Biochemical Characterization and Structural Basis of Reactivity and Regioselectivity Differences between Burkholderia thailandensis and Burkholderia glumae 1,6-Didesmethyltoxoflavin N-Methyltransferase

Article abstract of DOI:10.1021/acs.biochem.7b00476

Burkholderia glumae converts the guanine base of guanosine triphosphate into an azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of Burkholderia thailandensis and Burkholderia pseudomallei have a gene cluster encoding seven putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes are similar in sequence to BgToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, BthII1283 in B. thailandensis E264, is a predicted S-adenosylmethionine (SAM)-dependent N-methyltransferase that shows a low level of sequence identity to BgToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike BgToxA, BthII1283 catalyzes a single methyl transfer to N1 of 1,6-DDMT in vitro. In addition, we investigated the differences in reactivity and regioselectivity by determining crystal structures of BthII1283 with bound S-adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. BthII1283 contains a class I methyltransferase fold and three unique extensions used for 1,6-DDMT recognition. The active site structure suggests that 1,6-DDMT is bound in a reduced form. The plane of the azapteridine ring system is orthogonal to its orientation in BgToxA. In BthII1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in BgToxA, it is first directed toward an sp² orbital of N6 and then toward an sp² orbital of N1 after planar rotation of the azapteridine ring system. Furthermore, in BthII1283, N1 is hydrogen bonded to a histidine residue whereas BgToxA does not supply an obvious basic residue for either N6 or N1 methylation.

Full text of DOI:10.1021/acs.biochem.7b00476

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Article
Biochemical characterization and structural basis of reactivity
and regioselectivity differences of Burkholderia thailandensis and
Burkholderia glumae 1,6-didesmethyltoxoflavin N-methyltransferase
Michael K Fenwick, Khaled H. Almabruk, Steven E. Ealick, Tadhg P. Begley, and Benjamin Philmus
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00476 • Publication Date (Web): 30 Jun 2017
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Biochemical characterization and structural basis of reactivity and
regioselectivity differences of Burkholderia thailandensis and Burkholderia
glumae 1,6-didesmethyltoxoflavin N-methyltransferase
§
§
$
∥
Michael K. Fenwick , Khaled H. Almabruk , Steven E. Ealick , Tadhg P. Begley , and
Benjamin Philmus∥,*
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Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, College of
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Pharmacy, Oregon State University, Corvallis, OR 97331, and Department of Chemistry, Texas A&M
University, College Station, Texas 77843
This work is based upon research conducted at the Advanced Photon Source on the Northeastern
Collaborative Access Team beamlines, which are supported by award GM103403 from the NIH.
Use of the Advanced Photon Source is supported by the U.S. Department of Energy, Office of
Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. MacCHESS is supported by
NIH grant GM103485 at the Cornell High Energy Synchrotron Source.
‡
The coordinates of BthII1283 with bound SAH and with bound SAH and 1,6-DDMT have been
deposited in the Protein Data Bank under accession code 5UFN and 5UFM, respectively.
*
To whom correspondence should be addressed:
College of Pharmacy, Oregon State University, Corvallis, OR 97331
E-mail: benjamin.philmus@oregonstate.edu
Keywords: azapteridine, triazine, natural product, secondary metabolite
Running Title: 1,6-didesmethyltoxoflavin N1-methyltransferase
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ABBREVIATIONS
-DMT, 1-desmethyltoxoflavin (reumycin); 1,6-DDMT, 1,6-didesmethyltoxoflavin; 6-DMT, 6-
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desmethyltoxoflavin; D2O, deuterium oxide; DNA, deoxyribose nucleic acid; DTT,
dithiothreitol; ESI, electrospray ionization; GTP, guanosine triphosphate; HEPES, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid; HMBC, heteronuclear multiple bond correlation;
HPLC, high performance liquid chromatography; HRMS, high resolution mass spectrometry;
HsNTMT1, human α-N-terminal methyltransferase 1; HSQC, heteronuclear single quantum
coherence; IPTG, isopropyl β-D-1-thiogalactopyranoside; kU, kilounit; LB, lysogeny broth; Ni-
NTA, Ni-nitrilotriacetic acid; NMR, Nuclear magnetic resonance; OD600, optical density
measured at 600 nm; PCR, polymerase chain reaction; PDB, protein data bank; PEG,
polyethylene glycol; PES, polyethersulfone; RMSD, root mean square deviation; RMSD, root-
mean-squared deviation; rpm, revolutions per minute; SAH, S-adenosylhomocysteine; SAM, S-
adenosylmethionine; SEC-MALS, Size exclusion chromatography with multi-angle light
scattering; TLC, thin layer chromatography; Tris, tris(hydroxymethyl)aminomethane; UV,
ultraviolet; x g, times gravity.
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ABSTRACT. Burkholderia glumae converts the guanine base of guanosine triphosphate into an
azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of
Burkholderia thailandensis and Burkholderia pseudomallei have a gene cluster encoding seven
putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes
show sequence similarity to BgToxBCDE, which have been proposed to make 1,6-
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didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, BthII1283 in B.
thailandensis E264, is a predicted S-adenosylmethionine (SAM)-dependent N-methyltransferase
that shows low sequence identity to BgToxA, which sequentially methylates N6 and N1 of 1,6-
DDMT to form toxoflavin. Here we show that, unlike BgToxA, BthII1283 catalyzes a single
methyl transfer to N1 of 1,6-DDMT in vitro. In addition, we investigated the differences in
reactivity and regioselectivity by determining crystal structures of BthII1283 with bound S-
adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. BthII1283 contains a Class I
methyltransferase fold and three unique extensions used for 1,6-DDMT recognition. The active
site structure suggests that 1,6-DDMT is bound in a reduced form. The plane of the azapteridine
ring system is orthogonal to its orientation in BgToxA. In BthII1283 the modeled SAM methyl
group is directed towards the p-orbital of N1 whereas in BgToxA it is first directed towards an
sp2 orbital of N6 and then towards an sp2 orbital of N1 after planar rotation of the azapteridine
ring system. Furthermore, in BthII1283, N1 is hydrogen bonded to a histidine residue whereas
BgToxA does not supply an obvious basic residue for either N6 or N1 methylation.
INTRODUCTION
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The rice pathogen Burkholderia glumae BGR1 produces toxoflavin via expression
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of the gene cluster BgtoxABCDE.
We recently showed that BgToxA is an S-
adenosylmethionine (SAM)-dependent N-methyltransferase that catalyzes the sequential
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methylation of N6 and N1 of 1,6-didesmethyltoxoflavin (1,6-DDMT, 1) to form toxoflavin (2,
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Figure 1B). Crystal structures of BgToxA complexes show a 140° rotation between 1,6-DDMT
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and toxoflavin that places N6 and N1 at locations consistent with sequential methylation. The
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results support earlier work which proposed that 1,6-DDMT is an intermediate produced by
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BgToxB-E starting from guanosine triphosphate.
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Burkholderia thailandensis,
a growing source of exciting natural products, and its
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close relative Burkholderia pseudomallei, the causative agent of melioidosis in humans,
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not known to produce toxoflavin but some strains contain a gene cluster that has similarity to the
toxoflavin biosynthetic gene cluster. One such gene cluster containing 11 genes is found in B.
thailandensis E264 (BTH_II1275-1285, Figure 1A, Table S1) and encodes seven putative
biosynthetic proteins, BthII1279-1285. BthII1281, BthII1282, BthII1284, and BthII1285 show
sequence similarity and conservation to BgToxE, BgToxD, BgToxC, and BgToxB, respectively,
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Figure 1. Toxoflavin biosynthesis and toxoflavin-like gene cluster in B. thailandensis E264. (A) Diagrams of the
toxoflavin biosynthetic gene cluster found in B. glumae BGR1 (top) and related cluster identified in B. thailandensis
E264 (bottom). The numbers in the B. thailandensis cluster refer to the last two digits of the locus tag (BTH_II12XX)
assigned during genomic sequencing (e.g., 83=BTH_II1283). Homologues in the two clusters are depicted with
identically colored arrows and assigned the gene name used for B. glumae BGR1. Colors denote predicted or
experimentally verified gene functions: red, efflux or resistance; green, regulation; blue, biosynthesis; grey,
transposition; orange and pink, unknown function. (B) Proposed biosynthetic pathway of toxoflavin in B. glumae BGR1
and unknown molecule in B. thailandensis E264. The atom numbering scheme used in the manuscript can be seen
illustrated on 6-DMT (4).ꢀ
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suggesting that B. thailandensis produces 1,6-DDMT as a biosynthetic intermediate en route to a
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cryptic secondary metabolite as proposed for toxoflavin biosynthesis.
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The remaining three putative biosynthetic gene products (BthII1283, BthII1280, and
BthII1279) do not have homologs in the B. glumae cluster and suggest that toxoflavin is not the
produced natural product. BthII1280 is a putative O-methyltransferase (PDB code 3MCZ), and
BthII1279 is a putative member of the vicinal oxygen chelate (VOC) superfamily with a
predicted metal chelating triad consisting of two histidines and one glutamate (Figure S1).
BthII1283 is a putative SAM-dependent N-methyltransferase and is only 16 % identical to
BgToxA, which raises the strong possibility that BthII1283 catalyzes a different transmethylation
reaction.
In the present study, we initiated an investigation into the differences between the
toxoflavin-like gene cluster in B. thailandensis E264 and the toxoflavin gene cluster in B. glumae
BGR1 by examining the in vitro biochemical function of BthII1283 in the presence of 1,6-
DDMT and 1-DMT, and the active site of BthII1283 after soaking BthII1283/S-
adenosylhomocysteine (SAH) crystals with 1,6-DDMT. We find that in contrast to BgToxA,
BthII1283 catalyzes methyl transfer only to N1 of 1,6-DDMT, and our crystal structures provide
a structural basis for the differences in reactivity and regioselectivity.
MATERIALS AND METHODS
General. All chemicals and solvents were purchased either from Sigma Aldrich, Alfa Aesar or
Tokyo Chemical Industry Co., Ltd. (TCI). SAM was obtained as S-adenosyl-L-methionine
disulfate tosylate (Sigma-Aldrich, St. Louis, MO) and used without purification. NdeI, HindIII,
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T4 DNA ligase, Phusion DNA polymerase, and Escherichia coli BL21(DE3)-T1R were
purchased from New England Biolabs (Ipswich, MA) and used per the manufacturer’s
recommendations. E. coli DH5a was purchased from Invitrogen (Carlsbad, CA). Lysogeny
broth, Miller (LB) was purchased from EMD Millipore (Billerica, MA). All synthetic reactions
were performed under an argon or nitrogen atmosphere employing oven-dried glassware when
needed. 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. NMR spectra
were recorded on a Bruker 700 MHz spectrometer with a 5 mm proton/carbon cryo-probe with z-
axis gradient. 1H-NMR chemical shifts (δ) are expressed in ppm downfield from
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tetramethylsilane and are referenced to residual HDO in the D O NMR solvent (δ = 4.79 ppm),
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where s = singlet. H- C HMBC NMR spectra were recorded on a Bruker 700 MHz
spectrometer with complete proton decoupling and lacking the low-pass filter. The lack of a low-
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pass filter allows direct correlations to be observed as large double resonances due to JCH. The
UV absorptions were measured on an Eppendorf BioSpectrometer Kinetic and the cell used was
Eppendorf mCuvette G1.0 (Eppendorf, New Haven, CT). All microbial procedures were
performed using good microbial practices and aseptic technique. Kanamycin was used at 50
µg/mL for selection of plasmids in E. coli.
Creation of expression vector. The DNA fragment corresponding to BTH_II1283
(protein accession number ABC33990.1) was amplified from B. thailandensis E264 genomic
DNA by PCR using Phusion DNA polymerase and the oligonucleotides BTH1283Fwd (5¢-
TGGTGCCGCGCGGCAGCCATATGAGCGCCGCCGAACCGCA -3¢) and BTH1283Rev (5¢-
TCGAGTGCGGCCGCAAGCTTTCATCGGCGTCCACTCGCGTC -3¢) with an annealing
temperature of 58 °C. The restriction sites of NdeI and HindIII are indicated by the underlined
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bases while the italicized bases indicate random bases added to increase restriction enzyme
efficiency. The PCR amplicon was purified using the QIAquick PCR purification kit digestion
with NdeI and HindIII. The restricted fragment was purified using the QIAquick PCR
purification kit followed by ligation into pTHT, a modified pET-28 vector that supplies an N-
terminal fusion tag MGSDKIHHHHHHSSGENLYFQGH, which had been previously digested
with NdeI and HindIII, using T4 ligase. Chemically competent E. coli strain DH5α cells were
transformed with a portion of the ligation mixture and the transformation mixture was plated on
LB agar plates supplemented with kanamycin. Clones were grown overnight in LB media
containing kanamycin and isolated using the QIAprep mini spin kit. The inserted sequence was
verified by Sanger DNA sequencing resulting in plasmid pTBTH1283-2.
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Overexpression, purification and crystallization of BthII1283. E. coli BL21(DE3)-T1R cells
containing pTBTH1283-2 were grown overnight at 37 °C on LB agar plates containing
kanamycin. A single colony of each plate was inoculated into LB medium (5 mL) containing the
above antibiotic and cultured in a shaker at 200 rpm at 37 °C for 8 h. The seed culture (1 mL)
was transferred into LB medium (5 x 100 mL) in 5 x 500 mL Erlenmeyer flasks containing
kanamycin and grown at 37 °C until the OD600 reached 0.4. Then, the temperature was reduced
to 18 °C and after 30 min adaptation, IPTG (0.1 mM final concentration) was added to each
culture. After further growth for 16 h, the cells were harvested by centrifugation (2,200 x g, 20
min, 4 °C) and stored at -80 °C.
The frozen cells from 50 mL of culture were thawed on ice and then washed one time with
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cold sterile ddH O, followed by suspension in 5 mL lysis buffer [Tris-HCl (50 mM, pH 7.4),
NaCl (300 mM), imidazole (10 mM)]. The suspension was sonicated using a Qsonica sonicator
(9 mm probe, 30% amplitude, 15 sec, six times) on ice, then cell debris were removed by
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centrifugation at (19,745 x g, 4 °C for 20 min). The supernatant was applied to a Ni-NTA
agarose resin (0.5 mL, BioRad, Hercules, CA) by batch binding at 4 °C for 30 min followed by
pelleting the resin by centrifugation at 515 x g for 5 min. The supernatant was discarded and the
resin was washed two times with 10 mL washing buffer [Tris-HCl (50 mM, pH 7.4), NaCl (300
mM), imidazole (20 mM)]. BthII1283 was eluted from the resin with elution buffer [Tris-HCl
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(50 mM, pH 7.4), NaCl (300 mM), imidazole (250 mM)]. The protein solution was desalted
using an Econo-Pac 10DG Desalting column (BioRad) equilibrated with protein storage buffer
[Tris-HCl (25 mM, pH 7.4), glycerol (10% w/v)]. The protein concentration was determined
using an Eppendorf BioSpectrometer at (280 nm, 1 mm cell) with an extinction coefficient of
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For crystallization studies, BthII1283 was isolated from cell pellets derived from 2 L of
culture induced at an OD600 of approximately 1.0 and the following modifications. The cells
were suspended in lysis buffer [15 mM Tris, 500 mM NaCl, 0.1 mM SAH, and 2 mM
dithiothreitol (DTT), pH 7.4, supplemented with one protease inhibitor tablet (Invitrogen) and
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.6 kU of benzonase] and sonicated on ice. The cell debris was pelleted by centrifugation at
0,000 x g for 40 min and the supernatant was subjected to nickel chelate chromatography using
wash (20 mM Tris, 500 mM NaCl, 30 mM imidazole, 0.1 mM SAH, 2 mM DTT, pH 7.3) and
elution (20 mM Tris, 500 mM NaCl, 250 mM imidazole, 0.1 mM SAH, 2 mM DTT, pH 7.3)
buffers. The eluted protein was exchanged into wash buffer using an Econo-Pac 10DG desalting
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column (Bio-Rad) and was incubated at 4 °C for 24 h with tobacco etch virus protease (His -
tagged) for cleavage of the hexahistidine tag. The proteolytic reaction mixture was subjected to
subtractive nickel chelate chromatography, and the purified protein was exchanged into 5 mM
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HEPES, 25 mM NaCl, and 1 mM SAH, pH 7.0, concentrated to approximately 5 mg/mL, flash
frozen in liquid nitrogen, and stored at -80 °C.
Crystallization. BthII1283 and SAH were co-crystallized at 22 °C using the hanging drop
vapor diffusion method. Drops were formed by combining protein (approximately 5 mg/mL) and
reservoir solutions in a 1:1 ratio. Typical reservoir solutions consisted of 100 mM HEPES, pH 6–
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7, and 1-2 M (NH ) SO ; the (NH ) SO concentration was increased to 3.2 M prior to cryo-
cooling the crystals with liquid nitrogen. Crystals used for experimental phasing were soaked in
cryo-solutions supplemented with NaBr, with the concentration of NaBr increased gradually to
0.875 M. Crystals with bound 1,6-DDMT were obtained by soaking the crystals in cryo-solutions
supplemented with 25 mM 1,6-DDMT, 6 mM SAH, and 5 mM DTT.
Data Collection and Processing. Crystals of BthII1283 were irradiated with X-rays at
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00 K at beam line NE-CAT 24-ID-C of the Advanced Photon Source (APS) and beam lines A1
and F1 of the Cornell High Energy Synchrotron Source (CHESS). X-ray diffraction experiments
at NE-CAT 24-ID-C used X-rays having a wavelength of λ = 0.979 Å. Diffraction images were
recorded with 1°/s oscillations on a PILATUS 6MF detector positioned 260 mm from the crystal.
Experiments at A1 used X-rays having a wavelength of λ = 0.976 Å. Diffraction images were
recorded for 1° oscillations and 7 s exposures on an ADSC Q210 CCD detector positioned 175
mm from the crystal. Finally, experiments at F1 used X-rays having a wavelength near the Br
absorption edge (λ = 0.918 Å). Diffraction images were recorded for 1° oscillations and 5 s
exposures on an ADSC Q270 CCD detector positioned 250 mm from the crystal. X-ray images
2
2, 23
were processed using XDS or HKL2000.
statistics.
Table 1 gives a summary of the data collection
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Structure Determination and Refinement. Crystallographic phases were determined from
combined molecular replacement and single-wavelength anomalous diffraction phasing using
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4
25, 26
Phaser based on Br sites located using SHELXD.
A truncated form of a methyltransferase
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(
PDB ID 3CGG) was used as a search model. Density modification and auto-building were
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28
performed using PARROT and BUCCANEER, respectively. Iterative automated structure
2
9
refinement with translation, libration, and screw vibrational motion of partitioned chains was
3
0
31
performed using PHENIX and iterative manual model building was performed using COOT.
Table 2 summarizes the structure refinement statistics.
Size exclusion chromatography with multi-angle light scattering (SEC-MALS). The
32-34
oligomeric state of BthII1283/SAH was determined via SEC-MALS.
A frozen aliquot of
BthII1283/SAH prepared for crystallization was thawed and injected into a WTC-050S5 SEC
column connected to a DAWN HELEOS-II 18-angle light scattering detector and an Optilab T-
rEX refractive index detector (Wyatt Technology Corporation). The column was operated at
room temperature with a flow rate of 1 mL/min and was pre-equilibrated with a filtered solution
of 25 mM Tris, 75 mM NaCl, 0.1 mM SAH, and 10 mM DTT, pH 7.5. A 5 mg/mL sample of
monomeric bovine serum albumin (Sigma-Aldrich) was used as a molecular weight standard.
Analysis of chromatograms was performed using ASTRA V software.
Synthesis of 1,6-Didesmethyltoxoflavin (1,6-DDMT) and 1-desmethyltoxoflavin (1-DMT). The
3
5
synthesis of 1-DMT was performed as previously reported. The synthesis of 1,6-DDMT was
performed as previously described with the exception that for the last step 0.3 eq. of triphosgene
36
was used in place of phosgene.
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In vitro enzymatic activity assay. A reaction mixture containing 25 mM Tris-HCl buffer (pH
7
.4), 0.1 mM 1,6-DDMT, 1 mM SAM, 1 mM DTT, and 15 µM BthII1283 in a final volume of
00 µL was mixed gently by pipetting and incubated at 28 °C. After 1 h the protein was removed
1
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by passing the reaction mixture through a VWR centrifugal filter (Modified PES 10K, 500 µL).
All negative control reactions were performed in the same manner by replacing the omitted
reagent with water. A portion of the reaction (50 µL) was then analyzed by HPLC as described
below.
Enzymatic synthesis of toxoflavin. A reaction mixture containing 25 mM Tris-HCl buffer (pH
7
.4), 0.1 mM 1-DMT, 1 mM SAM, 1 mM DTT, and 15 µM BthII1283 in a final volume of 100
µL was mixed gently by pipetting and incubated at 28 °C. After 1 h the protein was removed by
passing the reaction mixture through a VWR centrifugal filter (Modified PES 10K, 500 µL) and
+
then was analyzed by HPLC as described below. HRMS for toxoflavin (ESI): m/z [M+H] calc.
7 8 5 2
for C H N O 194.0673, obs. 194.0669 (2 ppm error).
HPLC analysis of in vitro reaction mixtures. All enzymatic reactions were analyzed on an
Agilent 1100 HPLC instrument consisting of a vacuum degasser, quaternary pump, autosampler
o
o
(
cooled to 4 C), column thermostat (maintained at 30 C), and diode array detector using a
Supelcosil LC-18-DB column (15 cm x 4.6 mm, 5 µm, Supelco, Bellefonte, PA) where line A
was water, line C was 10 mM NH OAc, and line D was methanol at a flow rate of 1 mL/min.
4
The column was pre-equilibrated with 90% A/10% C and held at this composition for 2 min. The
composition was changed to 75% A/10% C/15% D over the next 13 min using a linear gradient.
The composition was then adjusted to 10% A/10% C/80% D over 2 min and this composition
was held for 5 min. The mobile phase was then changed to 90% A/10% C over 3 min and the
composition was held for 6 min prior to the next injection.
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Large scale enzymatic synthesis and partial purification of 6-desmethyltoxoflavin (6-DMT). A
reaction mixture of 50 mL containing potassium phosphate buffer (25 mM, pH 7.4), 0.1 mM 1,6-
DDMT, 1 mM SAM, 1 mM DTT, and 15 µM BthII1283 was mixed and incubated at 28 °C for 1
h then the protein was removed by passing the reaction mixture through GE Healthcare
centrifugal tubes (Vivaspin20, 5 kDa MWCO). The filtrate was concentrated under vacuum to
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.8 mL. The 1.8 mL filtrate was loaded to a PuriFlash 450 system (Interchim Inc., Los Angeles,
CA). Separation was achieved using an Uptisphere Strategy RP 10 µm 150 x 21.2 mm Prep-LC
column at flow rate 10 mL/min where line A was water, line C was 0.1% formic acid in water,
and line D was methanol. The column was pre-equilibrated with 90% A/10% C and upon
injection this composition was held for 2 min. The mobile phase was changed to 75% A/10%
C/15% D over the next 13 min using a linear gradient and then altered to 10% A/10% C/80% D
over 2 minutes. This composition was held for 5 min. Fractions containing the product were
pooled and then concentrated to 500 µL using a miVac concentrator (SP Scientific, Warminster,
1
PA) to give 0.9 mg of partially purified product. H NMR (700 MHz, D
2
O) d 8.77 (s, 1H, H-3),
+
3.89 (s, 3H, N-CH
3
). HRMS (ESI): m/z [M+H] calc. for C
6
H
6
N
5
O
2
180.0516, obs. 180.0512 (2
ppm error).
RESULTS AND DISCUSSIONꢀ
Phylogenetic analysis and in vitro biological activity. Phylogenetic analysis of BthII1283
showed that it closely clusters with other methyltransferases identified from the genomes of
Burkholderia pseudomallei and is related to methyltransferases identified in Xenorhabdus and
Pseudomonas strains, while BgToxA clustered in a different clade and is more closely related to
methyltransferases identified in different Xenorhabdus strains as well as Streptomyces strains
(
Figure S2). BthII1283 and the most related proteins were found in putative clusters with genes
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encoding proteins with similarity to ToxB, ToxC, ToxD and ToxE (Figure S2). This suggests
that BthII1283 utilizes 1,6-DDMT as a substrate for methylation. Due to the 16 % sequence
identity to BgToxA, we hypothesize that BthII1283 has the potential to methylate 1,6-DDMT
with a different regioselectivity.
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Incubation of 1,6-DDMT (1) yields a single product that generates a protonated
molecular ion of 180.0512, which is consistent with the molecular formula C
6 6 5 2
H N O (calc.
1
80.0516, 2 ppm error, Figure S3A,B), demonstrating that a single methyl group is transferred to
1
1,6-DDMT. Large-scale production, isolation and H NMR analysis of the partially purified
product in D O shows two singlets at 3.89 and 8.77 ppm (Figure S4). Further purification results
2
in conversion of the product back to 1,6-DDMT, so characterization was performed on the
partially purified product. The singlet at 3.89 ppm has an HSQC correlation to the carbon
resonance at 43.1 ppm, while the singlet at 8.77 ppm has an HSQC correlation to an aromatic
carbon resonance at 145.7 ppm. HMBC correlations from the aromatic signal show a correlation
to a carbon resonance at 146.7 ppm while the methyl singlet shows a correlation to a carbon
resonance at 152.2 ppm (Figure S5). Comparison of the observed chemical shifts to those
published for toxoflavin suggests that the methyl group was located at N1 to yield 6-DMT (4,
3
7
Figure 1B).
To provide further evidence that the methylation occurs at N1 we incubated 1-DMT with
BthII1283. Reumycin (1-DMT) is converted to a single product with an identical retention time
and UV-vis spectrum to a synthetic standard of toxoflavin and generated a protonated ion of
1
94.0669 (calc. 194.0673, 2 ppm error) demonstrating that the methylation occurred at N1
(
Figure S3C,D).
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38, 39
Overall structure of BthII1283. BthII1283 contains a Class I methyltransferase fold,
an N-terminal extension, and two internal extensions (Figure 2A, B). The N-terminal extension
contains a β-hairpin and an 18-residue α-helix connected by a seven residue loop, which
collectively form a ring-like structure. The first internal extension is located after the fifth β-
strand (β7) of the Class I methyltransferase fold and contains a β-hairpin that forms hydrogen
bonds with the β-hairpin in the N-terminal extension to make a four-stranded half β-barrel. The
second internal extension consists of the top eight residues of the β-hairpin formed by the final
two strands (β10 and β11) of the Class I methyltransferase fold, which protrude outwards. A
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ꢀ
Figure 2. Crystal structure of BthII1283. (A) Ribbon representation of BthII1283. The Class I methyltransferase fold, N-
terminal extension, first internal extension, and second internal extension are colored light blue, salmon, light green, and light
purple, respectively. SAH and 1,6-DDMT are shown as balls and sticks. (B) Structural topology diagram. Helices and β-strands
are shown as cylinders and large arrows, respectively. (C) Crystallographic asymmetric unit. Monomer A is on the left and
monomer B is on the right.ꢀ
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search of the Protein Data Bank shows that the variable regions of BthII1283 are unique but
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resemble those of some other methyltransferases. Of the ones characterized, human α-N-
terminal methyltransferase 1 (HsNTMT1) (PDB 5E1B; ~19 % identical in primary sequence to
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41
BthII1283), which catalyzes the methylation of the α-amino group of Ser1 in the Ran guanine
4
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nucleotide-exchange factor, shows the highest degree of structural similarity (Figure 3A).
A
notable difference is that the N-terminal extension of HsNTMT1 is shorter and lacks the 16-
residue segment in BthII1283 that contains a β-hairpin, which results in an exposed substrate-
binding cavity.
ꢀ
Figure 3. Structural comparison of BthII1283 with other N-methyltransferases. (A) Superimposition of HsNTMT1 onto
BthII1283. (B) Superimposition of BgToxA onto BthII1283. The Class I methyltransferase fold is colored light gray. The N-
terminal extension, first internal extension, and second internal extension of BthII1283 are colored salmon, light green, and light
purple, respectively. The corresponding regions in HsNTMT1 and BgToxA are colored dark red, dark green, and purple,
respectively. SAH bound to BthII1283 is shown as balls and sticks.ꢀ
The variable regions of BthII1283 show a slight resemblance to and occupy similar
locations as the variable regions of BgToxA. However, the N-terminal extension of BgToxA is
shorter and all-helical, the first internal extension of BgToxA is larger and contains a three-
stranded β-sheet, and the second internal extension of BgToxA has a bigger loop that contains
1
4
two short α-helicies (Figure 3B). Furthermore, the two internal extensions of BgToxA form a
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clamshell-like structure with the Class I methyltransferase fold that results in a substrate-binding
cavity with a side entrance, and the N-terminal extension undergoes disorder-order transitions
1
4
that open and close the cavity. In contrast, the N-terminal and internal extensions of BthII1283
form a cap over the Class I methyltransferase fold that encloses the substrate-binding cavity in
both BthII1283/SAH and BthII1283/SAH/1,6-DDMT structures. However, the β-hairpin in the
N-terminal extension lays directly over and interacts with 1,6-DDMT and its displacement would
result in an exposed substrate-binding cavity, similar to the structure of HsNTMT1, which lacks
the N-terminal β-hairpin. In addition, several residues in the N-terminal β-hairpin and the loop
that follows it display high B-factors (Figure 4). These results suggest that the substrate-binding
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ꢀ
Figure 4. B-factors of residues of BthII1283 based on average for main chain atoms. (A) Asymmetric unit of
BthII1283/SAH crystals with monomer A on the left and monomer B on the right. (B) Asymmetric unit of BthII1283/SAH/1,6-
DDMT crystals with monomer A on the left and monomer B on the right. The darkest shade of blue corresponds to the
minimum residue B-factor (10.5 in panel A and 14.4 in panel B), white corresponds to the average residue B-factor (17.8 in
panel A and 23.5 in panel B), and the darkest shade of red corresponds to a residue B-factor five or more standard deviations
above the average (59.9 in panel A and 68.3 in panel B). SAH and 1,6-DDMT are shown as balls and sticks.ꢀ
cavity of BthII1283 has a top entrance and that the N-terminal β-hairpin acts as a lid.
Crystal packing arrangement. The BthII1283 crystals belonged to the space group P6
1
and the asymmetric unit contains two BthII1283 monomers (A and B) that display twofold non-
crystallographic symmetry. The twofold axis runs between the α2 helices located at the protein-
protein interface (Figure 2C). The remainder of the interface is formed from interactions between
α3 and β10. Computer analysis of the interface suggests that this quaternary arrangement is
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disfavored when the ammonium sulfate ions from the structure are omitted. Size exclusion
chromatography with multi-angle light scattering (SEC-MALS) shows that BthII1283 elutes as a
monomer in a solution containing 25 mM Tris, 75 mM NaCl, 0.1 mM SAH, and 10 mM DTT at
pH 7.5 (Figure S6).
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SAH binding site. SAH binds to a conserved SAH/SAM-binding site in the Class I
methyltransferase fold and to the α-helix of the N-terminal extension (Figure 5). The C-terminal
end of the first β-strand of the fold (β
GXGXG (G74AG76RG78), and the C-terminal end of the second β-strand of the fold (β
followed by a conserved acidic residue (Asp95) that forms hydrogen bonds with the ribose
3
) is followed by a conserved nucleotide-binding motif,
4
) is
4
6
hydroxyls of SAH. The adenine moiety of SAH is interposed between the side chain of Cys137
and the side chain of Leu96. N7 and the amino group of the adenine moiety form hydrogen
bonds with the side chain amide of Gln141. N1 forms a hydrogen bond with the backbone NH of
Val120, and N3 forms a hydrogen bond with the backbone amide NH of Leu96. Finally, the
ꢀ
Figure 5. SAH-binding site of BthII1283. Stereoview (left) and schematic (right) representation of interactions of SAH (balls
and sticks) with residues of the Class I methyltransferase fold (light blue) and N-terminal extension (salmon). Dashed lines
represent potential hydrogen bonds. Water molecules are shown as red spheres in the stereo diagram and ‘W’ in the schematic
drawing.ꢀ
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amino group of the homocysteine moiety of SAH forms hydrogen bonds to the backbone
carbonyls of Gly74 and Asn135 and the carboxylate group of SAH makes a salt bridge with the
guanidinium group of Arg79.
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Active site structure. Electron density maps for BthII1283/SAH crystals soaked with 1,6-
DDMT show 1,6-DDMT bound to BthII1283 in the presence but not in the absence of DTT
(
Figure S7). However, different binding modes occur in monomer A and monomer B (Figure 6).
In both monomers, the azapteridine ring system is interposed between the side chain of Trp47
and the side chain of Phe168, with N1 being the closest nitrogen to Sδ of SAH (Sδ …N1, Sδ
…
N2, and Sδ …N8 distances are 3.8/3.7 Å, 4.0/4.2 Å, and 5.1/4.5 Å, respectively). In addition,
N6 forms a hydrogen bond with the carboxylate group of Asp10 (N6…Oδ2 distance is 2.6/2.9
ꢀ
Figure 6. 1,6-DDMT-binding site of BthII1283. Stereoview (left) and schematic (right) representations of interactions of 1,6-
DDMT with residues of the Class I methyltransferase fold (light blue), N-terminal extension (salmon), first internal extension
(light green), and second internal extension (light purple) in (A) monomer A and (B) monomer B of the asymmetric unit. SAH
and 1,6-DDMT are shown as balls and sticks. Dashed lines represent potential hydrogen bonds. Water molecules are shown as
red spheres in the stereo diagrams and ‘W’ in the schematic drawings. The side chain of Trp47 stacks against the azapteridine
ring system of 1,6-DDMT but is omitted for clarity.ꢀ
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Å), N4 forms a hydrogen bond to the hydroxyl group of Tyr219 (N4…OH distance is 3.0/2.8 Å),
and O5 hydrogen bonds to a water molecule and is located near the guanidinium group of
Arg212 (O5…NH2 distance is 3.4/3.5 Å).
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The main difference between the two binding modes occurs on the side of 1,6-DDMT
containing N1, O7, and N8. In monomer A, N1 forms a hydrogen bond with Nε2 of His140
(
(
N1… Nε2 distance is 2.8 Å), O7 forms a hydrogen bond with the hydroxyl group of Tyr185
O7…OH distance is 2.6 Å), and O7 and N8 are within hydrogen bonding distance of the
guanidinium group of Arg183 (O7…NH1 and N8…NH2 distances are 3.0 and 3.1 Å,
respectively) (Figure 6A). In monomer B, N1 is within hydrogen bonding distance of the
backbone carbonyl group of Gly136 (N1…O distance is 3.2 Å) and Nε2 of both His139 and
His140 (N1…Nε2 distances are 3.4 Å). N8 forms a hydrogen bond with Nε2 of His140 (N8…
Nε2 distance is 2.8 Å). O7 is within hydrogen bonding distance of the hydroxyl group of Tyr185
(O7…OH distance is 3.4 Å) and the guanidinium group of Arg183 (O7…NH1 distance is 2.8 Å),
which adopts two conformations (Figure 6B).
Substrate complex model. A model of the enzyme-substrate complex based on the
structure of BthII1283 monomer A shows that the methyl group of SAM is directed towards the
p-orbital of N1 of 1,6-DDMT and that His140 is well-positioned near N1 to act as a base (Figure
7
A). It is unclear if the binding mode of 1,6-DDMT in monomer B is biochemically significant
or is different due to crystallization artifacts or the tautomeric state of ,6-DDMT; the side chain
of Arg183 adopts two conformations (Figure 6B) and the N-terminal β-hairpin shows high B-
factors (Figure 4). Nevertheless, the structure shows that proton transfer between His140 and N8
of 1,6-DDMT is structurally feasible.
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The active site structure of both
BthII1283 monomers is consistent with the
results of the crystal soaking experiments,
which suggest that 1,6-DDMT is bound in a
reduced form (Figure 6). The hydroxyl group
of Tyr219 is 3.0/2.8 Å from N4 of 1,6-
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DDMT and 2.6/2.5 Å from a carboxylate
oxygen atom of Glu50, which suggests that
N4 is likely to be protonated. In monomer A,
N1 is 2.8 Å from Nε2 of His140 and Nε1 of
His140 is 2.9 Å from the carbonyl oxygen
atom of Leu184, which suggests that N1 is
also likely to be protonated. On the other
hand, the proximity of the guanidinium group
of Arg183 to N8 suggests that N8 is likely to
ꢀ
Figure 7. Stereoview representations of enzyme-substrate
complex models. (A) Model of BthII1283 active site
(monomer A) with bound SAM and 1,6-DDMT. (B) Model of
BgToxA active site with bound SAM and 1,6-DDMT (top) or
1
-DMT (bottom; model based on toxoflavin). In both panels,
SAM is modeled based on SAH coordinates. Dashed lines
represent potential hydrogen bonds.ꢀ
be deprotonated. In addition, O7 interacts closely with the guanidinium group of Arg183 and
forms a hydrogen bond with the hydroxyl group of Tyr185, which raises the possibility of
oxyanion stabilization. In monomer B, N8 is 2.8 Å from Nε2 of His140 and Nε1 of His140 is 2.8
Å from the carbonyl oxygen atom of Leu184, which suggests that N8 is likely to be protonated.
In addition, N1 is 3.2 Å from the carbonyl oxygen atom of Gly136, which suggests that N1 is
also likely to be protonated. The binding of the reduced form of 1,6-DDMT in the active site of
BthII1283 is consistent with the fact that DTT was required for full in vitro activity (Figure S8).
Here we propose that 1,6-DDMT (1) is first reduced to compound 3, which binds to enzyme with
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N8 deprotonated. The proton is abstracted from N1 to generate the dianion 7. This dianion then
undergoes methylation at N1 followed by dissociation from the enzyme active site and oxidation,
presumably through a reaction with molecular oxygen to generate 6-DMT (4). These results are
illustrated in the schematic drawings of Figure 6 and in the proposed mechanism (Figure 8).
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Figure 8. Proposed mechanism of methylation of 1,6-DDMT by BthII1283. First 1,6-DDMT is reduced to 3, which binds
to enzyme as the N8 anion. The proton is abstracted from N1 to generate dianion 7, which then undergoes methylation at N1
followed by dissociation from the enzyme active site and oxidation, presumably through a reaction with molecular oxygen to
generate 6-DMT (4).ꢀ
Methylation specificity. Both BgToxA and BthII1283 methylate 1,6-DDMT but result in
methyl groups at distinct sites producing regioisomers. BgToxA methylates N6 to produce 1-
14
DMT (reumycin) while BthII1283 methylates N1 to produce 6-DMT. Molecular models show
that positioning N6 in various proper orientations for methylation results in unfavorable
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interactions and clashes with active site residues of BthII1283 (Figure S9) consistent with the
specificity observed for the in vitro reaction.
BthII1283 and BgToxA both interpose 1,6-DDMT between a tryptophan side chain and a
0
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phenylalanine side chain but bind 1,6-DDMT in different orientations using a different set of
1
4
residues. In BgToxA, the plane of the azapteridine is orthogonal to its orientation in BthII1283
1
4
(Figure 7B). In addition, unlike BthII1283, BgToxA does not supply an obvious basic residue
for the catalysis of either N1 or N6 methylation. We proposed previously that 1,6-DDMT is
bound to BgToxA with N6 deprotonated and that tyrosine and asparagine residues stabilize an
O5 oxyanion. N1 is deprotonated in the likely solution form. Enzyme-substrate complex models
2
for BgToxA show that the methyl group of SAM is directed towards an sp lone pair on N6 and
2
then towards an sp lone pair on N1 after planar rotation of the azapteridine ring system (Figure
1
4
7
B). The binding of deprotonated forms of 1,6-DDMT and 1-DMT enables BgToxA to use a
similar active site geometry for both N6 and N1 methylations. BthII1283 only needs to catalyze a
single methylation and binding of a protonated form of 1,6-DDMT enables BthII1283 to perform
base-assisted methylation of N1.
Biological Significance. Early studies on the toxoflavin biosynthetic gene cluster
proposed that BgToxBCDE synthesize 1,6-DDMT and that BgToxA, the only methyltransferase
1
2
encoded by the gene cluster, methylates 1,6-DDMT twice to form toxoflavin. Our recent study
on BgToxA confirmed the latter prediction, revealed the order of the methyl transfer reactions,
1
4
and provided a structural basis of the dual methyltransferase activity. The high degree of
sequence identity between BthII1285, BthII1284, and BthII1282 of B. thailandensis and
BgToxBCD (as well as the moderate sequence identity between BthII1281 and BgToxE) led us
to investigate whether 1,6-DDMT could also be a potential biosynthetic intermediate in B.
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thailandensis. Our biochemical results show that BthII1283 methylates N1 of 1,6-DDMT and our
crystal structure of BthII1283 with bound 1,6-DDMT shows a high degree of structural
complementarity between the enzyme and substrate. These results strengthen the plausibility that
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,6-DDMT is a bona fide intermediate and hence also suggest that 1,6-DDMT is a branch point
to multiple natural products. The presence of genes encoding proteins with similarity to
ToxBCDE in the genomic regions surrounding the methyltransferases identified by BLAST
4
7
analysis suggests that further investigations will reveal the presence of multiple natural products
derived from GTP with 1,6-DDMT functioning as a key intermediate. However, to date we have
been unable to isolate a natural product associated with this cluster from B. thailandensis E264
despite growth in multiple media under a variety of conditions and comparison of the wild-type
strain to a strain containing a genetic disruption of BTH_II1282 (data not shown).
ACKNOWLEDGEMENTS
We gratefully acknowledge Dr. Kanagalaghatta R. Rajashankar of NE-CAT at the Advanced
Photon Source for assistance with phasing and the lab of Dr. Holger Sondermann at Cornell
University for access to and assistance with a SEC-MALS system. We would also like to
acknowledge Stephen Huhn and Dr. Patrick Reardon from the OSU NMR facility for help in
acquiring NMR spectra. This work was supported with research funds from the Oregon State
University, College of Pharmacy to BP.
SUPPORTING INFORMATION
Additional figures (Figures S1-S9) and data (Table S1) are supplied as Supporting information.
REFERENCES
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