Enzymatic production of (-)-indolactam V by LtxB, a cytochrome P450 monooxygenase

Article abstract of DOI:10.1021/np900481a

The P450 cytochrome monooxygenase gene, ltxB, was cloned and overexpressed in Escherichia coli as a 6xHis-tagged protein. The resulting recombinant LtxB was purified by Ni-NTA affinity chromatography and characterized biochemically. Purified LtxB demonstrated typical cytochrome P450 spectroscopic properties including substrate-induced transition from a low-spin (λ_max = 414 nm) to high-spin state (λ_max = 386 nm) upon incubation with N-methyl-L-valyl-L-tryptophanol. The catalytic activity of LtxB was verified by demonstrating the oxidation/cyclization of N-methyl-L-valyl-L- tryptophanol to (-)-indolactam V. LtxB shows a relaxed specificity for analogue substrates in which the valyl group is substituted for other aliphatic groups. The relaxed substrate specificity of LtxB, along with the relaxed specificity of the prenyltransferase, LtxC, allowed for the enzymatic production of a series of (-)-indolactam V and lyngbyatoxin analogues.

Full text of DOI:10.1021/np900481a

J. Nat. Prod. 2010, 73, 71–74
71
Enzymatic Production of (-)-Indolactam V by LtxB, a Cytochrome P450 Monooxygenase
Minh U. Huynh,† Matthew C. Elston,† Nick M. Hernandez,† David B. Ball,† Shin-ichiro Kajiyama,⊥ Kazuhiro Irie,§
William H. Gerwick,‡ and Daniel J. Edwards*,†
Department of Chemistry and Biochemistry, California State UniVersity, Chico, 400 W. 1st Street, Chico, California 95929
ReceiVed August 4, 2009
The P450 cytochrome monooxygenase gene, ltxB, was cloned and overexpressed in Escherichia coli as a 6xHis-tagged
protein. The resulting recombinant LtxB was purified by Ni-NTA affinity chromatography and characterized biochemically.
Purified LtxB demonstrated typical cytochrome P450 spectroscopic properties including substrate-induced transition
from a low-spin (λmax ) 414 nm) to high-spin state (λmax ) 386 nm) upon incubation with N-methyl-L-valyl-L-tryptophanol.
The catalytic activity of LtxB was verified by demonstrating the oxidation/cyclization of N-methyl-L-valyl-L-tryptophanol
to (-)-indolactam V. LtxB shows a relaxed specificity for analogue substrates in which the valyl group is substituted
for other aliphatic groups. The relaxed substrate specificity of LtxB, along with the relaxed specificity of the
prenyltransferase, LtxC, allowed for the enzymatic production of a series of (-)-indolactam V and lyngbyatoxin analogues.
(-)-Indolactam V (ILV)1 (Figure 1) is a potent activator of
various protein kinase C (PKC) isozymes and is the main phar-
macophoric component of the lyngbyatoxin2,3 and teleocidin4-6
natural products. Over the years ILV and analogues have been
extensively studied as synthetic targets for structure-activity
studies.7-11 Of particular importance is the recent identification of
ILV as a specific inducer of human embryonic stem cell differentia-
tion to pancreatic cell types.12 This newly identified activity for
ILV and related compounds has refocused attention on developing
efficient synthetic and/or biosynthetic routes to these compounds.
the recombinant C-terminal His-tagged LtxB protein. LtxB was
purified in a single step by Ni-NTA chromatography to yield 3
mg/L of culture volume of purified protein. Purity was assessed as
being greater than 90% by SDS-PAGE (Supporting Information).
LtxB showed the characteristic orange-red color of heme-containing
proteins and demonstrated a Soret band with λmax ) 414 nm. Upon
reduction of the heme Fe3+ to Fe2+ with dithionite, LtxB was able
to bind CO, generating a new Soret band with a λmax ) 444 nm,
characteristic of CO-bound ferrous heme (Supporting Information).
Small amounts of the P420 form of the enzyme were also observed
and varied slightly from batch to batch.
Since the isolation of the lyngbyatoxin biosynthetic gene
cluster,13 we have been investigating the biosynthetic enzymes
involved in the generation of lyngbyatoxin and its biosynthetic
precursor, ILV. The lyngbyatoxin biosynthetic gene cluster was
isolated from a Hawaiian strain of Lyngyba majuscula and
comprises three genes.13 The first gene, ltxA, encodes for a
nonribosomal peptide synthetase (NRPS) that condenses N-methyl-
L-Val and L-Trp and releases N-methyl-L-valyl-L-tryptophanol
(NMVT) via an NADPH-dependent reductive cleavage.14 The
second gene product, LtxB, is a cytochrome P450 monooxygenase.
Although this activity is consistent with the oxidation and cycliza-
tion of NMVT to ILV, biochemical verification of this activity has
yet to be reported. The last gene in the lyngbyatoxin pathway, ltxC,
encodes for a prenyltransferase that has been shown to attach a
geranyl group at C-7 of ILV to generate a quaternary carbon
center.13
Additional spectroscopic studies with LtxB verified that LtxB
underwent the expected transition from a low-spin (λmax ) 414 nm)
to high-spin state (λmax ) 386 nm) upon incubation with the NMVT
substrate. To determine the specificity of the NMVT-LtxB interac-
tion, we performed active site titration of LtxB with NMVT,
allowing the calculation of the dissociation constant as Kd ) 0.50
( 0.04 µM (Supporting Information). NMVT analogues substituted
in the valyl position were also evaluated for their ability to bind to
LtxB. Dipeptides with conservative substitutions at Val bound with
similar affinity to LtxB (Supporting Information). Other possible
substrates including L-tryptophanol, L-Trp, and N-methyl-L-phenyl-
Gly-L-Trp-ol were also tested for their ability to induce a spin-
state transition with LtxB, but did not induce spectroscopic shifts
at concentrations up to 200 µM. Because the binding of substrate
preceded electron transfer in the reaction cycle of cytochrome
P450s,15 the type I substrate binding behavior of LtxB toward
NMVT and close structural analogues was consistent with a
catalytically active cytochrome P450.
To explore the catalytic activity of LtxB, we tested the enzymatic
conversion of NMVT to ILV. In the assay we used NADPH as an
electron source and spinach ferredoxin and spinach ferredoxin
reductase as electron transfer agents. Incubation of LtxB with
NMVT led to the clear production of ILV, as demonstrated by
HPLC coelution and ESIMS [M + H]+ signals identical to an
authentic ILV standard (Figure 2B). The production of ILV was
absolutely dependent on the presence of LtxB, NADPH, spinach
ferredoxin, and spinach ferredoxin reductase (Figure 2B-E). A
control reaction with boiled LtxB also showed no activity (data
not shown). In addition, we tested the ability of LtxB to convert
alternative substrates to cyclized ILV analogues. Various N-methyl-
L-X-L-Trp-ol substrates were tested with LtxB, and small amounts
of cyclized products could be detected by HPLC analysis with UV-
diode array detection followed by ESIMS analysis. N-Methyl-L-
norvalyl-L-Trp-ol (Figure 2G) and N-methyl-L-X-L-Trp-ol (X )
Leu, Ile, norleucine) all gave cyclized product (see Table 1 for a
Here, we report on the function of LtxB as an N-arylation catalyst
in the generation of the nine-membered ring of ILV (Figure 1).
We also explored the substrate permissiveness of LtxB versus a
series of NMVT analogues with substitutions in the valyl position.
The relaxed substrate specificity of LtxB allowed for the production
of ILV analogues. Subsequently all ILV analogues produced by
LtxB were found to act as substrates for LtxC, leading to the
production of a series of new lyngbyatoxin analogues.
To experimentally verify the role of LtxB, we cloned the entire
ltxB gene into an E. coli overexpression plasmid and overproduced
* Author for correspondence. Tel: (530) 898-5226. Fax: (530) 898-5234.
E-mail:djedwards@csuchico.edu.
† California State University, Chico.
⊥ School of Biology-Oriented Science and Technology, Kinki University,
930 Nishimitani, Kinokawa, Wakayama, 649-6493, Japan.
§ Division of Food Science and Biotechnology, Graduate School of
Agriculture, Kyoto University, Kyoto 606-8502, Japan.
‡ Scripps Institution of Oceanography and Skaggs School of Pharmacy
and Pharmaceutical Sciences, University of California at San Diego, 9500
Gilman Dr., La Jolla, CA 92093.
10.1021/np900481a  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/10/2009

72 Journal of Natural Products, 2010, Vol. 73, No. 1
Notes
Figure 1. (A) Last two biosynthetic steps in the production of lyngbyatoxin. (B) Production of ILV and lyngbyatoxin analogues from
synthetic N-Me-L-X-L-Trp-ol compounds (norvaline analogue, R ) CH2CH2CH3; Leu analogue, R ) CH2CH(CH3)2; Ile analogue, R )
CH(CH3)CH2CH3; norleucine analogue, R ) CH2CH2CH2CH3; phenylglycine analogue, R ) phenyl group).
Table 1. Relative Reaction Yields for LtxB and LtxB/LtxC
Combination Reactions with N-Me-L-X-L-Trp-ol Substratesa
Figure 2. HPLC chromatograms demonstrating the enzymatic
activity of LtxB: (A) (-)-indolactam V chemical standard, (B)
enzymatic reaction of LtxB with NMVT, (C) control reaction
without LtxB, (D) control reaction without NADPH, (E) control
reaction without spinach ferredoxin reductase, (F) control reaction
without spinach ferredoxin, (G) enzymatic reaction of LtxB with
N-methyl-L-norvalyl-L-Trp-ol.
a The relative yield was defined as the ratio of the conversion of a
substrate to that of N-Me-L-Val-L-Trp-ol.
by the varying R groups on the NMVT analogues, and verification
by ESIMS supported the gross structures of these new products.
Successful generation of lyngbyatoxin and lyngbyatoxin analogues
was absolutely dependent on the presence of both LtxB and LtxC
(data not shown). Reactions in which the valine residue of NMVT
was substituted with leucine, isoleucine, norvaline, and norleucine
all gave similar or only slightly lower yields of lyngbyatoxin
analogues as compared to NMVT (Figure 3, Table 1). However,
the overall inefficient turnover of our in vitro P450 reaction system
did not allow us to perform detailed structural characterization or
biological testing of either the ILV or lyngbyatoxin analogue
reaction products.
In this report we confirm LtxB as the enzyme catalyzing
N-arylation of NMVT to form ILV, thus verifying its role in
lyngbyatoxin biosynthesis. We also show that both LtxB and LtxC
exhibit relaxed substrate specificity, allowing for the production
of several ILV and lyngbyatoxin analogues from synthetic dipeptide
comparison of the relative yields). No cyclized product could be
detected with N-methyl-L-phenylglycyl-L-Trp-ol.
We next tested the ability of an LtxB/LtxC coupled enzyme
system to convert NMVT and NMVT analogues to lyngbyatoxin-
like compounds (Figure 1B). When NMVT was incubated with
LtxB and LtxC, the production of lyngbyatoxin could be clearly
demonstrated by HPLC (Figure 3B). In the single enzyme system
LtxB showed conversion of various N-methyl-L-X-L-Trp-ol sub-
strates to the corresponding products; however, it was not clear if
the corresponding cyclized products would also act as substrates
for LtxC. HPLC analysis of reaction products in the LtxB/LtxC
enzyme system showed that all N-methyl-L-X-L-trp-ol compounds
that acted as substrates for LtxB were prenylated to the corre-
sponding lyngbyatoxin analogue by LtxC (Figure 3C-F). Reversed-
phase HPLC gave chromatographic peaks with retention times that
corresponded well with the expected structural changes imposed

Notes
Journal of Natural Products, 2010, Vol. 73, No. 1 73
and 5′-CCG CTC GAG CCA CTC AGC AGG TAA CT-3′ with the
Nde I and Xho I cloning sites underlined, respectively. The resulting
product was cloned into the NdeI-XhoI sites of pET20a. The ensuing
construct pET20a-LtxB was transformed into E. coli BL21(DE3). The
BL21(DE3) cells containing pET20a-LtxB were grown until an OD
0.6, at which time the growth temperature was lowered to 25 °C and
δ-aminolevulinic acid (0.5 mM) was added to the culture broth to
increase the production of the heme-containing LtxB. LtxB was
produced as a recombinant His6-tagged protein and purified by Ni-
NTA affinity chromatography using His-select resin according to the
manufacturer’s directions to yield an orange-red protein.
Spectroscopic Characterization of LtxB and Substrate Binding
Studies. The absorption spectra of purified LtxB with and without
substrate were obtained on a Hewlett-Packard 8453 spectrophotometer.
A reduced CO spectrum of LtxB was obtained by placing LtxB (5
µM) in a quartz cuvette (100 µL) with dithionite (5 mM) and bubbling
CO gas through the sample.19
The interaction of potential LtxB substrates was examined by
perturbation of the heme Soret band. LtxB (1 µM) in 50 mM Tris-HCl
(2.0 mL, pH 8.0) was placed in a 2 mL cuvette. After thermal
equilibration at 25 °C, a baseline was established between 350 and
450 nm. Next, sequential additions of a concentrated solution (1-3
µL) of N-methyl-L-X-L-Trp-ol dissolved in EtOH were added to the
sample cuvette to give a final ligand concentration in the range 0.2-20
mM. To a reference cuvette was added an equal volume of 100% EtOH
to account for any effect the solvent might have on the absorption
spectra. The LtxB spectrum without substrate was subtracted from each
spectrum with added substrate. From the resulting difference spectra
the ∆A386-418 values were calculated for each concentration of peptide.
The Kd values were estimated by fitting the ∆A386-418 versus [N-methyl-
L-X-L-Trp-ol] to a saturation curve in GraphPad.
Enzymatic Activity of LtxB. The production of ILV from NMVT
was carried out in a 500 µL reaction: LtxB (1 µM), N-methyl-L-X-L-
Trp-ol (0.5 mM), spinach ferredoxin (3.5 µM), spinach ferredoxin
reductase (0.1 units/mL), and NADPH (1 mM), in Tris-HCl buffer (50
mM, pH 8.0). For the production of lyngbyatoxin and its analogues
from N-methyl-L-X-L-Trp-ol, geranyl pyrophosphate (90 µM) and LtxC
(0.6 µM) were also added to the reaction mixture. Reactions were
incubated at 30 °C for 4 h. Reaction products were isolated by extraction
with EtOAc (3 × 500 µL). The EtOAc extractions were combined,
evaporated to dryness with a Savant SpeedVac system, and resuspended
in 50% MeOH/H2O solution. The products were analyzed by analytical
HPLC on a Hewlett-Packard Series 1100 instrument with a UV diode
array detector. Separation of (-)-indolactam products was carried out
on an Adsorbosphere Phenyl column (Alltech, 150 × 4.6 mm, 5 µm
pore size) with 40% MeOH in H2O with a flow rate of 0.9 mL/min
over 15 min. Separation of lyngbyatoxin products was done with a
50% MeOH/H2O to 100% MeOH gradient over 15 min with a flow
rate of 0.9 mL/min. Lyngbyatoxin and lyngbyatoxin analogue products
were detected by their absorbance at 280 nm. Product peaks were
collected and their masses determined by direct infusion in positive
ion mode on a ThermoFinnigan LCQ Advantage mass spectrometer.
Figure 3. Reversed-phase HPLC analysis of LtxB/LtxC chemoen-
zymatic reactions (see Experimental Section for complete descrip-
tion of reaction mixture): (A) lyngbyatoxin standard, (B) reaction
with N-methyl-L-Val-L-Trp-ol, (C) reaction with N-methyl-L-
norvalyl-L-Trp-ol, (D) reaction with N-methyl-L-norleucyl-L-Trp-
ol, (E) reaction with N-methyl-L-Leu-L-Trp-ol, (F) reaction with
N-methyl-L-Ile-L-Trp-ol.
compounds. The ILV analogues proposed to be produced in this
study have already been reported from microbial conversion
experiments in StreptoVerticillium blastmyceticum NA34-17.16
Although detailed chemical and biological testing of the reaction
products could not be performed due to the inefficient turnover of
LtxB, this study demonstrates proof-of-concept that the LtxB/LtxC
coupled system can in principle be used to generate new lyngb-
yatoxin analogues. We are currently exploring several refinements
of our system, such as construction of an LtxB/cytochrome P450
reductase protein fusion system as described for RhFRED.17,18 An
LtxB/RhFRED reductase fusion system may improve the conversion
efficiency, as well as eliminate the need for expensive spinach
ferredoxin/ferredoxin reductase. In addition, an NADPH generation
system could be adapted to lyngbyatoxin chemoenzymatic synthesis
to provide an efficient and lower cost reducing agent for the
cytochrome P450 reaction. These improvements may lead to more
efficient chemoenzymatic routes to the ILV and lyngbyatoxin class
of compounds.
Acknowledgment. This work was supported by a Cottrell College
Science Award of Research and by a CSUPERB SEED grant. We
would also like to acknowledge D. Clark at CSU Chico for assistance
with the ESIMS analysis of the lyngbyatoxin analogues.
Supporting Information Available: SDS PAGE gel of LtxB
purification, absorption spectra, and active site titration plots of LtxB
are available free of charge via the Internet at http://pubs.acs.org.
Experimental Section
References and Notes
Reagents and Chemicals. All N-methyl-L-X-L-Trp-ol substrates
were synthesized according to the literature procedure.16 δ-Aminole-
vulinic acid, spinach ferredoxin, spinach ferredoxin reductase, NADPH,
and His-Select chromatography resin were obtained from Sigma Life
Sciences (St. Louis, MO). Geranyl pyrophosphate was obtained from
Echelon Biosciences Incorporated (Salt Lake City, UT). Authentic (-)-
indolactam V was obtained from Axxora, LLC (San Diego, CA).
Authentic lyngbyatoxin A was isolated from Lyngbya majuscula
collected from Kahala Beach, Oahu, HI. Lyngbyatoxin A was purified
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NP900481A