Evolutionary divergence of sedoheptulose 7-phosphate cyclases leads to several distinct cyclic products

Article abstract of DOI:10.1021/ja3041866

Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectan

Full text of DOI:10.1021/ja3041866

Article
pubs.acs.org/JACS
Evolutionary Divergence of Sedoheptulose 7-Phosphate Cyclases
Leads to Several Distinct Cyclic Products
Shumpei Asamizu,‡ Pengfei Xie,†,‡ Corey J. Brumsted,‡ Patricia M. Flatt,¶,‡ and Taifo Mahmud*,‡
‡Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon 97331-3507, United States
S
* Supporting Information
ABSTRACT: Sedoheptulose 7-phosphate cyclases are enzymes
that utilize the pentose phosphate pathway intermediate,
sedoheptulose 7-phosphate, to generate cyclic precursors of
many bioactive natural products, such as the antidiabetic drug
acarbose, the crop protectant validamycin, and the natural
sunscreens mycosporine-like amino acids. These proteins are
phylogenetically related to the dehydroquinate (DHQ) synthases
from the shikimate pathway and are part of the more recently
recognized superfamily of sugar phosphate cyclases, which
includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-
scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like
proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes
catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for
aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone
synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity,
conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-
like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic
molecules.
Through a comparative bioinformatics analysis of sugar
phosphate cyclases we reported previously the identification of
a new clade of proteins that are similar to EEVS.3 These
proteins are widely distributed in cyanobacteria and fungi, such
as Nos2 (Npun_5600) from Nostoc punctiforme and Anb2
(Ava_3858) from Anabaena variabilis.3 More recently, through
seminal work of Balskus and Walsh, Npun_5600 has been
demonstrated to be responsible for the formation of desmethyl-
4-deoxygadusol (DDG), the precursor of the natural sunscreens
mycosporine-like amino acids.6 Interestingly, similar to EEVS,
Npun_5600 also utilizes sedoheptulose 7-phosphate as
substrate, although the formation of DDG from sedoheptulose
7-phosphate most likely occurs through a somewhat divergent
reaction mechanism.6
As part of our ongoing studies on the biosynthesis of
aminocyclitol-derived natural products, we recently identified
and characterized a pseudoglycosyltransferase enzyme (pseudo-
GT, VldE) that catalyzes the coupling between GDP-valienol
and validamine 7-phosphate in validamycin biosynthesis.7 To
assess the distribution of pseudo-GTs in nature, we carried out
bioinformatics analyses using the VldE sequence as a query and
found two cryptic gene clusters that contain VldE-homologues
(pseudo-GT20) in the genomes of Actinosynnema mirum DSM
43827 and Stigmatella aurantiaca DW 4/3-1 (Figure 1).8,9
INTRODUCTION
■
Sedoheptulose 7-phosphate, a phosphorylated monosaccharide
with seven carbon atoms and a ketone functional group, is an
intermediate in the pentose phosphate pathway (phosphogluc-
onate pathway). This pathway is critical in living organisms, as
its primary products include NADPH (used in reductive
reactions within cells), ribose 5-phosphate (used in the
biosynthesis of nucleotides), and erythrose 4-phosphate (one
of the precursors in the formation of aromatic amino acids). In
some organisms, sedoheptulose 7-phosphate is also involved in
secondary metabolism. It serves as the substrate of 2-epi-5-epi-
valiolone synthases (EEVS), enzymes that are involved in the
biosynthesis of C7N-aminocyclitol natural products, e.g., the
antidiabetic drug acarbose and the crop protectant validamy-
cin.1−3 EEVS are genetically related to the dehydroquinate
(DHQ) synthases from the shikimate pathway. The latter
enzymes catalyze the cyclization of 3-deoxy-D-arabino-
heptulosonate 7-phosphate (DAHP), another C7-sugar phos-
phate, to 3-dehydroquinic acid. Other related proteins include
aminodehydroquinate synthases (aminoDHQ), which cyclize
aminoDAHP to aminoDHQ in the biosynthesis of 3-amino-5-
hydroxybenzoic acid (3,5-AHBA),4 and 2-deoxy-scyllo-inosose
(DOI) synthases,5 which are involved in the biosynthesis of
many aminoglycoside antibiotics, e.g., kanamycin, neomycin,
butirosin, and spectinomycin. Based on their substrates and
catalytic functions, collectively these classes of enzymes form
the sugar phosphate cyclase (SPC) superfamily.3
Received: May 1, 2012
Published: June 28, 2012
© 2012 American Chemical Society
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Figure 1. Genome mining leading to the discovery of new genes that encode DHQ synthase-like proteins. The presence of DHQS genes (yellow
arrows) together with the putative pseudoglycosyltransferase (Ps-GT) genes (orange arrows) in the clusters raised questions as to whether DHQS
are involved in pseudosugar (cyclitol) formation.
Figure 2. Phylogenetic analysis of the superfamily of sugar phosphate cyclases. Boxed proteins are representatives of families of sugar phosphate
cyclases being studied in this paper. Maximum likelihood analysis was carried out using MEGA5 software with a WAG amino acid substitution
model, and a discrete gamma distribution was used to model evolutionary rate differences among sites. The robustness of the trees was assessed by
bootstrap analysis (100 replicates). The source and accession number of the proteins are listed in Table S1.
Based on the putative substrates for pseudo-GT en-
zymes,1,2,10−12 it is expected that the two clusters should also
contain pseudosugar biosynthetic genes, e.g., the 2-epi-5-epi-
valiolone synthase (EEVS) gene. However, initial inspections of
the clusters suggested that they do not contain the EEVS gene
but rather a putative dehydroquinate synthase (DHQS) gene,
which raised questions as to whether DHQS are also involved
in pseudosugar (cyclitol) formation.
Within this study, the gene products (Amir_2000 and
Staur_3140) were recombinantly prepared, and subsequent
enzymatic analysis revealed that these putative DHQS are
neither DHQS nor EEVS. Instead, they utilize sedoheptulose 7-
phosphate as substrate to form 2-epi-valiolone. The latter
compound has never been identified in nature before, but it
may well serve as an alternative precursor for aminocyclitol
biosynthesis. Herein we report the identification and character-
ization of these new proteins, which are designated as 2-epi-
valiolone synthases (EVS). We have also carried out
comparative genetic, biochemical, kinetic, and in situ NMR
analyses of the three different subsets of sedoheptulose 7-
phophate cyclases (EEVS, EVS, and DDGS). The results
provided further insights into their genetic diversity, conserved
amino acid sequences, and plausible catalytic mechanisms.
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Figure 3. Characterization of Amir_2000, Staur_3140, and related enzymes. A, SDS-PAGE of Ni-NTA purified cyclases. B, TLC analysis of the
cylases′ products with DAHP as substrate. C, TLC analysis of the cyclases′ products with sedoheptulose 7-phosphate as substrate. D, chemical
structures of 2-epi-5-epi-valiolone, 5-epi-valiolone, 2-epi-valiolone, valiolone, and desmethyl-4-deoxygadusol.
Co2+, however, did not give any product. On the other hand, a
RESULTS
■
parallel experiment with DHQS from E. coli (AroBEc) resulted
Bioinformatic Studies of Amir_2000 and Staur_3140.
in the expected 3-dehydroquinate product (Figure 3B),
As misannotations within the DHQS-like proteins are rather
eliminating the possibility of technical inadequacies, e.g.,
common, we carried out detailed bioinformatics studies of the
inappropriate assay condition or problems with product
genes, amir_2000 (from A. mirum) and staur_3140 (from S.
analysis; thus, suggesting that Amir_2000 and Staur_3140
aurantiaca). DHQS and DHQS-like proteins from both
may not be DHQS.
primary and secondary metabolic pathways show relatively
Characterization of Amir_2000 and Staur_3140 as
high sequence similarity (25−70% identity), and maximum
Sedoheptulose 7-Phosphate Cyclases. Whereas BLAST
likelihood analysis has revealed that protein similarity is highly
analysis revealed that the amino acid sequences of Amir_2000
correlated with the predicted enzyme function.3 To confirm
that amir_2000 and staur_3140 are indeed more closely related
to DHQS than to EEVS, we employed MEGA5 software with a
and Staur_3140 are more similar to DHQS (Amir_2000 and
Staur_3140 have 42% identity to DHQS from Desulfotomac-
ulum gibsoniae DSM 7213 and Dialister succinatiphilus YIT
WAG amino acid substitution model to generate a maximum
11850, respectively) than EEVS (Amir_2000 and Staur_3140
likelihood phylogenetic tree of the various DHQS-like proteins
have 35% and 31% identity to ValA, respectively), the fact that
(Figure 2).13,14 Input sequences were selected from GenBank
the genes are clustering with the putative pseudoglycosyl-
from each family of sugar phosphate cyclases including several
transferase genes suggests that these enzymes may be involved
putative DHQ synthase sequences such as Amir_2000 and
Staur_3140. E. coli glycerol dehydrogenase was used as an out-
in the formation of a cyclitol product. To explore this
possibility, we incubated the two proteins with sedoheptulose
7-phosphate, NAD+ and Co2+, or Zn2+. Sedoheptulose 7-
group. The results revealed that Amir_2000 and Staur_3140
are phylogenetically indeed more closely related to DHQS than
to EEVS.
Amir_2000 and Staur_3140 Are Not DHQ Synthases.
phosphate is the substrate for EEVS, whose product (2-epi-5-
epi-valiolone, EEV) is the common cyclic intermediate in many
aminocyclitol biosyntheses.1−3,15 Careful monitoring of the
DHQS are enzymes that catalyze the cyclization of DAHP to
DHQ, the first cyclic intermediate in the shikimate pathway.
This pathway is commonly present in fungi, bacteria, protozoa,
and plants for the synthesis of aromatic amino acids (e.g.,
phenylalanine, tyrosine, tryptophan), cofactors (e.g., pyrrolo-
quinoline quinone (PQQ), ubiquinone, folate), and natural
products (e.g., flavonoids, coumarins, tropanes, indole alkaloids,
lignans, and lignin). To investigate if Amir_2000 and
Staur_3140 are indeed DHQS, the genes were cloned in the
E. coli expression vector pRSET B (Invitrogen) and transferred
into E. coli BL21(DE3)pLysS. Expression of amir_2000 and
staur_3140 was induced by isopropyl-β-D-thiogalactopyrano-
side (IPTG), and the recombinant proteins (45.1 kDa and 47.7
kDa soluble polyhistidine-tagged proteins, respectively) were
purified using Ni-NTA columns (Figure 3A). Incubation of
these two proteins with DAHP, in the presence of NAD+ and
catalytic reactions using TLC and GC-MS revealed that both
Amir_2000 and Staur_3140 indeed convert sedoheptulose 7-
phosphate to a product with Co2+ being the preferred cofactor
(Figure S1). However, direct comparison of the products with
that of the well-characterized EEVS (ValA) from the
validamycin pathway showed that on TLC the compound has
an Rf value that is slightly lower than that of EEV, suggesting
that the product is not EEV (Figure 3C).
Synthesis of 2-epi-Valiolone and Characterization of
Amir_2000 and Staur_3140 Products. Based on X-ray
crystallographic studies, DHQS and DOIS have been proposed
to catalyze the cyclization of their respective sugar phosphate
substrates via aldol reactions. Because Amir_2000 and
Staur_3140 are highly similar to DHQS, we suspected that
they also adopt the same aldol mechanism. Consequently, the
most likely product for such a cyclization reaction with
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sedoheptulose 7-phosphate as substrate, besides EEV, is 2-epi-
valiolone (2EV). This is mechanistically plausible, as the aldol
cyclization can take place from either face of the ketone,
depending on the orientation of the intermediate within the
active site of the enzyme. To determine if this is the case, we
synthesized 2EV from (2S,3S,4S,5S)-2,3,4-tri-O-benzyl-5-C-
[(benzyloxy)methyl]-6,6-dichloro-1-oxo-2,3,4,5-cyclohexanete-
trol (2) (Scheme 1) and compared its physicochemical data
catalytic hydrogenation using wet 10% Pd/C as the catalyst
to give 2EV in a quantitative yield.
The synthetic 2EV was then used as an authentic sample for
the characterization of Amir_2000 and Staur_3140 products.
Preliminary comparative analysis using TLC revealed that the
enzyme products displayed a similar Rf value to that of the
synthetic compound. Further analysis of the products, after
trimethylsilyl derivatization, using GC-MS showed identical
retention time and fragmentation pattern as those of 2EV
(Figure S2), suggesting that the enzyme products are indeed
Scheme 1. Synthesis of 2-epi-Valiolone
1
2EV. This was further confirmed by comparison of the H
NMR spectra of Amir_2000 and Staur_3140 products with that
of the synthetic 2EV (Figure S3). All together, the data suggest
that Amir_2000 and Staur_3140 are neither DHQS nor EEVS,
but yet another subset of DHQS-like proteins, which we
identify as 2-epi-valiolone synthases (EVS).
Interestingly, in a prolonged incubation time at pH 7.4, the
product of Amir_2000 and Staur_3140 (2EV) is prone to
nonenzymatic epimerization. A similar conversion was also
observed with synthetically prepared 2EV when stored in an
aqueous solution. Comparative analyses by TLC and GC-MS
with synthetically prepared compounds suggest that the
epimerization product is valiolone (V) (Figure 3C). Epimeriza-
tion at C-2 from a β-configuration (mannose-like) to an α-
configuration (glucose-like) appears to occur more readily in
C7-cyclitols. Although in the validamycin pathway a dedicated
epimerase enzyme (ValD) is involved in the conversion of EEV
to 5-epi-valiolone (5EV), the conversion may also occur
nonenzymatically, albeit at a much lower rate.16
In Situ NMR Analysis of ValA, Amir_2000, Staur_3140,
Npun_5600, and Ava_3858. The discovery of Amir_2000
and Staur_3140 as 2-epi-valiolone synthases (EVS) expanded
the diverse groups of DHQS-like proteins. Together with the
EEVS and the DDGS, they represent the subgroup of
sedoheptulose 7-phosphate cyclases, which fascinatingly furnish
three distinct products, 2EV, EEV, and DDG, respectively.
with those of the enzyme products. 2 was prepared from
commercially available 2,3,4,6-tetra-O-benzyl-D-mannopyra-
nose (1) in five steps according to the method reported
previously.10 The compound was then converted, via a free-
radical reduction using Bu3SnH/AIBN, to 2,3,4,7-tetra-O-
benzyl-2-epi-valiolone (4). Finally, 4 was deprotected by
1
Figure 4. In situ H NMR analyses of ValA (A), Amir_2000 (B), and Npun_5600 (C) reactions.
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Figure 5. Superimposition of twenty low-energy structures of EEV (A), 5EV (B), 2EV (C), and V (D). The first two low-energy conformations of
2EV are shown in the ball-and-stick representation (E and F).
Therefore, it is intriguing to explore whether any of these
enzymes share a diffusible common intermediate(s) during or
after catalysis. To investigate this possibility, we set up in situ
NMR analyses of the enzymatic assays with EEVS (ValA), EVS
(Amir_2000 and Staur_3140), and DDGS (Npun_5600 and
Ava_3858) proteins. The catalytic function of Ava_3858 has
not previously been characterized; however, its high sequence
identity to Npun_5600 suggested that it is a DDGS. This
activity was confirmed in the present study (see below). ValA
was recombinantly prepared in E. coli and purified according to
the method we reported previously.2 The Npun_5600 and
Ava_3858 genes, amplified from the chromosomes of the
cyanobacteria N. punctiforme and A. variabilis, respectively, were
cloned in pRSET B and expressed in E. coli BL21(DE3)pLysS.
The recombinant proteins were purified by Ni-NTA columns.
To monitor the catalytic reactions of these five enzymes, we
geminal proton signals between 1.5 and 3.0 ppm [set A, 2.65
(dd, J = 1, 14.4 Hz, 6-Ha) and 2.89 (d, J = 14.4 Hz, 6-Hb); set
B, 1.83 (dd, J = 2.0, 14.6 Hz, 6-Ha) and 2.08 (d, J = 14.6 Hz, 6-
Hb)] (Figures 4B and S3B). None of these signals belong to
EEV or DDG, excluding the possibility that Amir_2000 and
Staur_3140 also produce those compounds. The signals are
also different from those of V [2.39 (d, J = 14.6 Hz, 6-Ha)] and
2.79 (d, J = 14.6 Hz, 6-Hb)].17 More importantly, identical two
1
sets of geminal proton signals were also observed in the H
NMR spectrum of the synthetic compound (Figure S3A),
confirming that the signals indeed belong to 2EV. Therefore,
we predicted that in an aqueous solution 2EV exists in two
stable conformational forms.
Computational Studies of the Valiolones. To confirm
the presence of two stable conformers of 2EV in an aqueous
solution, we performed computational modeling of 2EV
together with EEV, 5EV, and V using MacroModel Force
Field-based Molecular Modeling and the Jaguar ab initio
1
carried out time-course H NMR experiments in D2O/H2O.
The substrate (2 mg, as a barium salt) was dissolved in D2O
(152.5 μL) and potassium phosphate buffer (12.5 μL of 0.4 M
solution, pH 7.4), NAD+ (2.5 μL of 100 mM solution), and
Co2+ (2.5 μL of 20 mM solution) (all in H2O) were added. The
mixture was transferred into a Shigemi NMR tube, and the
reaction was started by adding the purified enzyme solution (80
Quantum Mechanics Programs (Schrodinger). Conformational
̈
searches were performed using AMBER* (water). Low-energy
structures found for each compound were selected and
subjected to unrestrained quantum mechanical geometry
minimization using Hybrid B3LYP/6-31G**-SCF [PBF-
(water)]. Optimizations were followed by single point energy
calculations to obtain more accurate energies for each structure.
Superimposition of twenty low-energy structures of EEV, 5EV,
and V revealed that the majority adopts a conserved chair
conformation (Figure 5A, 5B, and 5D). On the other hand,
2EV appears to adopt at least two different conformations in
water (Figure 5C). The quantum calculations found that the
two conformations were highly similar in enthalpy (within 0.2
kcal/mol), suggesting both conformers may coexist in solution
(Figure 5E and 5F). This is in complete agreement with the
1
μL). The reactions were carried out at 25 °C, and H NMR
measurements were performed under water suppression
conditions at 0 min (no enzyme), 30 min, 1 h, 3 h, and 5 h
(Figures 4 and S3). A decreased intensity of the proton signals
of the substrate sedoheptulose 7-phosphate and an increased
intensity of those of the products were observed in the NMR
spectra, confirming the distinct function of each of those
enzymes.
1H NMR spectrum of the ValA product showed a set of
geminal proton signals at 2.45 (d, J = 14.0 Hz, 6-Ha) and 2.91
(d, J = 14.0 Hz, 6-Hb) (Figure 4A), consistent with those
1
observed two sets of geminal proton signals in the H NMR
1
expected for EEV. On the other hand, H NMR spectra of
spectrum. The quantum calculations also revealed that V (31.8
kcal/mol) is thermodynamically more stable than 2EV (33.7
kcal/mol), which explains the tendency for nonenzymatic
epimerization of this compound. In contrast to the computed
structures of 2EV, which are highly variable (Figure 5C), all
twenty low-energy structures for V consistently adopt chair
conformations with the C-2, C-3, and C-4 hydroxy groups, as
well as the C-5 hydroxymethylene group, oriented in equatorial
positions (Figure 5D).
Kinetic Studies of ValA, Amir_2000, Staur_3140,
Npun_5600, and Ava_3858. To compare the affinity of
each of these enzymes toward the substrate sedoheptulose 7-
phosphate and determine their catalytic efficiency, we carried
DDG (the product of Npun_5600 and Ava_3858) showed
geminal proton signals at 2.41 (2H, d, J = 16.9 Hz, 4-Ha and 6-
Ha), 2.68 (2H, d, J = 16.9 Hz, 4-Hb and 6-Hb), and 3.50 (2H,
s, 7-H2) (Figures 4C and S3C). The overlapping C-4 and C-6
methylene proton signals in DDG suggest that the compound
exists in an equilibrium of two reversible enol forms. However,
no indication of the presence of diffusible common
intermediate(s) was observed in the 1H NMR spectra of
these reactions, thus, eliminating the possibility of non-
enzymatic conversions of any of these products.
Interestingly, cyclization of sedoheptulose 7-phosphate by
Amir_2000 and Staur_3140 gave a product(s) with two sets of
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out kinetic studies of ValA, Amir_2000, Staur_3140,
Npun_5600, and Ava_3858. The kinetic properties were
determined using the EnzChek Pyrophosphate Assay Kit18
(Molecular Probes) (Scheme S1). Each enzyme, in Tris-HCl
buffer, was preincubated with NAD+, CoCl2, 2-amino-6-
mercapto-7-methylpurine ribonucleoside (MESG), and purine
nucleoside phosphorylase (PNP) for 10 min at 28 °C.
Reactions were initiated by addition of sedoheptulose 7-
phosphate and monitored at A360 every 8 s. The apparent
kinetic values, obtained from Hanes-Woolf plots, are shown in
Table 1. The results suggest that despite their different catalytic
mechanisms and products, all tested proteins have relatively
similar substrate affinity and catalytic efficiency.
active site residues, except for those at positions R264 and
N268. In DOIS from the tobramycin (TbmA), kanamycin
(KanA), ribostamycin (RbmA), gentamycin (GntB), and
butirosin (BtrC) pathways,24,25 the arginine and asparagine
residues corresponding to position 264 and 268 in DHQS have
been altered to conserved glycine and glutamate residues,
respectively.
In EEVS (e.g., ValA, CetA, SalQ, PrlA) and DDGS (e.g.,
Npun_5600, Ava_3858), one-third of the active site residues
are consistently altered from those of DHQS including H275,
which is believed to play a critical role in enzyme catalysis.
Amino acid residues corresponding to DHQS K250, H275, and
K356 are highly conserved methionine, proline, and proline
residues, respectively, in EEVS and DDGS (Table 2). On the
other hand, the basic N268 in DHQS is a conserved aspartate
residue in EEVS and a conserved alanine residue in DDGS.
Interestingly, despite the fact that EVS are catalytically more
similar to EEVS and DDGS, genetically they demonstrate
higher similarity with DHQS. EVS retain high active site
residue conservation with DHQS, only differing at position
K356. In both Amir_2000 and Staur_3140, the lysine
corresponding to position 356 in DHQS has been altered to
a conserved arginine residue (Table 2).
Table 1. Apparent Kinetic Values for ValA, Amir_2000,
Staur_3140, Npun_5600, and Ava_3858
protein
ValA
Km (μM)
kcat (min−1
)
kcat/Km (μM−1•min−1
)
25.6 5.2
59.9 13.4
38.6 5.2
65.1 12.4
21.6 2.8
2.7 0.1
2.0 0.1
3.7 0.2
5.0 0.5
3.2 0.3
0.105 0.01
0.033 0.004
0.096 0.007
0.077 0.006
0.148 0.005
Amir_2000
Staur_3140
Npun_5600
Ava_3858
DISCUSSION
■
Identification of Sequences Characteristic to EVS.
Finally, to be able to recognize EVS proteins at the genetic
level, it is important to establish their sequence characteristics
that will enable us to distinguish them from DHQS and their
other close relatives. Previously, we established unique
signature sequences for each of the known sugar phosphate
cyclases (SPCs) that can be used to accurately annotate
sequences according to their function.3 Using a similar
approach we investigated the sequences of EVS to identify
characteristic residues that can differentiate EVS from DHQS
and other SPCs. Thus, amino acid sequences of representative
members of each family of SPCs (i.e., DHQS, aminoDHQS,
DOIS, EEVS, EVS, and DDGS) were compared using the
multiple alignment program ClustalW. The active site of
DHQS is formed within a cleft between the N- and C-terminal
domains and is lined by 14 amino acid residues. These active
site amino acid residues (Table 2) are identified based on the
X-ray crystal structure of the DHQS from Aspergillus nidulans.19
The family more related to DHQS is the aminoDHQS from the
rifamycin (RifG),20 ansamitocin (Asm47),21 mitomycin
(MitP),22 and geldanamycin (GdmO)23 pathways. Sequence
alignment of the binding pocket residues revealed that DHQS
and aminoDHQS retain high sequence conservation, only
differing at positions K197 and E260. Similarly, the DOIS
family is also closely related to DHQS, as both have conserved
Since the discovery of the AROM protein in Aspergillus nidulans
and Saccharomyces cerevisiae, a substantial list of DHQS has
been reported from other fungi, bacteria, apicomplexans, and
plants, suggesting that the shikimate pathway is a common
metabolic pathway in these organisms. However, in certain
host-associated bacteria, some of the shikimate pathway genes
appeared to be missing.26 Subsequently, through seminal work
by Floss and others,4,27 aminoDHQS, a closely related family of
DHQS, was found to be involved in the biosynthesis of
rifamycin, ansamitocin, geldanamycin, and mitomycin C. The
next well-studied DHQS-like proteins are DOIS,5,28,29 required
for the biosynthesis of aminoglycoside antibiotics, followed by
the EEVS that are involved in C7N-aminocyclitol biosyn-
thesis.1−3,12,15
The recent characterization of DDGS involved in the
biosynthesis of sunscreen compounds in cyanobacteria and
the characterization of EVS reported in this study further
underscore the uniquely diverse DHQS-like sugar phosphate
cyclases.3,6,30 Interestingly, except for aminoDHQS, whose
substrate is derived from a discrete pathway involving
kanosamine 6-phosphate and 3-amino-3-deoxy-D-fructose 6-
phosphate,4,31,32 the substrates for DHQS, EEVS, EVS, DDGS,
and DOIS are all directly derived from the pentose phosphate
pathway (Scheme 2). Particularly intriguing are the EEVS, EVS,
a
Table 2. Sequence Alignment of the Proposed Active Site Residues of Currently Known DHQS-like Sugar Phosphate Cyclases
protein
130
146
152
162
194
197
250
260
264
268
271
275
287
356
DHQS
aDHQS
DOIS
EEVS
EVS
R
R
R
R
R
R
D
D
D
D
D
D
K
K
K
K
K
K
N
N
N
N
N
N
E
E
E
E
E
E
K
R
K
K
K
K
K
E
D
E
E
E
E
R
R
G
R
R
R
N
N
E
H
H
H
H
H
H
H
H
H
P
H
H
H
H
H
H
K
K
K
P
R
P
K
K
M
K
D
N
A
H
P
DDGS
M
a
Multiple amino acid sequence alignment was conducted by ClustalW. Full-length amino acid sequences were aligned using the following
parameters: protein weight matrix = Blosum, gap open penalty = 15, gap extension penalty = 0.2. Numbering is based on DHQS domain of AROM
from A. nidulans. Proteins used for multiple sequence alignment are listed in Supplemental Table S1.
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a
Scheme 2. Most Substrates for DHQ Synthase-like Cyclases Are Directly Derived from the Pentose Phosphate Pathway
a
EEVS, EVS, and DDGS all utilize sedoheptulose 7-phosphate as substrate but yield three different cyclic products.
and DDGS that all utilize sedoheptulose 7-phosphate as
substrate but yield three different cyclic products. This raises
an important mechanistic question as to how three very closely
related enzymes utilize a common substrate but produce three
different products.
Further investigations of these enzymes at the molecular and
structural levels may help dissect the unique catalytic evolution
of this fascinating family of proteins.
Since our first report on the identification of EEVS in the late
1990s,1 dozens of EEVS and EEVS-like proteins have been
reported in the NCBI database. Mostly, they are of bacterial
origin, including marine actinomycetes and myxobacteria, but
genes encoding EEVS-like proteins are also found in fungal,
fish, and frog genomes. This is rather surprising, as only a
relatively limited number of C7N-aminocyclitol-containing
natural products have been reported in the literature and
most of them were from bacteria. Genome mining, however,
suggests the likelihood that aminocyclitol-containing com-
pounds are more common in nature than currently appreciated.
However, due to their physicochemical properties, e.g., high
polarity, they may be overlooked during the isolation of natural
products. On the other hand, EEVS or EEVS-like proteins may
also be involved in primary metabolism, providing biosynthetic
intermediates that will ultimately contribute to the structural or
physiological functions within the organisms.
Unlike EEVS, EVS appear to be less common in nature. A
preliminary inspection of the database resulted in the discovery
of only a few proteins that are homologous to EVS. This could
be due to the fact that the identification of EVS based on
sequence similarity is more challenging. EVS are highly similar
to DHQS, which by April 2012 have topped 6,875 entries in
the NCBI database. Our comparative analysis based on the
The catalytic mechanism of DHQS has been proposed to
proceed through a multistep process including alcohol
oxidation, phosphate β-elimination, carbonyl reduction, ring-
opening, and intramolecular aldol condensation.19 Similar
mechanisms were also proposed for DOIS, EEVS, and
DDGS.1,5,6 Therefore, it is tempting to assume that EVS also
adopt a similar mechanism, albeit the substrate may be oriented
differently within the enzyme active site pocket (Scheme 3). In
EEVS and EVS, the reaction is assumed to be initiated by
transient dehydrogenation of C-5 to a ketone, which sets the
stage for the elimination of phosphate, followed by reduction of
the C-5 ketone and ring-opening to produce the enol of a 6,7-
methyl ketone. The latter then undergoes intramolecular aldol
condensation to give either 2-epi-5-epi-valiolone or 2-epi-
valiolone, depending on the orientation of the aldol acceptor. In
DDGS,6 the reaction is also assumed to be initiated by
dehydrogenation of C-5 to a ketone, followed by the
elimination of phosphate. However, ring-opening and aldol
condensation should occur prior to enolization, dehydration,
reduction, and tautomerization to give desmethyl 4-deoxy-
gadusol (Scheme 3). However, how exactly these different
processes take place in EEVS, EVS, and DDGS remain obscure.
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Scheme 3. Proposed Catalytic Mechanisms for EEVS, EVS, and DDGS
predicted active site residues of Aspergillus nidulans DHQS
showed that only a single putative active site residue of EVS
differs from those in DHQS (Table 2). It remains to be
determined if this single active site difference is enough to cause
the change in substrate affinity between DHQS and EVS
enzymes. Other modifications near the identified active site
residues may play a more fundamental role in substrate
specificity within these two classes of enzymes, making
annotation of enzymatic function based on protein sequence
alone more difficult. In contrast, almost one-third of the active
site residues in EEVS and DDGS are different from those in
DHQS, making them readily distinguishable at the protein
sequence level. Thus, the characterization of Amir_2000 and
Staur_3140 reported here provide the foundation for further
understanding of this unique family of sedoheptulose 7-
phosphate cyclases.
with EVS but produce two other distinct products. Further
analysis of the products using in situ NMR and computational
calculations revealed that in an aqueous solution 2EV exists in
two stable conformational forms. Kinetic studies of EEVS, EVS,
and DDGS suggest that despite their different catalytic
mechanisms and products, they have relatively similar substrate
affinity and catalytic efficiency. The results not only highlight
the uniquely diverse DHQS-like sugar phosphate cyclases,
which may provide new tools for chemoenzymatic, stereo-
specific synthesis of various cyclic molecules, but will also
enable the identification of cryptic metabolic pathways involved
in the biosynthesis of biologically active secondary metabolites
with potential applications in medicine, agriculture, and other
industrial processes.
EXPERIMENTAL SECTION
■
General. All chemical reactions were performed under an argon or
nitrogen atmosphere employing oven-dried glassware. Analytical thin-
layer chromatography (TLC) was performed using silica plates (60 Å)
with a fluorescent indicator (254 nm), which were visualized with a
UV lamp and ceric ammonium molybdate (CAM) solution.
Chromatographic purification of products was performed on silica
gel (60 Å, 72−230 mesh). Proton NMR spectra were recorded on
Bruker 300 or 400 MHz spectrometers. Proton chemical shifts are
reported in ppm (δ) relative to the residual solvent signals as the
internal standard (CDCl3: δH 7.26; D2O: δH 4.79). Multiplicities in the
CONCLUSION
■
Using genome mining and biochemical studies we identified a
new subset of DHQS-like proteins in the actinomycete A.
mirum and the myxobacterium S. aurantiaca DW4/3-1 that
catalyze the conversion of sedoheptulose 7-phosphate to 2EV.
Comparative bioinformatic analyses revealed that at the amino
acid sequence level EVS is more similar to DHQS than to
EEVS and DDGS. The latter enzymes share the same substrate
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1H NMR spectra are described as follows: s = singlet, bs = broad
EcoRI digested PCR product was ligated into BamHI and EcoRI sites
of pRSET B to generate pRSET B-amir_2000.
singlet, d = doublet, bd = broad doublet, t = triplet, bt = broad triplet,
q = quartet, m = multiplet; coupling constants are reported in Hz.
Carbon NMR spectra were recorded on a Bruker 300 (75 MHz)
spectrometer with complete proton decoupling. Carbon chemical
shifts are reported in ppm (δ) relative to the residual solvent signal as
the internal standard (CDCl3: δC 77.16) or with sodium 2,2-
dimethylsilapentane-5-sulfonate (DSS) (δ 0.0) as an external standard.
Low-resolution electrospray ionization (ESI) mass spectra were
recorded on a ThermoFinnigan liquid chromatograph-ion trap mass
spectrometer, and high-resolution electrospray mass spectra were
recorded on a Waters/Micromass LCT spectrometer. Size exclusion
chromatography was done on Sephadex LH-20 (Pharmacia).
Molecular Phylogenetic Analysis by Maximum Likelihood
Method. For phylogenetic analysis, full-length amino acid sequences
were aligned using ClustalW with the following parameters: protein
weight matrix = Blosum; gap open penalty = 15; gap extension penalty
= 0.2. Maximum likelihood analysis was carried out using MEGA5
software with a WAG amino acid substitution model, and a discrete
gamma distribution was used to model evolutionary rate differences
among sites. The robustness of the trees was assessed by bootstrap
analysis (100 replicates).
Chemical Synthesis of 2-epi-Valiolone. 2,3,4,7-Tetra-O-ben-
zyl-2-epi-valiolone (4). To a solution of 2 (25 mg, 0.040 mmol) in
toluene (0.25 mL), tributyltin hydride (0.043 mL, 0.161 mmol) and
AIBN (2.6 mg, 0.016 mmol) were added, and the mixture was refluxed
for 2 h and then cooled to room temperature. The products were
extracted with EtOAc (3 mL), and the organic solution was washed
with 2 N HCl, sat. aq. NaHCO3, and brine. The organic solvent was
evaporated under reduced pressure, and the extract was purified over a
silica gel column (n-hexane/EtOAc 25:1-5:1) to give 4 (11 mg, 49%);
colorless syrup, 1H NMR (300 MHz, CDCl3) δ: 2.37 (d, J = 14.4 Hz,
6-Ha), 2.61 (s, 5-OH), 3.15 (d, J = 14.4 Hz, 6-Hb), 3.23 (d, J = 8.9 Hz,
7-Ha), 3.49 (d, J = 8.9 Hz, 7-Hb), 3.93 (dd, J = 7.6, 3 Hz, 3-H), 4.10
(d, J = 3 Hz, 4-H), 4.30 (d, J = 7.6 Hz, 2-H), 4.40−4.87 (PhCH2- × 4),
7.15−7.40 (C6H5 × 4). 13C NMR (75 MHz, CDCl3) δC: 44.9 (t, C-7),
72.1, 72.6, 73.3, 73.4 (all t, PhCH2 × 4), 74.0 (d, C-4), 77.4 (s, C-5),
79.5 (d, C-3), 81.0 (d, C-2), 127.7−128.4 (all d) and 137.2−137.9 (all
s, C6H5 × 4), 206.6 (s, C-1). LRMS (ESI-TOF) m/z 575 [M+Na]+.
HRMS (ESI-TOF) m/z 575.2391 (calcd for C35H36O6Na [M+Na]+:
575.2410).
Construction of an Expression Plasmid for Recombinant
Staur_3140. Stigmatella aurantiaca DW4/3-1 was grown on agar
plate media containing 1% Tryptone, 0.2% MgSO4 × 7H2O, 1.2%
HEPES, and 1.5% agar, pH adjusted to 7.2 with KOH, for 10 days at
30 °C and the grown cells were used to inoculate liquid media
containing 1% Tryptone, 0.2% MgSO4 × 7H2O, 0.4% soluble starch,
1.2% HEPES, pH 7.2 with KOH). After culture at 30 °C, 200 rpm for
4 days, cells were harvested and their genomic DNA was isolated. A
Staur_3140 gene was amplified by PCR using two primers (5′-GAA
GAT CTC GAC CCA ATG CCT TCC ACT G-3′ and 5′-CGG AAT
TCA TGT AGG CCG TGG ACG CGA G-3′, BglII and EcoRI are
underlined). BglII and EcoRI digested PCR product was ligated into
BamHI and EcoRI sites of pRSET B to generate pRSET B-Staur_3140.
Construction of an Expression Plasmid for Recombinant
Npun_5600 and Ava_3858. Frozen stocks of Nostoc punctiforme
ATCC 29133 and Anabaena variabilis ATCC 29413 were used to
inoculate Allen and Arnon liquid media, and the cultures were
statically grown with filtered air infused into the cultures using an air
stone.33 After culture at 28 °C for 21 days, 50 mL of culture (∼1 g of
cell mass) was harvested by centrifugation at 3,000 x g, and the
genomic DNA was isolated using a modified STE protocol. Briefly, the
cell pellet was frozen and ground in liquid nitrogen using a mortar and
pestle to fully disrupt the cell wall components. One gram of cell mass
was resuspended in 5 mL of STE buffer containing 20 mg of lysozyme
and incubated at 37 °C for 10 min. EDTA was added to a final
concentration of 0.1 M containing 1% SDS, 0.5 mg of RNase, and 0.15
mg Pronase, and the sample was incubated for an additional 15 min at
37 °C. The sample was extracted with phenol:chloroform and
precipitated with 0.3 M sodium acetate, pH 5.2 and 70% isopropanol.
Spooled DNA was washed with 70% ethanol, briefly air-dried, and
resuspended in sterile water. The npun_5600 (DDGS) gene was
́
amplified by PCR using two primers (5-GAA GAT CTG CAT ATG
AGT AAT GTT CAA GCA TCG T-3′ and 5′-CGG GGT ACC TCA
́
CAC TCC CAA TAG TTT GGA-3, BglII and KpnI are under-lined).
BglII and KpnI digested PCR product was ligated into BamHI and
KpnI sites of pRSET B to give pRSET B-npun_5600. The ava_3858
́
(DDGS) gene was amplified by PCR using two primers (5-GAA GAT
́
́
CTG CAT ATG AGT ATC GTC CAA GCA AAG-3 and 5-CGG
GGT ACC TTA TTT AAC ACT CCC GAT TAT T-3, BglII and
́
KpnI are underlined). BglII and KpnI digested PCR product was
ligated into BamHI and KpnI sites of pRSET B to give pRSET B-
ava_3858.
2-epi-Valiolone (5). To a solution of 4 (8 mg) in 95% aqueous
ethanol (0.80 mL) was added wet 10% Pd/C (8 mg), and the mixture
was stirred at room temperature under an H2 atmosphere for 2 h. The
suspension was passed through a Celite column to remove the catalyst
and then filtered through a membrane filter. The solvent was
Construction of an Expression Plasmid for Recombinant AroB.
The aroB gene from Escherichia coli (aroBEc) was amplified by PCR
1
́
from pJB14 (a gift from John W. Frost) using two primers (5-TGG
evaporated in vacuo to give pure 5 (2.7 mg); white solid, H NMR
ATG CTC GAG TAT GGA GAG G-3′ and 5′-CCT TTC GAA TTC
(300 MHz, CD3OD) δ: 1.83 (dd, J = 14.1, 2.1 Hz, 6-Ha1), 2.03 (d, J =
14.1 Hz, 6-Hb1), 2.47 (d, J = 14.1, 1.2 Hz, 6-Ha2), 2.87 (d, J = 14.1
Hz, 6-Hb2), 3.41 (d, J = 11.1 Hz, 7-Ha), 3.64 (d, J = 11.1, 7Hb),
3.98−4.02 (m, 3-H and 4-H), 4.31 (d, J = 3.3 Hz, 2-H). 13C NMR (75
MHz, CD3OD) δC: 44.1 70.4 (t, C-6), 71.3 (d, C-4), 73.7(d, C-2),
́
TCA CGC TGA-3, XhoI and EcoRI are underlined). XhoI and EcoRI
digested PCR product was ligated into the corresponding sites of
pRSET B to give pRSET B-aroBEc. DNA sequences of all PCR
amplified clones were confirmed by the Oregon State University
Center for Genome Research and Biocomputing (CGRB) core lab.
Expression of valA, amir_2000, npun_5600, ava_3858, and
aroBEc. Expression plasmids were used to transform E. coli BL21(DE3)
pLysS. Transformants were grown overnight at 37 °C on LB agar
plates containing 100 μg/mL ampicillin and 25 μg/mL chloramphe-
nicol. A single colony was inoculated into 3 mL of LB medium and
cultured at 37 °C for 6 h, and then 1 mL of seed culture was
transferred into 100 mL of LB medium in a 500 mL flask and grown at
28 °C until OD600 reached 0.3−0.5. Subsequently, the temperature
was reduced to 18 °C and after 1 h adaptation 0.1 mM IPTG was
added to induce production of the N-terminal hexahistidine-tagged
recombinant proteins. After further growth for 16−20 h, the cells were
harvested by centrifugation (5,000 rpm, 10 min, 4 °C) and stored at
−80 °C until used.
1
75.1 (s, C-5), 75.6 (d, C-3), 208.4 (s, C-1). H NMR (300 MHz,
D2O) δ: 1.85 (d, J = 15 Hz, 6-Ha1, 1H), 2.11 (d, J = 15 Hz, 6-Hb1,
1H), 2.67 (d, J = 14 Hz, 6-Ha2, 1H), 2.91 (d, J = 14 Hz, 6-Hb2, 1H),
3.48−3.86 (m, 7H), 3.9−4.2 (m, 3H), 4.52 (d, J = 3.9 Hz, 2-H, 1H).
LRMS (ESI-TOF) m/z 191 [M-H]−. HRMS (ESI-TOF) m/z
191.0554 (calcd for C7H11O6 [M-H]−: 191.0556).
Construction of ValA, Amir_2000, Staur_3140, Npun_5600,
Ava_3858, and AroBEc Expression Plasmids. Construction of an
expression plasmid for recombinant ValA from Streptomyces
hygroscopicus subsp. jinggangensis was reported in our previous paper.2
Construction of an Expression Plasmid for Recombinant
Amir_2000. Actinosynemma mirum DSM 43827 was grown on YMG
agar plate for 4 days, and the grown cells were used to inoculate YMG
liquid media. After incubation at 30 °C, 200 rpm for 2 days, cells were
harvested and their genomic DNA isolated. The amir_2000 gene was
Expression of Staur_3140. The expression plasmid was used to
transform E. coli BL21(DE3) pLysS. A transformant was grown
overnight at 37 °C on an LB agar plate containing 100 μg/mL
ampicillin and 25 μg/mL chloramphenicol. A single colony was
́
amplified by PCR using two primers (5-GAA GAT CTT ATG GAC
́
́
AGT CCC GCT GGT TAC C-3 and 5-CGG AAT TCA GGC TCA
TCG CAG CCT CAC C-3, BglII and EcoRI are underlined). BglII and
́
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inoculated into 3 mL of LB medium and cultured at 37 °C for 6 h, and
then 0.1 mL of seed culture was transferred into 100 mL of M9-
glucose medium in a 500 mL flask and grown at 15 °C for 2 days until
OD600 reached 0.3. Subsequently, 0.1 mM IPTG was added to induce
production of the N-terminal hexahistidine-tagged Staur_3140 protein.
After further grown for 24 h, the cells were harvested by centrifugation
(5,000 rpm, 10 min, 4 °C) and stored at −80 °C until used.
h, 0.5 h, 1 h, 3 h, 5 h. Temperature was set between 297.9 and 298.2 K,
and data acquisition was carried out with 64 scans for each time point.
Determination of the Km and kcat Values of ValA, Amir_2000,
Staur_3140, Npun_5600, and Ava_3858. A typical reaction mixture
(100 μL) contained Tris-HCl (50 mM, pH 7.0 for Amir_2000,
Staur_3140, Npun_5600, and Ava_3858, and pH 7.5 for ValA), NAD+
(200 μM), CoCl2 (50 μM), sedoheptulose 7-phosphate (0, 5, 10, 25,
50, 100, and 200 μM), enzyme (1 μM), 2-amino-6-mercapto-7-
methylpurine ribonucleoside (MESG) (0.2 mM), and purine nucleo-
side phosphorylase (PNP) (0.1 U). Optimal concentrations of the
cofactors (NAD+ and CO2+) were first determined to obtain a
saturated amount that would give Vmax with no inhibition of the
reaction. All components, except the substrate sedoheptulose 7-
phosphate, were incubated at 28 °C for 10 min, and then the reaction
was initiated by addition of the substrate. The reaction was monitored
at 360 nm every 8 s. Kinetic constants were derived from a Hanes-
Woolf plot.
Protein Purification for Enzyme Assay. Cell pellets from 50 mL of
culture were washed with binding (B) buffer (1 mL) (40 mM KPi, 300
mM NaCl, and 10 mM imidazole, pH 7.4) and centrifuged (8,000
rpm, 3 min, 4 °C). After removal of the supernatant, B buffer (1 mL)
was added, and the suspension was sonicated (8 W, 10 s, 5 times).
After centrifugation (14,500 rpm, 20 min, 4 °C), the supernatants (0.8
mL) were each mixed with B buffer-equilibrated Ni-NTA resin
(QIAGEN) (0.2 mL) and incubated for 1 h at 4 °C. After incubation,
the mixtures were centrifuged (4,000 rpm, 3 min, 4 °C), and the
supernatants were discarded. One mL of washing (W) buffer [40 mM
KPi, 300 mM NaCl, and 20 mM imidazole (50 mM imidazole in case
of ValA and Staur_3140), pH 7.4] was added, and the mixture was
centrifuged (4,000 rpm, 3 min, 4 °C). This washing step was repeated
three times. Subsequently, elution (E) buffer (0.5 mL) (40 mM KPi
pH 7.4, 300 mM NaCl, and 500 mM imidazole, pH 7.4) was added
and incubated at 4 °C for 20 min to elute the desired proteins. This
elution step was repeated twice. Eluted proteins were dialyzed against
dialysis (D) buffer (1 L) (10 mM KPi, 0.1 mM DTT, and 1 mM NaF,
pH 7.4) 3 times for 3 h each.
Protein Purification for Kinetic Studies Using Enzchek Phosphate
Assays. Enzyme purification for kinetic determinations using the
Enzchek phosphate assay kit (Invitrogen) was done similar to the
procedure mentioned above except that B2 buffer (40 mM HEPES,
300 mM NaCl, 10% glycerol, and 10 mM imidazole, pH 7.4) was used
for binding, W2 buffer [40 mM HEPES, 300 mM NaCl, 10% glycerol,
and 20 mM imidazole (50 mM imidazole in case of ValA and
Staur_3140), pH 7.4] for washing, E2 buffer (40 mM HEPES, 300
mM NaCl, 10% glycerol, and 500 mM imidazole, pH 7.4) for elution,
and D2 buffer [10 mM Tris-HCl, 0.1 mM DTT, pH 7.0 (pH 7.5 in
case of ValA)] for dialysis. The Ni-NTA purified proteins were
analyzed by SDS-PAGE and concentrated by ultrafiltration using
Amicon YM-10 (Millipore). Protein concentration was determined
using the Bradford assay (BIO-RAD) with BSA as standard.
Enzyme Assays for TLC and GC-MS Analyses. Each reaction
mixture (20 μL, or 40 μL in case of reactions for GS-MS analysis)
contains potassium phosphate buffer (20 mM, pH 7.4), NAD+ (1
mM), CoCl2 (0.2 mM), sedoheptulose 7-phosphate (5 mM), and
enzyme (10−20 μM). The mixture was incubated at 30 °C for a total
of 6 h. For TLC analysis, aliquots were taken at 2 h, 4 h, and 6 h. For
GC-MS analysis, aliquots were taken at 3 h.
GC-MS Sample Preparation for ValA, Amir_2000, and
Staur_3140 Reaction Products. Reaction mixtures (40 μL) were
lyophilized, and Sil-A (Sigma-Aldrich) (∼80 μL) were added to the
lyophilized samples and let stand for 20 min. The solvent was
evaporated with argon gas, and the products were extracted with
hexanes (100 μL) and subjected to GC-MS analysis.
GC-MS Condition. A HP GC-5890 series II and MS-5971 (Hewlett-
Packard) system, HP-5 ms (0.25 μm, Agilent technologies) and
CycloSil-beta (0.25 μm, Agilent technologies) columns were used for
analysis. Temperature gradient was set as 7 °C/min from 70 °C at 2
min until 300 °C for HP-5 ms column and 5 °C/min from 50 °C at 2
min until 240 °C for CycloSil-beta column.
High-Resolution Mass Spectral Analysis of Amir_2000 Product.
HRMS (ESI-TOF) m/z 191.0548 (calcd for C7H11O6 [M − H]−:
191.0556).
ASSOCIATED CONTENT
* Supporting Information
■
S
Proteins and accession numbers used in alignment and
phylogenetic studies (Table S1), TLC analysis of reaction
products (Figure S1), GC/MS analysis of tetrasilylated 2-epi-
valiolone standard and Amir_2000, Staur_3140, and ValA
1
reaction products (Figures S2−S3), H NMR spectrum of 2-
epi-valiolone standard and in situ 1H NMR analyses of
Staur_3140, and Ava_3858 reactions (Figure S4), kinetic data
(Figure S5), 1H and 13C spectra of synthetic compounds
generated in this study (Figures S6−S10). This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Taifo.Mahmud@oregonstate.edu
■
Present Addresses
†Scripps Florida.
¶Department of Chemistry, Western Oregon University.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Heinz G. Floss for his comments and
assistance in the preparation of this manuscript, Rolf Muller for
■
̈
providing Stigmatella aurantiaca DW4/3-1, Kerry McPhail and
Justyna Sikorska for technical assistance. The project described
was supported by grant R01AI061528 from the National
Institute of Allergy and Infectious Diseases and by the Herman-
Frasch Foundation.
REFERENCES
■
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