A Single PLP-Dependent Enzyme PctV Catalyzes the Transformation of 3-Dehydroshikimate into 3-Aminobenzoate in the Biosynthesis of Pactamycin

Article abstract of DOI:10.1002/cbic.201300153

Natural amino donation: A PLP-dependent aminotransferase PctV, encoded in the pactamycin biosynthetic gene cluster, was found to catalyze the formation of 3-aminobenzoate from 3-dehydroshikimate with L-glutamate as the amino donor. The PctV reaction comprises a transamination and two dehydration reactions. This is the first report of a simple 3-ABA synthase in nature.

Full text of DOI:10.1002/cbic.201300153

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DOI: 10.1002/cbic.201300153
A Single PLP-Dependent Enzyme PctV Catalyzes the
Transformation of 3-Dehydroshikimate into 3-
Aminobenzoate in the Biosynthesis of Pactamycin
Akane Hirayama,[b] Tadashi Eguchi,*[b] and Fumitaka Kudo*[a]
Scheme 1. Biosynthesis of pactamycin (asterisks indicate incorporation pattern of 13C from [6-13C]glucose).
A variety of different aminobenzoates have been identified as
characteristic building blocks for the construction of structural-
ly unique and biologically important natural products.[1] In this
structural class, 2-aminobenzoate (anthranilate) and 4-amino-
benzoate (p-aminobenzoate, PABA) are well-known com-
pounds that are derived from chorismate, which is a key inter-
mediate in the biosynthesis of the aromatic amino acids l-phe-
nylalanine and l-tyrosine. Although the pathways underlying
the biosynthesis of 2- and 4-aminobenzoates are well under-
stood, the pathway responsible for the formation of simple 3-
aminobenzoate (3-ABA) has not been characterized, even
though 3-amino-5-hydroxybenzoate (AHBA) synthase in rifamy-
cin biosynthesis[2] and enzymes for 3-amino-4-hydroxybenzoate
(3,4-AHBA) synthesis in grixazone biosynthesis[3] have been ex-
tensively investigated. The existence of a pathway for the for-
mation of 3-ABA was suggested by the biosynthesis of pacta-
mycin. Pactamycin is a potent cytotoxic agent produced by
Streptomyces pactum, and consists of a characteristic five-mem-
bered aminocyclitol ring decorated with 3-aminoacetophenone
(3-AAP), 6-methylsaliciate (6-MSA), and N,N-dimethylcarbamoyl
groups (Scheme 1).[4] Feeding of isotope-labeled compounds
such as glucose, acetate, and methionine to a culture of
S. pactum revealed that the 3-AAP moiety of pactamycin could
be derived from 3-dehydroquinate (DHQ) or 3-dehydroshiki-
mate (DHS) in the shikimate pathway, based on the labeling
pattern of pactamycin/pactamycate with [1-13C]glucose, [6-
13C]glucose, and [U-13C]glucose (Scheme 1).[5] Given that the
methyl group (C-8“) of the 3-AAP moiety is derived from the
methyl group of acetate, 3-amino[ring-U-14C]benzoate was fed
to a culture of S. pactum, and the results showed incorporation
of radioactivity into pactamycate.[5b] In addition, a feeding ex-
periment involving the addition of 3-amino-5-fluorobenzoate
to S. pactum culture resulted in the production of 5’’-fluoropac-
tamycin and 5’’-fluoropactamycate.[6]
[a] Prof. Dr. F. Kudo
In contrast, the glycosyltransferase PctL, which is encoded in
the pactamycin biosynthetic gene cluster, catalyzes N-acetyl-
glucosaminyl transfer to 3-AAP to make an N-glycosidic link-
age.[7] It was speculated that the resulting N-glycoside under-
went a cyclization reaction to a cyclopentane ring via an imini-
um intermediate, with the process being mediated by a specific
oxidative enzyme encoded in the pct biosynthetic gene cluster.
The presence of 3-AAP as an intermediate in the pactamycin
biosynthesis, however, was unclear, and thus in the current
Department of Chemistry, Tokyo Institute of Technology
2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551 (Japan)
E-mail: fkudo@chem.titech.ac.jp
[b] A. Hirayama, Prof. Dr. T. Eguchi
Department of Chemistry and Materials Science
Tokyo Institute of Technology
2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551 (Japan)
E-mail: eguchi@cms.titech.ac.jp
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300153.
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study, feeding experiments were performed with deuterium-
labeled 3-AAP as well as 3-ABA.
PctV,[7,9] are encoded in the pactamycin biosynthetic gene clus-
ter, and it was speculated that either of these could be respon-
sible for the hypothesized transamination of DHQ or DHS.[10]
These enzymes were heterologously expressed in Escherichia
coli and subsequently purified to homogeneity (Figure S2). The
aminotransferase activities of PctC and PctV were initially ana-
lyzed by detecting reduction in the signal corresponding to
the pyridoxal 5’-phosphate (PLP)–aldimine complex (400 nm)
and formation of a signal corresponding to pyridoxamine 5’-
phosphate (PMP; 320 nm) with proteinogenic amino acids
(apart from proline) in the presence of PLP. For both enzymes,
l-glutamate was found to be the best substrate to afford PMP
(Figure S3). DHQ and DHS were then added to the above enzy-
matic mixtures, and the resulting products were analyzed by
HPLC. The results revealed that the PctV reaction with DHS
and l-glutamate afforded 3-ABA as a product (Figure 2),
whereas the PctC reactions with DHQ, DHS, and l-glutamate
did not give any detectable product (data not shown). The kcat
and Km values for PctV with DHS were estimated as (0.39Æ
0.04) minÀ1 and (2.1Æ0.4) mm, respectively, at pH 7.5 and 288C
(Figure S4). These results demonstrate that PctV strictly recog-
nizes DHS as a substrate and catalyzes the amino transfer reac-
tion, thereby forming a PMP–DHS Shiff-base intermediate that
subsequently undergoes two dehydration reactions to afford
3-ABA (Scheme 2). It is likely that the first dehydration could
be catalyzed by the enzyme, because electron flow from a
lone pair of a nitrogen atom in the pyridine ring of PLP assists
the dehydration at C-4 of DHS. The second dehydration could
be assisted by deprotonation of the H-6 proton, although it
cannot be excluded that aromatization toward 3-ABA from the
presumed intermediate could occur spontaneously. When DHQ
was used as a substrate for PctV, only a trace amount of 3-ABA
was formed, presumably from a small amount of contaminat-
ing DHS in the DHQ sample. Taken together, these results sug-
gest that PctV strictly recognizes DHS as its substrate and that
it catalyzes the amino transfer and dehydration reactions. The
DHS dehydratase AsbF is known to catalyze the dehydration of
DHS to give 3,4-dihydroxybenzoate in petrobactin biosynthe-
sis.[11] In the AsbF reaction, a manganese ion is required to sta-
bilize an intermediate. In contrast, the PctV reaction did not ex-
hibit any requirement for the addition of a metal ion (data not
shown). This indicates that the dehydration process in the PctV
reaction is not dependent on a metal cation as a Lewis-acid
catalyst.
In previous reports,[8] the nitration reactions of deuterium-
labeled acetophenone and benzoic acid were carried out in
mixtures of H2SO4 and HNO3, to give 3-nitroacetophenone and
3-nitrobenzoate, respectively. These compounds were then
reduced by refluxing in concentrated HCl in the presence of
SnCl2 or by catalytic hydrogenation with Pd/C, to give 3-amino-
acetophenone ([D4]3-AAP) and 3-aminobenzoate ([D4]3-ABA),
respectively (see Figure S1 in the Supporting Information). Sub-
sequent feeding experiments involving the addition of [D4]3-
AAP and [D4]3-ABA to the pactamycin producer S. pactum
NBRC 13433, allowed efficient incorporation of [D4]3-ABA into
1
pactamycate, based on H NMR analysis of the isolated com-
pounds, whereas the analysis revealed that [D4]3-AAP had not
been incorporated (Figure 1). Surprisingly, when [D4]3-ABA was
Figure 1. 1H NMR spectra (500 MHz, CD3OD) of pactamycate isolated from
the culture with A) [D4]3-AAP or B) [D4]3-ABA. Arrows in B) identify signifi-
cant reduction in the four signals derived from four protons of the 3-AAP
moiety in pactamycate.
fed to the culture, only a trace amount of unlabeled pactamy-
cate was produced. These results clearly demonstrate that 3-
ABA is a direct biosynthetic intermediate of pactamycin,
whereas 3-AAP is not. Given that the glycosyltransferase PctL
recognizes 3-AAP as a substrate, it follows that its natural sub-
strate must bear a close resemblance to 3-AAP, for example
the CoA or ACP thioester derivatives of 3-(3-aminophenyl)-3-
oxopropanoic acid, which would be hydrolyzed and decar-
boxylated to give the 3-AAP moiety of pactamycin in the bio-
synthetic pathway.
A similar enzymatic reaction scheme was found in the reac-
tion mechanism of 3-amino-5-hydroxybenzoate (AHBA) syn-
thase, RifK. This enzyme catalyzes the dehydration of 5-amino-
5-deoxy-3-dehydroshikimate (5-aminoDHS).[2a] In the RifK reac-
tion, the amino group of 5-amino-DHS is trapped with PLP as
a PLP–aminoDHS Shiff-base intermediate, with subsequent de-
hydration followed by stereospecific deprotonation of the H-6
proton (or vice versa), thereby leading to AHBA (Scheme 2).
The PctV and RifK reactions are therefore apparently pseudo-
symmetrical, to afford 3-ABA and AHBA, respectively. It is note-
worthy that PctV catalyzes three chemical reactions: a transami-
nation and two dehydration reactions. In contrast, RifK only
catalyzes the dehydration of 5-amino-DHS, although RifK does
To develop an understanding of the missing biosynthetic
link between 3-ABA and the primary metabolites (possibilities
include DHQ and DHS in the shikimate pathway), we initially
attempted to characterize an aminotransferase that specifically
recognized DHQ or DHS as substrate and introduces an amino
group to the six-membered carbocyclic ring. It was envisaged
that subsequent dehydration reactions would convert the ami-
nated intermediate to 3-ABA. Two aminotransferases, PctC and
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catalyze the transamination of UDP-3-ketoglucose to afford
UDP-kanosamine (UDP-3-amino-3-deoxyglucose) during the
early stages of 4-amino-DHAP (4-amino-3,4-dideoxy-d-arabino-
heptulosonate-7-phosphate) biosynthesis.[12]
PctV shows high similarity (~40%) to the glutamate-1-semi-
aldehyde aminomutase (GSAM) family of proteins, which are
known to be inhibited by gabaculine (3-amino-2,3-dihydroben-
zoate). Gabaculine binds covalently to PLP to form a PMP–ga-
baculine aldimine adduct, which functions as a time-depen-
dent inhibitor of GSAM.[13] Because gabaculine is structurally
similar to 3-ABA, PctV would likely be inhibited by gabaculine,
potentially with a time-dependent inhibition profile similar to
that observed against GSAM. It would also be interesting to
determine how PctV distinguishes DHS from DHQ, because the
transamination and triple dehydration of DHQ to afford 3-ABA
could be reduced to a single enzymatic step. Presumably, a par-
ticular spatial structural relationship between the carboxylate
and carbonyl group on the six-membered ring is crucial for
substrate recognition. The high incorporation ratio of [D4]3-
ABA into pactamycate suggests that the 3-ABA-forming re-
action is critical for the biosynthesis of this unique secondary
metabolite; thus PctV should efficiently catalyze not only the
transamination but also the double dehydration at the active
site to afford 3-ABA, although the dehydration reactions could
simply occur spontaneously as described above. Detailed bio-
chemical studies with this unique 3-ABA synthase, PctV, are
currently underway in our laboratory.
PctC shows high similarity (~40%) to members of the DegT/
DnrJ/EryC1/StrS family of aminotransferase, to which RifK (the
AHBA synthase) belongs. It was therefore initially envisaged
that PctC is responsible for the transamination of DHS and
subsequent dehydration reactions. PctC, however, did not cata-
lyze the expected transamination of DHQ or DHS, even though
it showed aminotransfer activity when l-glutamate was used
as an amino donor for PLP. Pactamycin contains four nitrogen
atoms, and two of these atoms should be derived from N-ace-
tylglucosamine and carbamoyl phosphate. In the current study,
we have clearly demonstrated that PctV is responsible for the
introduction of the nitrogen atom of the AAP moiety of pacta-
mycin from l-glutamate. The remaining nitrogen atom of the
five-membered carbocyclic moiety should therefore be intro-
duced by PctC aminotransferase, presumably after carbocycle
formation.
We also found that 3-AAP is not a true biosynthetic inter-
mediate for pactamycin. It is therefore necessary to find the
natural substrate for the glycosyltransferase PctL, to develop
an understanding of the carbocycle formation. Functional anal-
ysis towards identification of candidate enzymes that recog-
nize 3-ABA as a substrate is currently underway in our labora-
tory.
In summary, a unique 3-ABA synthase, PctV, has been identi-
fied as a new member of the family of aminobenzoate-forming
enzymes in natural product assembly lines. A PctV homologue
was found in the genome of Streptomyces acidiscabies 84-104
(79% identity), which contains a very similar pactamycin bio-
synthetic gene cluster. However, no other highly homologous
protein has been found in the current gene database. In
Figure 2. Activity of PctV with DHS and Glu (HPLC analysis, 223.7 nm).
A) PctV (49 mm) with 2.0 mm DHS, 20 mm l-Glu, and 1.2 mm PLP in 50 mm
Tris buffer (pH 7.5) containing 10% glycerol; 15 h reaction. B) Authentic 3-
ABA. C) and D) Spectra for conditions in A) but without PLP and l-Glu, re-
spectively. Inserts show UV spectra for the peaks at 15.8 min (3-ABA).
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Scheme 2. Proposed reaction mechanisms for A) PctV (3-ABA formation) and B) RifK (AHBA formation).
future, the amino acid sequence of PctV should therefore be
[2,4,5,6-D4]3-Nitroacetophenone (464 mg, 2.74 mmol) was placed in
a 30 mL flask with concentrated HCl (3.8 mL), and the resulting sus-
pension was stirred at ambient temperature. SnCl2·2H2O (1.98 g,
8.78 mmol) was then slowly added to the flask in a portion-wise
manner, and the resulting mixture was heated at reflux for 1.5 h.
The reaction mixture was then poured into a small amount of ice/
water, the pH was adjusted to pH 12 with aqueous NaOH (50% w/
w), and the mixture was extracted with CHCl3. The organic layer
was evaporated, and the residue (260 mg) was purified by silica-
gel chromatography (hexane/ethyl acetate 3:1) to give [2,4,5,6-
D4]3-aminoacetophenone (186 mg, 49%). 13C NMR (125 MHz,
CDCl3): d=26.7 (s), 113.7 (t, J=24 Hz), 114.0 (s), 118.5 (t, J=25 Hz),
118.7 (s), 119.3 (t, J=24 Hz), 119.6 (s), 128.9 (t, J=24 Hz), 138.2 (s),
146.7 (s), 198.5 ppm (s).
indicated as a 3-ABA synthase in gene annotation.
Experimental Section
Materials and instruments: All molecular biology, recombinant
DNA manipulation, and enzyme treatments were performed ac-
cording to standard protocols.[14] Restriction enzymes and ligase
were purchased from Takara (Shiga, Japan). DNA oligonucleotide
primers were synthesized by Fasmac (Kanagawa, Japan). Kubota
3700 (Kubota, Tokyo, Japan) and Himac SCR20B (Hitachi, Tokyo,
Japan) centrifuges were used. UV/Vis analysis and absorbance
measurements were recorded on a UV-2450 spectrophotometer
(Shimadzu, Kyoto, Japan). NMR analysis was performed on a DRX-
500 spectrometer (Bruker, Rheinstetten, Germany).
Synthesis of [2,4,5,6-D4]3-aminobenzoic acid (Figure S1):
[2,3,4,5,6-D5]Benzoic acid (502 mg, 3.95 mmol; Sigma–Aldrich,
99 atom% D) was placed in a 30 mL Erlenmeyer flask with concen-
trated H2SO4 (1.5 mL), and the resulting solution was cooled to
À788C. A cooled (À788C) mixture of concentrated HNO3 (0.5 mL)
and concentrated H2SO4 (1.0 mL) was then slowly added to the re-
action in a drop-wise manner from a Komagome pipette. The reac-
tion solution was then stirred manually at 15 min intervals at ambi-
ent temperature to dissolve any solids. After 2 h, the reaction mix-
ture was poured into crushed ice (15 g) in an Erlenmeyer flask, and
the resulting precipitate (466 mg) was collected by suction filtra-
tion before being purified by silica-gel chromatography (CHCl3/
CH3OH 10:1) to give pure [2,4,5,6-D4]3-nitrobenzoic acid (450 mg,
67%).
Synthesis of [2,4,5,6-D4]3-aminoacetophenone (Figure S1):
[2,3,4,5,6-D5]Acetophenone (1.06 g, 8.48 mmol; Sigma–Aldrich.,
99 atom% D) was placed in a 30 mL Erlenmeyer flask with concen-
trated H2SO4 (2.9 mL), and the resulting suspension was cooled to
À788C. A cooled (À788C) mixture of concentrated HNO3 (0.9 mL)
and concentrated H2SO4 (2.0 mL) was then slowly added to the
mixture in a drop-wise manner from a Komagome pipette. The re-
sulting solution was stirred manually at 15 min intervals at ambient
temperature to dissolve any solids. After 1 h, the reaction mixture
was poured into crushed ice (35 g) in an Erlenmeyer flask, and the
resulting precipitate was collected by filtration to give pure
[2,4,5,6-D4]3-nitroacetophenone (470 mg, 33%).
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[2,4,5,6-D4]3-Nitrobenzoic acid (437 mg, 2.55 mmol) was placed in
a 50 mL two-necked flask together with 1,4-dioxane (7 mL) and
Pd/C (10%, 126 mg), and the resulting mixture was agitated under
hydrogen at atmospheric pressure for 5 h. Upon completion of the
reaction (as determined by TLC), the mixture was filtered through
a pad of Celite, and the filtrate was collected and evaporated to
remove solvent. The resulting crude residue (357 mg) was recrystal-
lized from water (7 mL) to obtain pure [2,4,5,6-D4]3-aminobenzoic
acid (246 mg, 68%). 13C NMR (125 MHz, CD3OD): d=115.5 (t, J=
24 Hz), 115.8 (s), 118.5 (t, J=25 Hz), 119.0 (t, J=24 Hz), 128.1 (t, J=
24 Hz), 131.1 (s), 147.6 (s), 169.1 ppm (s).
gated into the corresponding sites of a pET28 vector. The resulting
plasmids (pctC/pET28 and pctV/pET28) were introduced into E. coli
BL21(DE3) for expression. Transformed E. coli (pctC/pET28/BL21
and pctV/pET28/BL21) was cultured in LB medium containing ka-
namycin (30 mgmLÀ1 ), and expression was induced at the mid-log
phase (absorbance at 600 nm=0.5–0.7) with isopropyl b-d-thioga-
lactopyranoside (IPTG, 0.1 mm). Culture growth continued over-
night (158C, 200 rpm agitation), and cells were collected by centri-
fugation (5000g, 48C, 20 min).
The cell pellets were then suspended in 10 times their weight of
buffer A (Tris buffer (50 mm, pH 7.5) containing glycerol (10% w/
w)), and the cells were disrupted by sonication (5ꢁ1 min, inter-
spersed with 1 min periods of cooling on ice). The resulting sus-
pension was centrifuged (10000g, 48C, 20 min), and the super-
natant (cell-free extract) was then loaded onto a TALON affinity
column (Takara) for PctC purification. After washing with buffer A
(20 mL, containing imidazole (20 mm)), PctC was eluted with the
same buffer containing 200 mm imidazole. For PctV purification,
the supernatant of the cell-free extract was loaded onto a His60 Ni
Superflow affinity column (Takara). After washing with of buffer A
(20 mL, containing of imidazole (60 mm)), PctV was eluted with
the same buffer containing 200 mm imidazole. The collected frac-
tions were analyzed by 12.5% SDS-PAGE, and the fractions contain-
ing the pure proteins were passed through a PD-10 column (GE
Healthcare) to remove imidazole (Figure S2). The resulting pure
protein was stored on ice until use. Protein concentrations were
estimated by the Lowry method (absorbance in solution at
750 nm).
Feeding experiments: S. pactum NBRC 13433 was maintained on
a slant of Maltose-Bennett’s agar (yeast extract (0.1%), beef extract
(0.1%), NZ amine (0.2% type A), maltose (1.0%), and agar (2.0%))
at 288C. A scraping from the slant was used to inoculate seed
medium (100 mL; Tryptone/Yeast medium: Tryptone (0.5%) and
yeast extract (0.3%)) in a 500 mL baffled flask equipped with
a cotton plug, and the culture was incubated (24 h, 308C) with
shaking (200 rpm). This preculture (1 mL) was used to inoculate
producing media (100 mL; soluble starch (3%), soya flake A (1.5%),
yeast extract (0.2%), corn steep liquor (0.5%), NaCl (0.3%),
MgSO4·7H2O (0.6%), CoCl2·6H2O (0.0005%), and CaCO3 (0.3%)) in
a 500 mL baffled flask equipped with a cotton plug. The main cul-
ture was incubated (5.5 days, 308C) with shaking (200 rpm).
[2,4,5,6-D4]3-Aminoacetophenone (105 mg) was dissolved in DMSO
(525 mL), and the resulting solution (100 mL) was added to each of
the 100 mL portions of culture (total 500 mL culture, with a final
concentration of 1.4 mm) after 24 h of the main culture. [2,4,5,6-
D4]3-Aminobenzoate (100 mg) was dissolved in DMSO (500 mL),
and 100 mL of the resulting solution was added to each of the
100 mL portions of culture (total 500 mL culture, with a final con-
centration of 1.4 mm) after 24 h of the main culture.
Enzymatic assay for PctV and PctC: To determine the best amino
donor for PctC and PctV, a solution of proteinogenic amino acids
(15 mL, 10 mm; excluding proline), was mixed (total 150 mL) with
PctC (135 mL, 8.3 mm) or PctV (135 mL, 3.3 mm) in the presence of
PLP (60 mm). Absorption (300–500 nm) was monitored by using a
Shimadzu UV-2450 spectrophotometer at 90 s intervals for 15 min
(Figure S3). To observe changes in absorbance more clearly, l-
glutamate (75 mL, 10 mm) was also mixed (total 150 mL) with PctV
(75 mL, 31 mm) in the presence of PLP (300 mm)).
The culture broth was then centrifuged (6000g, 30 min, 48C), and
the supernatant was extracted with ethyl acetate (3ꢁ500 mL). The
combined organic layers were then dried with Na2SO4, and the sol-
vent was removed in vacuo. The residue (204 mg from the 3-AAP
culture; 92 mg from the 3-ABA culture) was then purified by silica-
gel column chromatography (CHCl3/CH3OH, 15:1) with Silica Gel
60N (100–210 mm; Kanto Chemical Co.,Tokyo, Japan) to obtain pac-
tamycate (5.0 mg from the 3-AAP culture; 5.4 mg from the 3-ABA
culture).
To detect the transamination activity of PctV with DHS, PctV
(33.8 mL, 73 mm) was reacted in the presence of PLP (1.2 mm), l-
glutamate (20 mm), and DHS (2 mm) in buffer A at 288C for 15 h
(total 50 mL). The reaction was quenched by addition of methanol,
and the supernatant was then centrifuged (10000g, 30 min) before
being filtered through Millex LG filters (Millipore). The filtrate was
then injected into an Elite LaChrom L-2455 HPLC instrument (DAD
Detector and L-2130 Pump; Merck/Hitachi) equipped with a Luna
PFP(2) column (3mm, 100 ꢂ, 150ꢁ4.6 mm; Phenomenex, Torrance,
CA). The elution conditions were CH3CN (5%) in NaH2PO4 buffer
(0.1m) with a flow rate 0.4 mLminÀ1 at RT.
Cloning and purification of PctV and PctC: Cosmids cpac8 and
cpac8-11,[7] which were obtained from a previous experiment, were
used as templates for PCR amplification of the pctC (primers PctC-
N 5’-CCcata tgCGGT ACGAGC CCTG-3’ and PctC-C 5’-CTCGGG
GGAGCt CCGTAC G-3’) and PctV (primers PctV-N 5’-GAGGTT Ccatat
gGGTGC CAACGA TG-3’ and PctV-C 5’-CGGAtC CGGGCG AGCAGC
TC-3’) genes, respectively (introduced NdeI/SacI and NdeI/BamHI
restriction sites underlined). PCR conditions: 30 cycles of 988C for
10 s, 558C for 5 s, 728C for 90 s; primeSTAR polymerase (0.1 mL;
Takara, Otsu, Japapn), primeSTAR buffer (Mg2+ plus; 2 mL, 5ꢁ;
Takara), dNTPs (0.8 mL, 2.5 mm each), DMSO (0.5 mL (pctC only)), pri-
mers (0.3 mL of 100 mm each), template (1.0 mL), adjusted to 10 mL
with water. The resulting PCR product was subjected to an Ex Taq
polymerase reaction [728C for 3 min; Ex Taq polymerase 0.1 mL,
10ꢁ buffer 0.5 mL, dNTP (0.2 mL, 2.5 mm each), PCR product 4.3 mL)]
to attach 3’ A overhang, and the resulting DNA fragment was ligat-
ed to T-vector pMD19 (Takara) before being introduced to E. coli
DH5a, and was then cultured in lysogeny broth (LB) containing
ampicillin (100 mgmLÀ1). Several clones were cultured, and plasmid
DNA was extracted. After confirming the DNA sequence, the plas-
mid was digested with the appropriate restriction enzymes and li-
To determine PctV substrate specificity for DHS or DHQ, PctV
(29 mL, 17 mm) was reacted (total 50 mL) in the presence of PLP
(0.1 mm), l-glutamate (200 mm), and DHS or DHQ (0.2 mm) in Tris
buffer (10 mm, pH 7.5) at 288C for 24 h.
To investigate PctC activity with DHS and DHQ, PctC (33.8 mL,
27 mm) was reacted (total 50 mL) with a mixture containing PLP
(1.2 mm), l-glutamate (20 mm), and DHS (2 mm) in buffer A at
288C for 18 h. No other conditions investigated (including different
enzyme and substrate concentrations) gave the desired reaction
product.
For steady state kinetics of PctV with DHS, PctV (35 mL, 143 nm)
was reacted (total 50 mL, performed in triplicate) in the presence of
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PLP (0.1 mm), l-glutamate (200 mm), and DHS 1.25, 2.5, 5.0, 7.5,
10.0, or 20.0 mm) in Tris buffer (10 mm, pH 7.5) at 288C for 30 min.
The reaction was quenched by addition of methanol, the mixture
was centrifuged (10000g, 30 min), and the supernatant was evapo-
rated to remove methanol and water. The residue was dissolved in
water (10 mL) and subjected to HPLC analysis (as described above).
The elution conditions were trifluoroacetate (0.1%) and CH3CN
(7.5%) in H2O, at a flow rate 0.5 mLminÀ1 at RT.
Acknowledgements
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We thank Prof. Kazunobu Matsushita at Yamaguchi University in
Japan for providing the DHQ and DHS materials. This work was
supported in part by Grants-in-Aid for Scientific Research on In-
novative Areas from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT).
Keywords: aminobenzoate · biosynthesis · dehydroshikimate ·
natural products · pactamycin
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Received: March 15, 2013
Published online on June 6, 2013
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