleavage was
monitored via SDS-PAGE with Coomassie blue visualization.
After cleavage was complete (∼16 h), BacB was once again
subjected to gel filtration chromatography. The fractions were
analyzed by SDS-PAGE, and aliquots of BacB were flash frozen
and stored at −80 °C. BacB concentration was determined
using an extinction coefficient of 24 410 M−1 cm−1 at 280 nm.
Assignment of 1H and 13C Spectra of Unlabeled
Chorismate and Prephenate. Separately, 3 mg of chorismic
acid and potassium prephenate (converted from barium
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prephenate as previously described14) were dissolved in 500 μL
of 10 mM potassium phosphate buffer pH 8.0 and purified by
loading onto a 100 × 10 mm Hypercarb HPLC column equi-
librated in 10 mM potassium phosphate buffer, pH 8.0. Com-
pounds were eluted with a linearly increasing gradient of aceto-
nitrile. Chorismate elution was monitored via UV absorbance at
275 nm. Prephenate elution was monitored by LC/MS run in
positive ion detection mode with water + 0.1% formic acid as
the mobile phase and acetonitrile + 0.1% formic acid as the
eluent. HPLC fractions containing the desired compound (total
volume of 3 mL) were frozen and lyophilized to dryness. Dried
compound and associated potassium phosphate salt were
dissolved in 300 μL of D2O and placed in a 5 mm D2O
matched Shigemi tube for NMR analysis. Spectral assignments
decoupled 13C NMR spectra collected with an interscan delay
time of 150 s to allow full relaxation of the quaternary carbons.25
[13C]-Chorismate samples were prepped for NMR exactly as
described above for unlabeled chorismate.
Conversion of [13C]-Chorismate to [13C]-Prephenate
and NMR Assignment. 2.1 mg of [13C]-chorismate was
incubated with 145 μg of chorismate mutase in 3 mL of 50 mM
potassium phosphate buffer pH 8.0 for 15 h at 21 °C. Reaction
progress was monitored by the disappearance of the UV
absorbance of chorismate. The reaction was quenched via addi-
tion of acetonitrile to 30% v/v and vortexing. The quenched
reaction was frozen and lyophilized to dryness. Lyophilized
components were resuspended in 500 μL of 10 mM potassium
phosphate buffer pH 8.0 and precipitated protein was pelleted
via centrifugation at 16000g for 10 min. The supernatant
containing the [13C]-prephenate was removed and purified as
described above for unlabeled prephenate. 3 mL of prephenate
containing fractions was frozen and lyophilized to dryness. The
lyophilized prephenate and salt were resuspended in 300 μL
of D2O and placed in a 5 mm D2O matched Shigemi tube
1
1
1
were made via analyses of H, 13C, H−1H-gCOSY, H−13C-
1
gHSQC, and H−13C-gHMBC spectra. Proton spectra were
referenced to residual H2O (4.79 ppm) while 13C spectra were
referenced to the methyl carbon of acetonitrile at (1.47 ppm).22
(Acetonitrile was spiked into the NMR sample at 0.3% v/v after
all necessary spectra were acquired, and an additional 13C experi-
ment was performed to obtain the reference so as not to con-
taminate the original spectra.) Unless otherwise stated, water
suppression (via presaturation) was utilized in all proton spectra
collected.
1
for NMR analysis. H and 13C NMR data were referenced as
described above.
Conversion of [13C]-Prephenate to [13C]-ex-H2HPP and
NMR Assignment. To produce [13C]-ex-H2HPP via non-
enzymatic isomerization of [13C]-en-H2HPP, 2 mg of [13C]-
prephenate (purified as described above) was incubated with 10
μM BacA in 3 mL of 50 mM potassium phosphate buffer pH
8.0 for 24 h at 21 °C. The conversion of prephenate (no UV
absorbance) to en-H2HPP (λmax = 258 nm) and then to ex-
H2HPP (λmax = 295 nm) was monitored by UV absorbance.14
The completed reaction was quenched and purified, and NMR
data were acquired exactly as described above for prephenate,
except UV absorbance at 295 nm (rather than LC/MS) was
used to identify ex-H2HPP elution in the purification.
Production and purification of [13C]-ex-H2HPP isomers 1
and 2 generated from enzymatic BacB isomerization of [13C]-
en-H2HPP were performed as described above for the non-
enzymatic isomerization reaction, except 5 mg of [13C]-prephenate
was used in a reaction volume of 5 mL and 10 μM BacB was
added to the reaction. NMR data were acquired exactly as
described above for prephenate.
Production of [13C]-Chorismate from [13C]-Glucose.
Aerobacter aerogenes 62-1 (also known as Klebsiella pneumoniae
62-1) was obtained from our lab’s strain inventory from
∼199023 (also available from ATCC #25306). This strain
(which lacks chorismate mutase activity) and the associated
method for the overproduction of chorismate were developed
by Gibson and colleagues.11,24 Medium A (for the growth of
A. aerogenes 62-1) and medium B (for the production of chorismate)
were made exactly as previously described, except glucose was not
added to medium B.24 500 mL of medium A was inoculated with a
single colony of A. aerogenes 62-1 in a 2800 mL wide-mouth
baffled flask. The culture was shaken at 200 rpm at 30 °C until the
OD600 nm measured 1.0. The cells were pelleted at 3000g at 4 °C
for 15 min. The supernatant was discarded, and the cell pellet was
gently washed with 250 mL of medium B lacking glucose. The
cells were again pelleted via centrifugation as described above. The
supernatant was once again discarded, and the cell pellet was
resuspended in 250 mL of fresh medium B along with 2 g of either
1-[13C]-glucose or 5-[13C]-glucose (99% enrichment) that had
been dissolved in 10 mL of medium B and filtered through a 0.22
μm PES syringe filter. The culture was transferred to a 2800 mL
wide-mouth baffled flask and incubated at 30 °C for 15 h while
shaking at 200 rpm. The cells were pelleted by centrifugation and
discarded.
Production, Purification, and NMR Assignments of
Unlabeled ex-H2HPP Isomers. The production and
purification procedure described above for [13C]-ex-H2HPP
isomers was repeated with unlabeled prephenate to obtain
samples of unlabeled ex-H2HPP isomers 1 and 2 for NMR data
collection. Full NMR assignments were made using the same
methods as described above for unlabeled chorismate and pre-
phenate. Nuclear Overhauser effect (NOE) data reporting on
the spatial distance between protons were obtained by acquir-
The supernatant containing the excreted chorismate was
filtered through a 0.22 μm PES filter and loaded in 50 mL
aliquots onto a 100 × 21.2 mm Hypercarb HPLC column
equilibrated in 10 mM potassium phosphate buffer, pH 8.0.
The bound compound was eluted via a linearly increasing
gradient of acetonitrile. UV absorption at 275 nm was
monitored to detect chorismate elution. Fractions containing
pure chorismate were initially indentified by a UV-absorbance
wavelength scan (λmax = 275 nm, data not shown) and then
1
ing a H−1H-NOESY with a mixing time of 500 ms. Mass
spectra of the isomers were obtained via LC/MS in negative ion
detection mode using H2O + 0.1% ammonium hydroxide as the
mobile phase and acetonitrile + 0.1% ammonium hydroxide as
the eluent.
Reactions comparing the production of ex-H2HPP isomers 1
and 2 between BacA alone (with nonenzymatic diene
isomerization) and BacAB in tandem were set up to contain
500 μM prephenate and 15 μM of each enzyme (incubated for
24 h at 21 °C). Reactions were quenched by addition of
acetonitrile to 30% v/v, then frozen, and lyophilized to dryness.
Dried mixtures were resuspended in 10 mM potassium phos-
phate buffer pH 8.0 and loaded onto a 100 × 2.1 mm analytical
1
verified by H NMR spectroscopy (Figures S2B and S4A).
Fractions were pooled, frozen, lyophilized to dryness, and
stored at −80 °C. Chorismate concentrations were determined
by UV absorbance (ε275 nm = 2630 M−1 cm−1).10 The 13C enrich-
1
ment of chorismate was determined by integration of H NMR
spectra (satellite peaks of 13C-linked protons) and inverse-gated
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13C-enrichment at an sp2 center, readily distinguishable by
NMR spectroscopy. In case the BacA enzyme is not completely
enantioselective, the ratio of [13C] enrichment at the sp3/sp2
carbons in the BacB product would provide the degree of
selectivity in the double-bond isomerization. Because the
double bonds in the 1,4-diene ring system of prephenate are
prochiral, a chemical synthesis to introduce [13C] at one of the
double bonds (but not the other) with absolute stereochemical
control seemed daunting.
In contrast, the immediate metabolic precursor chorismate,
convertible to prephenate by the 3,3-electrocyclic rearrange-
ment catalyzed by chorismate mutase,26 presented a more
appealing target. While chemical synthesis of chorismate
labeled at particular carbons with 13C could be feasible,27 we
turned instead to a microbial biosynthetic route.
The biosynthetic path of glucose carbons into chorismate has
been known for decades starting with the pioneering studies of
Sprinson and colleagues;28,29 randomization at the level of the
C3 triose phosphates during glycolysis gives doubling of label in
shikimate and then tripling of label via the enolpyruvyl group
found in chorismate even from singly labeled forms of glucose.25
As a control to establish the methodology in our group, we
fermented the readily available and relatively inexpensive 1-[13C]-
glucose with the classical Aerobacter aerogenes 62-1 blocked mutant
strain24 that accumulates chorismate in the medium. We saw
enrichment at carbons 2, 6, and 9 in the NMR spectrum of
chorismate as previously reported25 (Figure S2), validating the
approach. However, this labeling pattern would not solve the
BacA regiochemistry question as it would go on to label C5 and
C5′ of prephenate equally (and then carbons 3, 5, and 9 of
Δ3,Δ5-ex-H2HPP) (see Figure S4 for chorismate and
prephenate numbering).
Hypercarb column equilibrated in 10 mM potassium phosphate
buffer pH 8.0. Compounds were eluted with a linearly increasing
gradient of acetonitrile and elution monitored via UV absorbance
at 295 nm. The 295 nm HPLC traces were integrated using the
Karat 32 HPLC software from Beckman Coulter.
Kinetics of ex-H2HPP Isomer Formation by BacAB.
Kinetics of ex-H2HPP isomer production from tandem BacAB
incubation with prephenate were measured from a reaction
containing 1 mM prephenate and 5 μM BacAB. The reaction
was incubated at 21 °C, and reaction time points were taken by
quenching 25 μL reaction aliquots with 50 μL of acetonitrile
and vortexing. The quenched reactions were stored at −80 °C
until all time points were quenched. Quenched aliquots were
dried in vacuo and resuspended in 175 μL of 10 mM potassium
phosphate buffer pH 8.0. The resuspended aliquots were
analyzed by HPLC identically to the BacA vs BacAB reactions
described in the previous paragraph.
Equilibration of 3E-7R- and 3Z-7R-ex-H2HPP with BacB
(and BacA). Production and purification of unlabeled ex-
H2HPP isomers were accomplished as described above for the
production of [13C]-ex-H2HPP-isomers via tandem BacAB
incubation, except 5 mg of unlabeled potassium prephenate was
used as the starting material. The concentration of each ex-H2HPP
isomer was determined by UV absorbance (ε295 nm = 15 300 M−1
cm−1 14). The reaction mixture for the equilibration time course
consisted of 3 μM BacB (or BacA) in 50 mM potassium
phosphate buffer pH 8.0. The reaction was initiated via addition of
400 μM ex-H2HPP (either purified isomer 1 or 2). A control
reaction was also setup containing no BacB to monitor non-
enzymatic equilibration. Time points were taken by quenching
25 μL aliquots of the reaction with 50 μL of acetonitrile and
vortexing. Quenched time points were stored at −80 °C until
aliquots for all time points were collected. Samples were then dried
in vacuo and resuspended in 75 μL of 10 mM potassium
phosphate buffer pH 8.0. Resuspended samples were analyzed by
HPLC exactly as described above for the BacA vs BacAB
comparison reactions. The fraction of isomer 2 was calculated by
dividing the integral value of the peak of isomer 2 by the sum of
the integral values of the peaks of isomers 1 and 2 for each run.
The data were fit to a first-order exponential decay equation using
GraphPad Prism.
Instead, we reasoned that 5-[13C]-glucose (a limiting reagent
at the commercial price of $1950/g) would be a useful starting
material to get to an appropriately labeled sample of prephenate
by carrying out such fermentations. From 2 g of 5-[13C]-
glucose in a 250 mL fermentation, we obtained 56 mg of
chorismate after purification on a preparative HPLC column.
As anticipated, the chorismate sample had three 13C atom
enrichments of 57%, 70%, and 56% in the chorismate carbon
NMR spectrum (Figures 3 and 4A), at carbons 1 (135.0 ppm),
5 (130.5 ppm), and 8 (153.9 ppm), respectively (see Figure
S5A and Table S1 for full chorismate assignments). The carbon
spectra were acquired with inverse-gated decoupling and a
sufficiently long interscan delay time (150 s with no decoupler)
to allow full carbon relaxation to achieve the full intensities of
each of the carbon lines.25 13C enrichment was calculated from
a combination of the 13C-linked proton satellite peaks of C5 in
RESULTS AND DISCUSSION
■
Generation of [13C]-Chorismates from [13C]-Glucose
Isotopomers. Our approach to determine which prochiral
double bond in prephenate is isomerized during nonaromatiz-
ing decarboxylation by BacA was to use carbon magnetic
resonance to distinguish between sp3 and sp2 carbon centers in
the BacA product Δ4,Δ8-en-H2HPP, since large shifts in the
resonance positions were formerly seen in the 13C NMR spectrum
(for example, between C6 (127.01 ppm) and C8 (31.32 ppm)14).
In turn, coupling of the BacA product to BacB action to generate
the more stable Δ3,Δ5-ex-H2HPP product would also allow the
same carbon NMR approach. The acceleration of conversion of
BacA-generated Δ4,Δ8-endocyclic-H2HPP to the thermodynami-
cally favored Δ3,Δ5-exocyclic-H2HPP by BacB also prevents
nonenzymatic breakdown of the endo isomer to phenylpyruvate
and so provides quantitative flux to Δ3,Δ5-ex-H2HPP.
1
the H spectrum (Figure S4A) and signals from the inverse-
gated decoupled carbon spectrum (Figure 4A).
Conversion of 1,5,8-[13C]-Chorismate to 2,4,6-[13C]-
Prephenate. To convert this labeled [13C]-chorismate sample
to prephenate, we turned to chorismate mutase, which we
cloned and overproduced from the B. subtilis bacilysin
producer. Trial studies with unlabeled chorismate established
that quantitative rearrangement of the chorismate scaffold to
the prephenate scaffold occurred on a scale useful for carbon
NMR, and the prephenate could be purified, isolated, and
characterized (data not shown). Subsequent incubation of 2.1
mg of the 1,5,8 [13C]-chorismate with 145 μg of pure
chorismate mutase gave prephenate with the indicated carbon
NMR resonances (Figure 4B). During the course of the 3,3-
rearrangement, C1 of chorismate becomes C4 of prephenate,
For example, if one had a sample of 6-[13C]-prephenate as
substrate for BacA (and then tandem processing by BacB),
isomerization of the pro-R double bond would yield an sp3-
labeled carbon in Δ3,Δ5-ex-H2HPP while isomerization at the
pro-S double bond (Figures 1B and 3) would instead yield the
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Figure 4. Inverse-gated decoupled carbon spectra of (A) 1,5,8-[13C]-chorismate, (B) 2,4,6-[13C]-prephenate, (C) 2,4,8-[13C]-ex-H2HPP isomer 1,
and (D) 2,4,8-[13C]-ex-H2HPP isomer 2. 1,5,8-[13C]-Chorismate was obtained from fermentation of 5-[13C]-glucose with A. aerogenes 62−1; 2,4,6-
[13C]-prephenate from CM action on 1,5,8-[13C]-chorismate; and 2,4,8-[13C]-ex-H2HPP isomers 1 and 2 from tandem BacAB action on 2,4,6-[13C]-
prephenate.
C5 becomes C6, and C8 becomes the C2 ketone. Figure 4B
validates that the prephenate product has 13C enrichment at
carbons 2 (204.4 ppm), 6 (128.2 ppm), and 4 (49.2 ppm) (see
Figure S5B and Table S2 for full prephenate NMR assignments).
Because of the symmetry of the cyclohexadiene ring of pre-
phenate, carbons 6 and 6′ cannot be resolved in the NMR. Thus,
without knowing the origin of this triply labeled 13C-prephe-
nate sample, one could not tell from the carbon NMR spectrum
that only one of the two prochiral carbons, C6 and C6′, contained
the 13C enrichment. That differentiation can only be revealed by
subsequent reactions that can distinguish the two prochiral olefins,
(e.g., the nonaromatizing decarboxylase/isomerase BacA).
isomerization allowed HPLC purification of Δ3,Δ5-ex-H2HPP.
The carbon NMR spectrum (Figure S3A) of this compound,
which turns out to be the 3E-olefinic isomer as detailed in the
following paragraphs, shows three peaks, at 30.9 (C8), 156.8
(C4), and 196.1 (C2) ppm vs a CH3CN standard. By com-
parison with our assignment of the carbon NMR spectrum of
unlabeled material (Figure S5D and Table S4), these resonances
correspond to carbons 2, 4, and 8 of Δ3,Δ5-ex-H2HPP. The
diagnostic resonance is the 13C enrichment at the sp3 carbon 8 of
ex-H2HPP. There is no detectable enrichment at the sp2 C6.
Therefore, it is clear that BacA is an enantioselective
decarboxylation and isomerization catalyst. As decarboxylation
proceeds, the electrons released in the C4−COO bond cleavage
move into the ring, creating the new double bond, and the
electrons in the original pro-R double bond are used to pick up
a proton (path 1 of Figure 1B) at C6 of prephenate. From the
Processing of 2,4,6-[13C]-Prephenate by BacA: Assign-
ment of Product Stereochemistry at C7 from Action of
BacA. Tandem incubation of the triply labeled [13C]-prephenate
with purified B. subtilis enzyme BacA followed by nonenzymatic
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Figure 5. (A) HPLC separation (295 nm UV trace) of ex-H2HPP isomers 1 and 2 produced via action of BacA alone (5%/95% of isomers 1/2) or
1
tandem BacAB action (26%/74% of isomers 1/2) on prephenate after 24 h incubation at 21 °C. (B) H NMR of unlabeled ex-H2HPP isomer 1.
(C) 1H NMR of unlabeled ex-H2HPP isomer 2. ex-H2HPP isomers in (B) and (C) were produced by the tandem action of BacAB on prephenate.
perspective of C7 this is the R-isomer. No 7S-OH product
appears to be generated.
chemical shifts) for isomer 1 that is absent in isomer 2. Thus,
isomer 1 is assigned as the 3Z-7R diastereomer and isomer 2
the 3E-7R diastereomer of ex-H2HPP. (Complete NMR spectral
data for isomer 1 are presented in Figure S5C and Table S3.)
Although both 3Z- and 3E-ex-H2HPP isomers are present
after long incubations containing BacA and BacB, kinetic
analysis (Figure 7A) indicates that the 3E isomer is formed first
(at about a 50/1 E/Z ratio) and the 3Z-geometric isomer grows
in slowly. The initial product was confirmed as ex-H2HPP
(λmax = 295 nm) and not en-H2HPP (λmax = 258 nm) by UV
absorption14 (data not shown). The 3Z isomer then grows in
over a period of ∼17 h under the particular experimental con-
ditions. Comparing the 17 and 40 h time points suggest an
equilibrium has been approached with a ratio of ∼3/1 of the
E/Z-isomers of ex-H2HPP.
BacB-Mediated Equilibration of 3E-7R- and 3Z-7R-ex-
H2HPP. From the trace in Figure 7A, it appeared that BacB
could in fact catalyze equilibration between the initially formed
3E and the late-appearing 3Z isomers. As shown in Figure 7B
starting from 85% pure Z-isomer, BacB alone catalyzed the
conversion of Z- to E-isomer until equilibrium was reached.
(4% nonenzymatic conversion of the Z-isomer was observed as
background during the 24 h interval.) Going in the opposite
direction, when BacB was added to 85% pure E-isomer it was
converted to the Z-isomer until equilibrium was reached
(Figure 7C). (0.1% nonenzymatic conversion of the E-isomer
was observed as background during the 24 h interval.) For the
incubation of BacB with both the Z- (isomer 1) and E-isomer
(isomer 2) of ex-H2HPP, equilibrium was obtained at the same
3/1 ratio of E/Z geometric isomers (Figure 7D) as was observed
for the reaction from prephenate with BacAB (Figure 7A).
Detection and NMR Assignment of Geometric
Isomers of Δ3,Δ5-H2HPP from Action of BacB. When
BacB was added to the BacA incubations with the 2,4,6-[13C]-
prephenate to accelerate the allylic isomerization of the en-
H2HPP to the more stable ex-H2HPP product, in contrast to
the single product peak detected with BacA and nonenzymatic
isomerization, the BacAB tandem incubation gave rise to two
peaks separable by HPLC as shown in Figure 5A. Because of
the substantial polarity of the H2HPP isomers, a Hypercarb
column proved to be the support on which isomer separation
could be achieved. These are labeled as isomer 1 and isomer 2
because they have the same molecular mass (observed m/z
181.0503; calculated m/z 181.0506) by LC/MS analysis. Also
as seen in Figures 4C and 4D they have comparable carbon
NMR spectra, indicating the pro-R double bond has moved
(BacA action as expected) and both isomers are 7R-alcohol
forms of ex-H2HPP.
It seemed likely that isomer 1 and 2 might be the 3E/3Z
geometric isomers around the exocyclic double bond in ex-
H2HPP. The first indication confirming this was the proton
NMR spectra (Figures 5B,C). Isomer 1 and isomer 2 have
equivalent resonances for alcoholic H7 and the methylene
hydrogens 8a and 8b. The dispersion for protons 3, 5, and 6
are radically different and the splitting pattern for the methylene
hydrogens 9a and 9b are also distinct, consistent with different
geometries at the 3,4-olefin. NOESY spectra are shown in
Figure 6A for isomer 1 and Figure 6B for isomer 2. Most
diagnostic is the NOE cross-peak between the olefinic H3 and
one of the H9 resonances (indistinguishable due to identical
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1
Figure 6. H−1H-NOESY spectra of ex-H2HPP isomer 1 (A) and isomer 2 (B) produced from the tandem action of BacAB on prephenate. (A)
NOE interaction between protons 3 and 9 of isomer 1 (cross-peak is boxed) identify the Δ3-olefin as possessing “Z” geometry. (B) NOE interaction
between protons 3 and 5 could not be distinguished due to overlapping resonances. However, a NOE cross-peak between protons 3 and 9 is absent,
indicating “E” geometry for the Δ3-olefin.
Figure 7. (A) HPLC traces (295 nm) showing the kinetics of ex-H2HPP isomer 1 and 2 formation from the tandem action of 5 μM BacAB on 1 mM
prephenate at 21 °C. (B) HPLC traces (295 nm) showing the equilibration of 400 μM ex-H2HPP isomer 1 with 3 μM BacB at 21 °C. (C) HPLC
traces (295 nm) showing the equilibration of 400 μM ex-H2HPP isomer 2 with 3 μM BacB at 21 °C. (D) Kinetics of equilibration of ex-H2HPP
isomers 1 and 2 with BacB. Data were fit to a first-order exponential decay equation yielding t1/2 values of 3.6 and 10.4 h for the equilibration of
isomers 1 and 2, respectively.
Pure BacA has no ability to interconvert the 3E and 3Z-ex-H2HPP
isomers (data not shown).
A possible route from the 3E-ex-H2HPP initial product to the
3Z-olefin regioisomer is shown in Figure 8. BacB must be able
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Figure 8. Tandem action of BacAB with prephenate initially yields only the E-isomer of ex-H2HPP. BacB is then able to equilibrate between the
E- and Z-isomers of ex-H2HPP via a proposed dienolate intermediate generated by abstraction of a proton from C9.
to act on the 3E product isomer by abstraction of one of the
acidic C9 methylene hydrogens as a proton to yield the C9
carbanion, which has the indicated dienolate as a resonance
contributor. The dienolate can rotate around the C3−C4 single
bond to give a mixture of E- and Z-dienolates. Protonation at
C9 would yield either the 3E- or 3Z-ex-H2HPP conjugated
products, depending on dienolate composition. At 3 μM
[BacB] and 400 μM [ex-H2HPP], the half-time for
equilibration was found to be 3.6 h in the Z- to E-direction.
The 133/1 ratio of substrate to enzyme suggests about 0.3
equilibration events per minute under the given conditions.
From BacA incubations in the absence of BacB and at initial
time points with BacB the 3E-/3Z-isomer ratio is about 50/1,
indicating a kinetically favored path to 3E. The question arises
why BacB is retained in the biosynthetic pathway, accelerating
the flux by 103 to the 3E-ex-H2HPP regioisomer. It may be that
the subsequent equilibration of up to 25% of the 3Z-7R-ex-
H2HPP product by BacB suggests that one or more of the
downstream enzymes in the bacilysin (or related biosynthetic
pathways) pathway may care about the E/Z olefin geometry.
That is the subject of future efforts now that the isomers have
been detected, structures assigned, and kinetics of equilibration
determined.
3E and 3Z isomers around the 3,4-exocyclic double bond. We
anticipate this reflects the reversible ability to generate the
dienolate intermediates in the BacB active site and reprotonate
at C9. BacB speeds up the 1,3-allylic isomerization some 103
over the nonenzymatic rate, which otherwise yields the E-olefin
at about a 50/1 kinetic ratio over the Z-isomer, far away from
the 3/1 thermodynamic ratio.
These findings on BacA and BacB set the stage for evaluation
of subsequent issues of stereochemistry and mechanism in
these nonaromatizing pathways arising from decarboxylation of
prephenate to anticapsin/bacilysin and other dihydroaromatic
amino acids. As prephenate is processed by BacA and BacB, the
C5HC6H (pro-R) olefin gets saturated to a CH2−CH2 pair
of carbon atoms. Protonation occurs first at C8 of en-H2HPP
and then at C9 as ex-H2HPP is formed. Because both C8 and C9
are methylene groups in the BacB product, in tetrahydrotyro-
sine, and then in anticapsin, the stereochemistry is cryptic when
incubations are conducted in H2O. In D2O it should next be
possible to determine chirality and thus orientation of the active
site BD+ groups relative to the plane of the cyclohexa-
diene ring of bound substrates in BacA and BacB, respectively,
as constraints on mechanism and comparisons to how
canonical prephenate aromatizing decarboxylases work.
The mix of E- and Z-geometries in the Δ3,Δ5-ex-H2HPP
generated by BacB is also cryptic in the overall pathway since
that olefin gets reduced in the next step by delivery of a hydride
ion from NADH to C4. However, that E- vs Z-olefin geometry
could influence the stereochemistry at C4 in the reductive step
catalyzed by YwfH (Figure 1A), and evaluation of that outcome
will also be a subject of future efforts. Currently, only natural
products with tetrahydrotyrosine of the 4S-configuration have
been assigned.20,30 However, other natural products containing
tetrahydrotyrosine with unassigned stereochemistry at C4 are
known,31,32 and how stereochemistry is controlled at that
center during biosynthesis is a mystery.
Ultimately stereochemical and mechanistic studies need to be
coupled with BacA structural studies to give insights into how
electrons released into the cyclohexadiene ring on decarbox-
ylation of prephenate are diverted out of the canonical aro-
matization manifold and quenched instead by regioselective
protonation at C6 in this newly appreciated enzyme subfamily.
CONCLUSIONS
■
This study establishes that BacA is indeed an enantioselective
pro-R olefin isomerase during prephenate nonaromatizing
decarboxylation. It is likely that the selective isomerization of
the pro-R olefin in prephenate by BacA reflects an active site
orientation where the enzyme−substrate complex with bound
prephenate brings a side chain conjugate acid (BH+) adjacent
to C6 of prephenate in the active site. Determination of the 7R-
stereochemistry in Δ3,Δ5-ex-H2HPP, and by extension its
predecessor Δ4,Δ8-en-H2HPP with no detectable 7S-isomer,
indicates a high degree of olefin selectivity. This occurs as BacA
concomitantly and surprisingly suppresses the aromatization
fate that is characteristic of canonical prephenate decarboyx-
lases in biosynthetic pathways to phenylalanine.
BacB, the next enzyme in the bacilysin biosynthetic pathway,
takes the Δ4,Δ8- endocyclic-H2HPP and first generates the 3E-
geometric isomer. BacB will then catalyze the formation of the
3Z- from the 3E-olefin and proceed to equilibrate between the
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(8) Chmara, H. (1985) Inhibition of Glucosamine Synthase by
Bacilysin and Anticapsin. J. Gen. Microbiol. 131, 265−271.
(9) Chmara, H., Zahner, H., and Borowski, E. (1984) Anticapsin, an
Active-Site Directed Irreversible Inhibitor of Glucosamine-6-Phos-
phate Synthetase from Escherichia-Coli. J. Antibiot. 37, 1038−1043.
(10) Edwards, J., and Jackman, L. (1965) Chorismic Acid. A Branch
Point Intermediate in Aromatic Biosynthesis. Aust. J. Chem. 18, 1227−
1239.
(11) Gibson, M., and Gibson, F. (1964) Preliminary Studies on
Isolation and Metabolism of Intermidiate in Aromatic Biosynthesis -
Chorismic Acid. Biochem. J. 90, 248−256.
(12) Knaggs, A. (2001) The Biosynthesis of Shikimate Metabolites.
Nat. Prod. Rep. 18, 334−355.
(13) Steinborn, G., Hajirezaei, M., and Hofemeister, J. (2005) Bac
Genes for Recombinant Bacilysin and Anticapsin Production in
Bacillus Host Strains. Arch. Microbiol. 183, 71−79.
ASSOCIATED CONTENT
* Supporting Information
■
S
SDS-PAGE analysis of chorismate mutase purification; additional
13C and 1H NMR spectra of 13C labeled and unlabeled chorismate,
prephenate, 3Z-7R-ex-H2HPP, and 3E-7R-ex-H2HPP; and tabu-
lated NMR spectral data for unlabeled chorismate, prephenate, 3Z-
7R-ex-H2HPP, and 3E-7R-ex-H2HPP. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Christopher_Walsh@hms.harvard.edu; Ph:
617.432.1715; Fax: 617.432.0483.
■
Funding
(14) Mahlstedt, S. A., and Walsh, C. T. (2010) Investigation of
Anticapsin Biosynthesis Reveals a Four-Enzyme Pathway to
Tetrahydrotyrosine in Bacillus Subtilis. Biochemistry. 49, 912−923.
(15) Mahlstedt, S., Fielding, E. N., Moore, B. S., and Walsh, C. T.
(2010) Prephenate Decarboxylases: A New Prephenate-Utilizing
Enzyme Family that Performs Nonaromatizing Decarboxylation En
Route to Diverse Secondary Metabolites. Biochemistry. 49, 9021−9023.
(16) Crawford, J. M., Mahlstedt, S. A., Malcolmson, S. J., Clardy, J.,
and Walsh, C. T. (2011) Dihydrophenylalanine: A Prephenate-
Derived Photorhabdus Luminescens Antibiotic and Intermediate in
Dihydrostilbene Biosynthesis. Chem. Biol. 18, 1102−1112.
(17) Van Vleet, J., Kleeb, A., Kast, P., Hilvert, D., and Cleland, W. W.
(2010) (13)C Isotope Effect on the Reaction Catalyzed by Prephenate
Dehydratase. Biochim. Biophys. Acta, Proteins Proteomics 1804, 752−
754.
(18) Hermes, J., Tipton, P., Fisher, M., Oleary, M., Morrison, J., and
Cleland, W. (1984) Mechanisms of Enzymatic and Acid-Catalyzed
Decarboxylations of Prephenate. Biochemistry 23, 6263−6275.
(19) Rajavel, M., Mitra, A., and Gopal, B. (2009) Role of Bacillus
Subtilis BacB in the Synthesis of Bacilysin. J. Biol. Chem. 284, 31882−
31892.
(20) Ishida, K., Christiansen, G., Yoshida, W. Y., Kurmayer, R.,
Welker, M., Valls, N., Bonjoch, J., Hertweck, C., Boerner, T.,
Hemscheidt, T., and Dittmann, E. (2007) Biosynthesis and Structure
of Aeruginoside 126A and 126B, Cyanobacterial Peptide Glycosides
Bearing a 2-Carboxy-6-Hydroxyoctahydroindole Moiety RID E-9795−
2011. Chem. Biol. 14, 565−576.
(21) Gray, J., Golinellipimpaneau, B., and Knowles, J. (1990)
Monofunctional Chorismate Mutase from Bacillus-Subtilis - Purifica-
tion of the Protein, Molecular-Cloning of the Gene, and Over-
expression of the Gene-Product in Escherichia-Coli. Biochemistry 29,
376−383.
(22) Gottlieb, H., Kotlyar, V., and Nudelman, A. (1997) NMR
Chemical Shifts of Common Laboratory Solvents as Trace Impurities.
J. Org. Chem. 62, 7512−7515.
(23) Liu, J., Quinn, N., Berchtold, G., and Walsh, C. (1990)
Overexpression, Purification, and Characterization of Isochorismate
Synthase (Entc), the 1st Enzyme Involved in the Biosynthesis of
Enterobactin from Chorismate. Biochemistry 29, 1417−1425.
(24) Gibson, F. (1964) Chorismic Acid - Purification + some
Chemical + Physical Studies. Biochem. J. 90, 256−261.
(25) Rajagopalan, J., and Jaffe, E. (1993) Microbial-Production and Nmr
Characterization of [2,6,9-C-13]Chorismate and [1,3,5,8-C-13]-
Chorismate. Bioorg. Med. Chem. Lett. 3, 1453−1456.
This work was supported in part by National Institutes of
Health Grants AI042738 and GM49338 (C.T.W.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. Sarah A. Mahlstedt, Stuart W. Haynes, Timothy
A. Wencewicz, and Steven J. Malcolmson for helpful advice and
discussions. We again thank TAW and SJM for careful reading
of the manuscript.
ABBREVIATIONS
■
B. subtilis, Bacillus subtilis; E. coli, Escherichia coli; A. aerogenes,
Aerobacter aerogenes; H2HPP, dihydro-4-hydroxyphenylpyru-
vate; H4HPP, tetrahydro-4-hydroxyphenylpyruvate; H4Tyr,
tetrahydrotyrosine; IPTG, isopropyl-β-D-galactopyranoside;
SDS-PAGE, sodium dodecyl sulfate−polyacrylamide gel
electrophoresis; LC/MS, liquid chromatography/mass spec-
trometry; NMR, nuclear magnetic resonance; gCOSY, gradient
homonuclear correlation spectroscopy; gHSQC, gradient
heteronuclear single-quantum coherence; gHMBC, gradient
heteronuclear multiple bond coherence; NOESY, nuclear
Overhauser effect spectroscopy; FPLC, fast protein liquid
chromatography; HPLC, high performance liquid chromatog-
raphy; PCR, polymerase chain reaction; NADH, reduced
nicotinamide adenine dinucleotide; CM, chorismate mutase;
D2O, deuterium oxide.
REFERENCES
■
(1) Walker, J., and Abraham, E. (1970) Isolation of Bacilysin and a
New Amino Acid from Culture Filtrates of Bacillus-Subtilis. Biochem. J.
118, 557−561.
(2) Hilton, M., Alaeddinoglu, N., and Demain, A. (1988) Synthesis of
Bacilysin by Bacillus-Subtilis Branches from Prephenate of the
Aromatic Amino-Acid Pathway. J. Bacteriol. 170, 482−484.
(3) Roscoe, J., and Abraham, E. (1966) Experiments Relating to
Biosynthesis of Bacilysin. Biochem. J. 99, 793−800.
(4) Chmara, H., Woynarowska, B., and Borowski, E. (1981)
Epoxypeptide Antibiotic Tetaine Mimics Peptides in Transport to
Bacteria. J. Antibiot. 34, 1608−1612.
(5) Perry, D., and Abraham, E. (1979) Transport and Metabolism of
Bacilysin and Other Peptides by Suspensions of Staphylococcus-
Aureus. J. Gen. Microbiol. 115, 213−221.
(6) Chmara, H., Smulkowski, M., and Borowski, E. (1980) Growth
Inhibitory Effects of Amidotransferase Inhibition in Candida-Albicans
by Epoxypeptides. Drugs Exp. Clin. Res. 6, 7−14.
(26) Lee, A., Stewart, J. D., Clardy, J., and Ganem, B. (1995) New
Insight into the Catalytic Mechanism of Chorismate Mutases from
Structural Studies. Chem. Biol. 2, 195−203.
(27) Gustin, D., and Hilvert, D. (1999) Chemoenzymatic Synthesis
of Isotopically Labeled Chorismic Acids. J. Org. Chem. 64, 4935−4938.
(28) Srinivasan, P., Shigeura, H., Sprecher, M., Sprinson, D., and
Davis, B. (1956) Biosynthesis of Shikimic Acid from D-Glucose. J. Biol.
Chem. 220, 477−497.
(7) Kenig, M., Vandamme, E., and Abraham, E. (1976) Mode of
Action of Bacilysin and Anticapsin and Biochemical Properties of
Bacilysin-Resistant Mutants. J. Gen. Microbiol. 94, 46−54.
3250
dx.doi.org/10.1021/bi300254u | Biochemistry 2012, 51, 3241−3251

Biochemistry
Article
(29) Herrmann, K. (1995) The Shikimate Pathway - Early Steps in
the Biosynthesis of Aromatic-Compounds. Plant Cell. 7, 907−919.
(30) Baldwin, J., Adlington, R., and Mitchell, M. (1993) Stereo-
controlled Enantiospecific Synthesis of Anticapsin - Revision of the
Configuration. J. Chem. Soc., Chem. Commun., 1332−1335.
(31) Namikoshi, M., Sun, F., Choi, B., Rinehart, K., Carmichael, W.,
Evans, W., and Beasley, V. (1995) 7 More Microcystins from Homer-
Lake Cells - Application of the General-Method for Structure
Assignment of Peptides Containing Alpha,Beta-Dehydroamino Acid
Unit(s). J. Org. Chem. 60, 3671−3679.
(32) Harada, K., Mayumi, T., Shimada, T., Suzuki, M., Kondo, F., and
Watanabe, M. (1993) Occurrence of 4 Depsipeptides, Aeruginopep-
tins, Together with Microcystins from Toxic Cyanobacteria.
Tetrahedron Lett. 34, 6091−6094.
3251
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