Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli

Article abstract of DOI:10.1021/acs.biochem.7b01205

The methylation of unactivated carbon and phosphorus centers is a burgeoning area of biological chemistry, especially given that such reactions constitute key steps in the biosynthesis of numerous enzyme cofactors, antibiotics, and other natural products of clinical value. These kinetically challenging reactions are catalyzed exclusively by enzymes in the radical S-adenosylmethionine (SAM) superfamily and have been grouped into four classes (A-D). Class B radical SAM (RS) methylases require a cobalamin cofactor in addition to the [4Fe-4S] cluster that is characteristic of RS enzymes. However, their poor solubility upon overexpression and their generally poor turnover has hampered detailed in vitro studies of these enzymes. It has been suggested that improper folding, possibly caused by insufficient cobalamin during their overproduction in Escherichia coli, leads to formation of inclusion bodies. Herein, we report our efforts to improve the overproduction of class B RS methylases in a soluble form by engineering a strain of E. coli to take in more cobalamin. We cloned five genes (btuC, btuE, btuD, btuF, and btuB) that encode proteins that are responsible for cobalamin uptake and transport in E. coli and co-expressed these genes with those that encode TsrM, Fom3, PhpK, and ThnK, four class B RS methylases that suffer from poor solubility during overproduction. This strategy markedly enhances the uptake of cobalamin into the cytoplasm and improves the solubility of the target enzymes significantly.

Full text of DOI:10.1021/acs.biochem.7b01205

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Article
Enhanced Solubilization of Class B Radical S-adenosylmethionine
Methylases by Improved Cobalamin Uptake in Escherichia coli
Nicholas Lanz, Anthony J. Blaszczyk, Erin McCarthy, Bo
Wang, Roy Wang, Brianne Jones, and Squire J. Booker
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01205 • Publication Date (Web): 03 Jan 2018
Downloaded from http://pubs.acs.org on January 3, 2018
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Enhanced Solubilization of Class B Radical S-adenosylmethionine Methylases by Improved
Cobalamin Uptake in Escherichia coli
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, , #
, #
Nicholas D. Lanz, † § Anthony J. Blaszczyk, † Erin L. McCarthy, † Bo Wang, ‡ Roy X. Wang, ‡
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, , , *
Brianne S. Jones, ‡ and Squire J. Booker† ‡ ┴
Departments of †Biochemistry and Molecular Biology and of ‡Chemistry, and the ┴Howard
Hughes Medical Institute, The Pennsylvania State University, University Park, PA 16802
§Present address. Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and
Research, U.S. Food and Drug Administration, Silver Spring, MD, 20993
#These two individuals contributed equally
Correspondence and requests for materials should be addressed to S. J. B. (squire@psu.edu)
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ABSTRACT
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The methylation of unactivated carbon and phosphorus centers is a burgeoning area of biological
chemistry, especially given that such reactions constitute key steps in the biosynthesis of numerous
enzyme cofactors, antibiotics, and other natural products of clinical value. These kinetically challenging
reactions are catalyzed exclusively by enzymes in the radical Sꢀadenosylmethionine (SAM) superfamily
and have been grouped into four classes: A, B, C, and D. Class B radical SAM (RS) methylases require a
cobalamin cofactor in addition to the [4Feꢀ4S] cluster that is characteristic of RS enzymes. However, their
poor solubility upon overexpression and their generally poor turnover has hampered detailed in vitro
studies of these enzymes. It has been suggested that improper folding, possibly caused by insufficient
cobalamin during their overproduction in Escherichia coli, leads to formation of inclusion bodies. Herein,
we report our efforts to improve the overproduction of class B RS methylases in a soluble form by
engineering a strain of E. coli to take in more cobalamin. We cloned five genes (btuC, btuE, btuD, btuF,
and btuB) that encode proteins that are responsible for cobalamin uptake and transport in E. coli and
coexpressed these genes with those that encode TsrM, Fom3, PhpK, and ThnK, four class B RS
methylases that suffer from poor solubility during overproduction. This strategy markedly enhances
cobalamin uptake into the cytoplasm and improves the solubility of the target enzymes significantly.
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INTRODUCTION
Radical Sꢀadenosylmethionine (SAM) methylases constitute one of the largest subfamilies of the
radical SAM (RS) superfamily.1ꢀ3 These enzymes methylate unactivated carbon and phosphorus centers,
many of which are found in a growing number of natural products that display antibiotic or antineoplastic
activity, as well as enzyme cofactors and other cellular metabolites, such as bacteriochlorophyll (enzyme
cofactor),4 pactamycin (antitumor agent and antibiotic),5 mitomycin C (antitumor agent),6 moenomycin A
(phosphoglycolipid antibiotic),7 fosfomycin (broadꢀspectrum antibiotic),8ꢀ11 thienamycin (βꢀlactam
antibiotic),12 gentamicin (aminoglycoside antibiotic),13 clorobiocin (aminocoumarin antibiotic),14
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fortimicin (aminoglycoside antibiotic),15,
thiostrepton (thiopeptide antibiotic)17 polytheonamide
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(secondary metabolite),18, 19 quinomycin (peptide antibiotic),20 and chondrochloren (antibiotic)21 (Figure
1). They have been organized into four classes based on cofactor requirement, architecture, and
mechanism of action.2, 3 The best studied of the RS methylases are RlmN and Cfr, which use a cysteine
dyad to methylate C2 and C8 of adenosine 2503 in ribosomal RNA or C2 of adenosine 37 in specific
transfer RNAs (tRNAs).22ꢀ27 Class B RS methylases require a cobalamin cofactor, and methylate both sp2ꢀ
and sp3ꢀhybridized carbon centers as well as phosphinate phosphorus atoms. Class C enzymes uniquely
methylate, methenylate, or cyclopropanate sp2ꢀhydridized carbon centers that are typically found in
complex natural products.28ꢀ30 Class D RS methylases are the least characterized. Unlike class A, B, and C
enzymes, which transfer a C1 unit from SAM, class D enzymes employ an alternative source of the C1
unit, hypothesized to be a derivative of methylenetetrahydrofolate. A fifth class of RS methylases has also
recently been suggested. These class E enzymes appear to methylate a sulfido ion in a metallocluster
intermediate during the maturation of the M cluster of nitrogenase.31
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Figure 1. Various natural products that contain methyl moieties installed by cobalaminꢀdependent RS
methylases. Compounds with structures shown represent products of class B RS methylases that have
been studied in vitro. One arrow indicates the actual product from an in vitro reaction, while two arrows
indicate the final natural product whose biosynthesis involves a class B RS methylation step.
Class B enzymes are the most abundant RS methylases and appear to be the most versatile. Moreover,
their importance in the biosynthesis of numerous natural products of clinical value has led to efforts to
isolate and characterize them and their mechanisms of catalysis. However, they have been resistant to
robust mechanistic analysis because of poor solubility upon overproduction and/or low activity.2 Three of
the first class B RS methylases to be the subject of in vitro reports—PhpK from Kitasatospora
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phosalacinea, GenK from Micromonospora echinospora, and Fom3 from Streptomyces wedmorensis—
were all purified from inclusion bodies and then refolded and reconstituted with iron, sulfide, and
methylcobalamin (MeCbl) to generate their ironꢀsulfur (FeꢀS) and cobalamin cofactors. Characterization
of the enzymes by UVꢀvis and electron paramagnetic resonance (EPR) spectroscopies, when conducted,
showed them to exhibit limited cofactor incorporation, and their activities were poor if at all existent. A
fourth class B methylase, TsrM from Streptomyces laurentii, was the first to be isolated from the soluble
fraction of E. coli crude extract.32 Although yields of pure protein were not reported, the enzyme was of
modest quality as judged by its activity and UVꢀvisible spectrum. In subsequent studies of the enzyme, a
construct encoding TsrM fused to the protein SUMO was used for overproduction in E. coli cultured in a
specialized medium containing ethanolamine as the sole source of nitrogen and carbon, conditions that
have been suggested to enhance cobalamin uptake in E. coli.33 Using this strategy, low milligram
quantities of homogeneous enzyme could be purified from 32 L of E. coli culture, allowing for
spectroscopic studies of the enzyme to be conducted.34 A similar strategy proved successful for purifying
soluble ThnK35—a class B RS methylase involved in the biosynthesis of thienomycin—and PoyC, a class
B RS methylase involved in the biosynthesis of polytheonamide.18, 36
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In this work, we extend our efforts to isolate soluble class B RS methylases in larger yields by coꢀ
expressing RS methylase genes along with btuC, btuE, btuD, btuF, and btuB from E. coli, which, except
for btuE, encode proteins that are involved in cobalamin uptake and trafficking. These genes were cloned
into a pBAD42 vector under the control of an arabinoseꢀinducible PBAD promoter, and the resulting
pBAD42ꢀBtuCEDFB vector was used to transform E. coli containing pDB1282—which harbors genes
involved in FeꢀS cluster biosynthesis—and a pETꢀbased vector containing the target RS methylase. Upon
expression of the btu genes, a substantial increase in the intracellular cobalamin concentration—
particularly that of hydroxocobalamin (OHCbl) and adenosylcobalamin (AdoCbl) (Figure 2)—is
observed. Furthermore, coexpression of either the tsrM, fom3, thnK, or phpK genes with the btu genes
leads to a marked increase in soluble purified proteins.
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Figure 2. Structures of (a) hydroxocobalamin, (b) adenosylcobalamin, (c) methylcobalamin, and (d)
cobinamide.
Materials and Methods
Materials
DNA primers were obtained from IDT DNA (Coralville, IA). Enzymes and other reagents for cloning
and nucleic acid manipulation were obtained from New England Biolabs (Ipswich, MA). OHCbl, MeCbl,
and AdoCbl were purchased from SigmaꢀAldrich (St. Louis, MO). Ferric chloride was obtained from
EMD Biosciences (Gibbstown, NJ). Bradford reagent for protein concentration determination as well as
the bovine serum albumin (BSA) standard was purchased from Pierce, Thermo Fisher Scientific
(Rockford, IL). Dithiothreitol (DTT),
Lꢀ(+)ꢀarabinose, isopropyl βꢀDꢀ1ꢀthiogalactopyranoside (IPTG),
ampicillin, spectinomycin, and kanamycin were purchased from Gold Biotechnology (St. Louis, MO).
SUMO express protease was purchased from Lucigen (Middleton, WI). All other materials and reagents
have been previously reported,34, 37 or were of the highest available quality.
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General Methods. The polymerase chain reaction (PCR) was conducted using either a Stratagene
Robocycler Gradient 40 or a BioRad S1000 thermocycler. DNA isolation kits were purchased from
MachereyꢀNagel (Bethlehem, PA). The pRham NꢀHis SUMOꢀTsrM (pRhamꢀSUMOꢀTsrM) construct
was created using the Expresso Rhamnose SUMO cloning system (Lucigen; Middleton, WI). The P1vir,
∆btuR BW25113 strain, pBAD42, and pCP20 were purchased from the Yale Coli Genetic Stock Center. A
Cary 50 spectrometer from Varian was used to record UVꢀvisible spectra using the WinUV software
package to control the system. All anaerobic experiments were conducted in a Coy (Grass Lakes, MI)
anaerobic chamber.
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Cloning of the btuCEDFB Operon. The genes encoding the cobalamin transport system of E. coli
were cloned into a pBAD42 plasmid under the control of the PBAD promoter using semiꢀnested restriction
sites to allow for the genes to be inserted sequentially. Three of the five genes, btuC, btuE, and btuD, are
located in an operon in the E. coli genome. The remaining genes, btuF and btuB, are found in unrelated
operons. The btuCED operon was amplified from E. coli K12 genomic DNA using the PCR. The
forward primer, 5′ꢀCGC GGC GTC GAA TTC ATG CTG ACA CTT GCC CGC CAA CAA CAG CGꢀ
3′, contained a nine baseꢀpair GC clamp, an EcoRI restriction site (underlined), and the first 29 base pairs
of btuC. The reverse primer, 5′ꢀ CGC GGC GTC TCT AGA CGG TAG GAC CTC GAG TCA GAT
GGT CGA AAT CAG CAT TCT GTG ACCꢀ3′, contained a nine baseꢀpair GC clamp, an XbaI restriction
site (underlined), an arbitrary nine baseꢀpair sequence, an XhoI restriction site (bolded), and the last 30
baseꢀpairs of btuD. The PCR product was digested with EcoRI and XbaI and ligated into the pBAD42
vector digested with the same enzymes.
The btuF gene was amplified using a forward primer, 5′ꢀCGC GGC GTC CTC GAG ATG GCT
AAG TCA CTG TTC AGG GCG CTG GTCꢀ3′, containing a nine baseꢀpair GC clamp, an XhoI
restriction site (underlined), and the first 30 baseꢀpairs of btuF. The reverse primer, 5′ꢀCGC GGC GTC
TCT AGA CGG TAG GAC GGT ACC TCA ATC TAC CTG TGA AAG CGC ATT ACA GAGꢀ3′,
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contained a nine baseꢀpair GC clamp, an XbaI restriction site (underlined), an arbitrary nine baseꢀpair
sequence, a KpnI restriction site (bolded), and the last 30 baseꢀpairs of the btuF gene. The PCR product
was digested with XhoI and XbaI and ligated into the pBADꢀbtuCED construct after it was digested with
the same enzymes.
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The btuB gene was amplified using a forward primer, 5′ꢀCGC GGC GTC GGT ACC ATG ATT AAA
AAA GCT TCG CTG CTG ACG GCG ꢀ3′, containing a nine baseꢀpair GC clamp, a KpnI restriction site
(underlined), and the first 30 baseꢀpairs of btuB. The reverse primer, 5′ꢀ CGC GGC GTC TCT AGA
TTA GAA GGT GTA GCT GCC AGA CAA GGT G ꢀ3′, contained a nine baseꢀpair GC clamp, an XbaI
restriction site (underlined), and the last 28 baseꢀpairs of the btuB gene. The PCR product was digested
with KpnI and XbaI and ligated into the pBADꢀbtuCEDꢀbtuF construct after it was digested with the
same enzymes.
Spacing the Genes for Expression as an Operon. The resulting pBADꢀbtuCEDꢀbtuFꢀbtuB construct
was modified by sequential siteꢀdirected mutagenesis to remove the interstitial XhoI and KpnI restriction
sites using the primer, 5′ꢀGGT CAC AGA ATG CTG ATT TCG ACC ATC TGA TG GCT AAG TCA
CTG TTC AGG GCG CTG GTCꢀ3′, and its reverse complement to remove the XhoI site, and the primer,
5′ꢀC TGT AAT GCG CTT TCA CAG GTA GAT TGA TG ATT AAA AAA GCT TCG CTG CTG ACG
GCGꢀ3′, and its reverse complement to remove the KpnI site. The stop codon of the 5′ gene and the start
codon of the 3′ gene overlap by one baseꢀpair in both cases (underlined) for optimal operon expression.38,
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Optimizing Ribosome Binding Sites. To increase the translation efficiency of the encoded genes, the
associated ribosome binding sites (rbs) were optimized by siteꢀdirected mutagenesis. Two ribosome
binding sites are present within the construct, one encoded by the pBAD42 vector 5′ of the btuC gene and
a second in the >60 baseꢀpair intergenic region between btuC and btuE. Both rbs were converted to 5′ꢀ
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TAAGGAGGT–X5– ATGꢀ3′, where the final three bases are the start codon of the following gene. The
resulting plasmid was designated pBAD42ꢀBtuCEDFB.
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Overexpression of the btuCEDFB Operon. The btuCEDFB operon was expressed in the BL21 (DE3)
strain of E. coli in M9ꢀethanolamine medium, M9ꢀminimal medium, or LB medium, each of which was
supplemented with 2.5 µM hydroxocobalamin. The M9ꢀethanolamine media utilizes ethanolamine as the
sole carbon and nitrogen source and, contrary to M9ꢀminimal medium, is supplemented with OHCbl,
trace metals, and all 20 amino acids, but lacks glucose and ammonium chloride. Cultures were incubated
at 37 °C until they reached an OD600 of 0.3, at which point gene expression was induced with 0.2% (w/v)
arabinose. The cultures were incubated at 37 °C for an additional 4 h and harvested by centrifugation. To
test the ability of btuCEDFB to increase cobalamin uptake, growth studies were also conducted with an
empty pBAD42 vector under the same conditions described above.
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Cloning of the phpK and fom3 Genes. The phpK gene from Kitasatospora phosalacinea and the fom3
gene from Streptomyces fradiae were codonꢀoptimized for expression in E. coli and synthesized by
GeneArt (ThermoFisher, Carlsbad, CA). The genes were delivered in a pMKꢀRQꢀBb vector between a 5′
BsaI restriction site and a 3′ XhoI restriction site. The genes were excised from the pNMKꢀRQꢀBb vector
with BsaI and XhoI and then subcloned into a pSUMO vector digested with the same enzymes. The
resulting constructs encode PhpK or Fom3 fused to the Cꢀterminus of SUMO, which contains an Nꢀ
terminal hexahistidine tag. The correct DNA sequences were verified and designated pSUMOꢀPhpK and
pSUMOꢀFom3.
Analysis of Operon Overexpression. Harvested cells were introduced into an anaerobic chamber,
resuspended in buffer containing 50 mM HEPES, pH 7.5, 300 mM KCl, 10% glycerol, and 1 mM βꢀ
mercaptoethanol, and lysed by sonication. The lysate was centrifuged at 25,000 × g for 1 h at 4 °C. All
subsequent studies were conducted in dim light in an anaerobic chamber to avoid photolysis of
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cobalamin. The supernatant was treated with an equal volume of 200 mM H2SO4 to precipitate the protein
component and release the cobalamin cofactor. The samples were centrifuged again for 15 min at 18,000
× g before analysis by liquid chromatography with tandem mass spectrometry (LCꢀMS/MS).
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LCꢀMS/MS Analysis of Cobalamin Content. LCꢀMS/MS analysis of the cobalamin forms in E. coli
was performed using an Agilent 3470 QQQ MS attached to an Agilent 1290 Infinity II LC system.
Samples were separated on an Agilent Technologies Zorbax ExtendꢀC18 RRHD column (2.1 mm × 50
mm, 1.8 ꢁm particle size) equilibrated in 95% solvent A (0.1% formic acid) and 5% solvent B
(acetonitrile). An isocratic gradient was maintained for 0.4 min, which was followed by a gradient of 5–
20% solvent B from 0.4 to 0.8 min. Another gradient from 20ꢀ42% solvent B from 0.8 to 1.6 min was
applied before returning to 5% solvent B from 1.6 to 1.9 min. A flow rate of 0.35 mL/min was maintained
throughout the chromatographic procedure. The column was allowed to reꢀequilibrate for 0.5 min under
initial conditions before subsequent sample injections. OHCbl elutes at approximately 1.16 min under
these conditions, while AdoCbl and MeCbl elute at 1.51 and 1.71 min, respectively.
OHCbl, MeCbl, and AdoCbl were detected by MS/MS multipleꢀreaction monitoring (MRM) using
Agilent Jet Spray electrosprayꢀionization in positive mode (AJSESI+). OHCbl was detected in the +2
chargeꢀstate at an m/z of 664.9, which corresponds to the loss of a water molecule. Transitions of
664.9ꢀ635.8 and 664.9ꢀ147.0 were used to quantify hydroxocobalamin. Transitions of 673.0ꢀ665.6
and 673.0ꢀ147.0 for MeCbl, and 790.6ꢀ665.6 and 790.6ꢀ147.0 for AdoCbl were used to quantify
those molecules. The AJSESI+ source was equilibrated with a nitrogen gas temperature of 300 °C and
flow rate of 5.0 L/min, a nebulizer pressure of 45 PSI, and a capillary voltage of 3500 V. The optimal
fragmentor voltages for OHCbl, MeCbl, and AdoCbl were determined to be 120 V, 104 V, and 108 V,
respectively.
Transition
(m/z)
Collision Energy
(V)
664.9ꢀ635.8
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Hydroxocobalamin
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664.9ꢀ147.0
673.0ꢀ665.6
673.0ꢀ147.0
790.6ꢀ665.6
790.6ꢀ147.0
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Methylcobalamin
Adenosylcobalamin
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Standards (~10 µM) of OHCbl, MeCbl, and AdoCbl were prepared in water, and their concentrations
were determined by boiling them in 100 mM potassium cyanide and measuring the resulting absorbance
at 367 nm (ɛ367 = 30,800 Mꢀ1 cmꢀ1).40 A standard curve for analysis by LC–MS/MS contained OHCbl,
MeCbl, and AdoCbl at concentrations of 31 nM – 64 µM in twoꢀfold increments, and 100 µM tyrosine,
which was used as the internal standard (IS) for quantitative analysis.
Overexpression and Purification of Class B RS Methylases in the Presence of the btuCEDFB Operon.
Genes encoding TsrM and Fom3 were expressed as previously described,34 with the following two
amendments: the BL21 (DE3) strain of E. coli was used rather than the RosettaBlue (DE3) pLysS strain,
and the pBAD42ꢀBtuCEDFB plasmid, which confers resistance to spectinomycin, was maintained in the
strain by supplementation of the media with 50 µg/mL spectinomycin. Purification of the SUMOꢀtagged
enzyme was performed as previously described.34, 41 Briefly, harvested cells were resuspended and lysed
inside an anaerobic chamber. Target proteins were purified by an initial immobilized metal affinity
chromatography (IMAC) step using NiꢀNTA, followed by cleavage of the SUMO tag using the Ulp1
protease overnight on ice. The cleavage reaction was reapplied to a NiꢀNTA column to remove the
hexahistidineꢀtagged Ulp1 protease, the SUMO tag, and any nonꢀspecifically bound contaminating
proteins. The flowꢀthrough containing the native protein was collected and analyzed for purity by SDSꢀ
PAGE. The concentration was determined by the Bradford assay using bovine serum albumin, fraction V
(BSA) as a standard, as previously described.42
Insertion of Gly Linker into pSUMOꢀPhpK. A codon for glycine was inserted between the gene
encoding the SUMO tag and the phpK gene using siteꢀdirected mutagenesis to allow for efficient cleavage
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of the resulting fusion protein by Ulp1. The forward primer, 5′ꢀCGC GAA CAG ATT GGA GGT GGA
ATG AAA CAT TGCꢀ3′, and its reverse complement were used to insert the glycine linker codon
(underlined) into the pSUMOꢀPhpK construct.
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Overproduction and Purification of PhpK. E. coli BL21 (DE3) was transformed sequentially with the
plasmids pSUMOꢀGlyꢀPhpK, pDB1282, and pBAD42ꢀBtuCEDFB. A starter culture was inoculated from
a single colony and incubated for 15 h at 37 °C while shaking at 250 rpm. Overexpression of the phpK
gene was conducted at 37 °C in 16 L of autoꢀinduction media supplemented with OHCbl (1.3 µM).43
Each 6 L flask of culture media was inoculated with 1 mL of starter culture. At an OD600 of ~0.3, 50 µM
FeCl3 and 150 µM cysteine were added to the media and expression of the genes encoded on pDB1282
and pBAD42ꢀBtuCEDFB was induced with 0.2% (w/v) of ꢀarabinose. Once cultures reached an OD600
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of ~1.0, flasks were placed on ice for 20 min. Cultures were then incubated at 18 °C for 16 h before the
cells were collected by centrifugation at 7,000 × g for 12 min. Purification of PhpK was performed as
described above for TsrM and Fom3.
Overexpression and Purification of ThnK. E. coli BL21 (DE3) cells containing pDB1282 were
transformed with the plasmid pET24bꢀThnK (gift from the lab of Dr. Craig Townsend, Johns Hopkins
University) and pBAD42ꢀBtuCEDFB. Overexpression of the thnK gene was conducted in ethanolamine
media, as described above. Purification of ThnK was performed as described above. However, because it
was not overproduced as a fusion protein with SUMO, it was not treated with the SUMO protease.
Rather, ThnK was purified further by sizeꢀexclusion chromatography using an ÅkTA FPLC system fitted
with an S200 column and housed in an anaerobic chamber.
Determination of Protein Concentration. The concentrations of Fom3 and PhpK were determined by
the Bradford assay using BSA as a standard. An amino acid analysis was conducted to determine
correction factors for Fom3 and PhpK, as has previously been described.44 It was determined that the
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Bradford assay overestimates the concentration of Fom3 and Phpk by factors of 1.18 and 1.17,
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Determination of Stoichiometry of Bound Cobalamin. The concentration of proteinꢀbound cobalamin
was determined using a previously described procedure.40 Each protein sample was boiled in 0.1 M
potassium cyanide for 5 min to convert all forms of cobalamin to dicyanocobalamin. The concentration of
dicyanocobalamin was calculated using the known extinction coefficient at 367 nm (ɛ367 = 30,800 Mꢀ1 cmꢀ
1).
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ꢀbtuR BL21(DE3) Strain Construction. The E. coli BW25113 strain with a kanamycin cassette
replacing the btuR gene (ꢀbtuR:kan) was obtained from the Keio collection. P1 transduction was
performed using established methods to move the genetic marker from the BW25113 strain to E. coli
BL21 (DE3).45 Briefly, the host strain (BW25113 ꢀbtuR:kan) was cultured in LB media overnight at 37
°C with shaking at 250 rpm for ~14 h. The cells (1 mL) were then used to inoculate 200 mL of LB media
supplemented with 0.2% glucose and 5 mM CaCl2. 1 mL of a 1:100 dilution of P1vir, obtained from the
Yale Coli Genetic Stock Center, was added to the culture (100 mL) and incubated for 4 h at 37 °C with
shaking at 250 rpm. The OD600 was monitored every 30 min until the culture cleared, as evidenced by
visible cell debris and a significant drop in the optical density. The culture was then treated with 5% (v/v)
chloroform, vortexed, and centrifuged for 30 min at 3,500 × g at 4 °C. The supernatant was removed,
carefully avoiding cell debris. The new lysate was stored at 4 °C supplemented with 5% (v/v) chloroform.
To perform the transduction, the recipient strain, E. coli BL21(DE3), was first cultured at 37 °C with
shaking at 250 rpm overnight. The following day, 200 µL of culture was aliquoted into four
microcentrifuge tubes and centrifuged at 14,000 × g for 5 min. The supernatant was removed and the cells
were resuspended in 100 µL λꢀCa++ buffer (10 mM TrisꢀHCl, pH 7.9, 20 mM MgSO4, and 5 mM CaCl2).
Varying amounts (0 µL, 10 µL, 50 µL and 100 µL) of P1vir lysate were added to each of the tubes, which
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were then incubated for 30 min at 37 °C without shaking. LB media (1 mL) was added to each of the
tubes and supplemented with 100 mM sodium citrate. The samples were then incubated for an additional
hour at 37 °C and then plated on LB + Kan (25 µg/mL) agar with an additional 100 µL of 1 M citrate
spread on top of the plate. The plate was incubated at 37 °C overnight. The resulting colonies were
purified by reꢀstreaking, and then tested for sensitivity to P1 phage. The T7 tester phage from Novagen
was used to confirm the presence of the DE3 lysogen in the strain according to the manufacturer’s
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instructions.
Following transduction, chemically competent ꢀbtuR:kan BL21(DE3) cells were
transformed with the pCP20 plasmid—which encodes a temperatureꢀsensitive origin of replication and
the FLP recombinase—in order to excise the kanamycin cassette.46 A culture was inoculated with a single
colony and incubated overnight at 43 °C and 180 rpm to induce FLP recombinase expression and select
for loss of pCP20. A 106 dilution of the culture was plated to select for individual colonies and incubated
at 30 °C overnight to prevent partial loss of plasmids from colonies containing cells that did not lose the
pCP20 plasmid. The following day, individual colonies were patched on three different plates in the
following order: LB + kanamycin (25 µg/mL), LB + ampicillin (100 µg/mL), and LB to select for
colonies that had lost the kanamycin cassette as well as the pCP20 plasmid, yielding the ꢀbtuR
BL21(DE3) strain.
Cloning of the tsrM Gene into the pRhamꢀSUMO Vector. The tsrM gene from Streptomyces laurentii
was amplified from a plasmid encoding TsrM by the PCR using the following forward and reverse
primers, respectively: 5′ꢀ CGC GAA CAG ATT GGA GGT ATG CTC CGT AAA GGC ACC GTC GCꢀ
3′ and 5′ꢀ GTG GCG GCC GCT CTA TTA CCG GAC GGC CTC CGC GAG CTCꢀ3′. Following
purification by gelꢀelectrophoresis, Ecloni 10G chemically competent cells (Lucigen) were coꢀ
transformed with the preꢀprocessed pRhamꢀSUMO vector and the purified PCR product. The sequence
of the resulting tsrMꢀSUMO construct was verified by DNA sequencing at the Penn State University
Nucleic Acid Facility. Chemically competent ꢀbtuR BL21 (DE3) cells were coꢀtransformed with
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pDB1282 (AmpR) encoding FeꢀS cluster biosynthesis machinery, pBAD42ꢀBtuCEDFB (SpecR), encoding
cobalamin uptake machinery, and the pRham NꢀHis SUMOꢀtsrM construct (KanR). A list of all the strains
used in this study can be found in Table S1.
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Overexpression of the tsrM Gene in the ꢁbtuR BL21 Strain of E. coli. The ꢂbtuR BL21 strain of E.
coli was coꢀtransformed with pRham NꢀHis SUMOꢀtsrM together with the pDB1282 and pBAD42ꢀ
BtuCEDFB plasmids. Bacterial cultures were incubated at 37 °C in 16 L of LB media supplemented with
1.3 µM OHCbl and allowed to shake at 180 rpm. At an OD600 of ~0.3, expression of the genes encoded on
the pDB1282 and pBAD42ꢀBtuCEDFB plasmids was induced with 0.2% (w/v)
cultures were supplemented with 25 µM FeCl3 and 75 µM cysteine. At an OD600 of ~0.6, the cultures
were placed on ice for 20 min prior to induction of expression with 0.2% (w/v) ꢀrhamnose, and were
Lꢀarabinose, and the
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supplemented with an additional 25 µM FeCl3 and 75 µM cysteine. Cultures were incubated for 16 h at 20
°C prior to harvesting cells by centrifugation at 7,000 × g for 12 min.
TsrM and Fom3 Activity Determinations. All reactions were conducted under anaerobic conditions
and contained the following in a final volume of 170 µL: 20ꢀ50 mM HEPES, pH 7.5, 10ꢀ100 mM KCl,
5% glycerol, 1ꢀ2 mM SAM, 1ꢀ2 mM substrate, 100 µM tyrosine, enzyme, and a reducing agent. In
reactions containing TsrM (1 µM), the flavodoxin reducing system (25 µM flavodoxin, 10 µM flavodoxin
reductase, and 1 mM NADPH) was used, while in reactions containing Fom3 (9 µM), methyl viologen (1
mM) and NADPH (2 mM) were used as reductant. All reactions were quenched by addition of H2SO4 to a
final concentration of 100ꢀ200 mM. Standard curves for product quantification by LCꢀMS/MS contained
100 µM tyrosine and varying concentrations of substrate (HEPꢀCMP or Trp), SAH, SAM, and product (2ꢀ
methyltryptophan or HPPꢀCMP).
Quantification of Product Formation by LCꢀMS/MS. Quantification of 5'ꢀdA, SAH, and MeTrp was
performed using methods similar to those previously described.34, 41 Quenched reaction mixtures were
separated on an Agilent Zorbax ExtendedꢀC18 RRHD column (2.1 mm × 50 mm, 1.8 µm particle size).
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In the Fom3 reaction, HPPꢀCMP was separated using a Zorbax SBꢀAq RRHD column (2.1 mm × 50 mm,
1.8 µm particle size) equilibrated in 99% solvent A (40 mM ammonium acetate, pH 6.0, 5% methanol)
and 1% solvent B (methanol). A gradient of 1ꢀ5% solvent B was applied from 0.4 to 0.7 min, followed by
a gradient from 5ꢀ60% solvent B from 0.7 to 1.4 min, which was followed by a gradient from 60%ꢀ1%
solvent B from 1.4 to 2.1 min. The column was allowed to reꢀequilibrate for 0.5 min under initial
conditions before subsequent sample injections. HPPꢀCMP was detected in the +1 charge state using
AJSESI+ with MRM, and the 446.1 ꢀ 112 and 446.1 ꢀ 94.9 transitions were used to quantify the
molecule.
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Synthesis of HEPꢀCMP.
Scheme 1. Steps involved in the synthesis of HEPꢀCMP.
o
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Reactions and conditions: a) ((2ꢀbromoethoxy)methyl)benzene, 170 C, 2 h; b) 6 M HCl, 110 C,
overnight; c) cytidine monophosphate, CDI, TEA, DMF, then 3, DMF, rt, 2 d.
step a:
The neat mixture of triethylphosphite (1.7 g, 10.2 mmol, 1 equiv) and ((2ꢀbromoethoxy)methyl)benzene
(2.2 g, 10.2 mmol, 1 equiv) was heated at 170 oC for 2 h. After cooling to room temperature, the reaction
mixture was purified by silica gel flash chromatography (hexanes : ethyl acetate = 1:1) to give 2.0 g of
compound 2 as a colorless syrup in 71% yield. 1H NMR (500 MHz, CDCl3) δ 7.38 – 7.27 (m, 5H), 4.53
(s, 2H), 4.14 – 4.04 (m, 4H), 3.76 (dt, J = 12.0, 7.4 Hz, 2H), 2.16 (dt, J = 18.6, 7.4 Hz, 2H), 1.31 (t, J =
13
7.1 Hz, 6H) (Figure S1); C NMR (126 MHz, CDCl3) δ = 137.88, 128.42, 127.74, 73.03, 64.21, 61.68,
61.63, 27.63, 26.52, 16.42, 16.37 (Figure S2); 31P NMR (202 MHz, CDCl3) δ = 28.62 (Figure S3).
step b:
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Compound 2 (1.0 g, 3.7 mmol, 1 equiv) was suspended in HCl (6 M, 30 mL), and the resulting mixture
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was refluxed at 110 C overnight. After cooling to room temperature, the reaction mixture was washed
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with diethyl ether (30 mL), and the aqueous phase was dried in vacuo. The resulting residue was reꢀ
suspended in water (10 mL), and the pH was adjusted to 8 with triethylamine. The resulting solution was
lyophilized to give 725 mg of compound 3 as white wax in 87% yield. Compound 3 was pure enough for
further reaction, and no further purification was performed. 1H NMR (500 MHz, D2O) δ 3.78 – 3.66 (m,
2H), 3.09 (q, J = 7.3 Hz, 5H), 1.95 – 1.81 (m, 2H), 1.17 (t, J = 7.3 Hz, 8H) (Figure S4); 13C NMR (126
MHz, D2O) δ = 56.84, 46.61, 31.36, 30.33, 8.19 (Figure S5); 31P NMR (202 MHz, D2O) δ = 23.39
(Figure S6).
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step c:
To a stirred suspension of cytidine 5’ꢀmonophosphate (CMP, 161.6 mg, 0.5 mmol, 1 equiv) in the coꢀ
solvent of DMF (10 mL) and triethylamine (TEA, 10 mL) was added carbonyldiimidazole (CDI, 243.2
mg, 1.5 mmol, 3 equiv) in one portion, and the resulting mixture was stirred at room temperature for 2 h.
Another portion of CDI (81.1 mg, 0.5 mmol, 1 equiv) was added, and the reaction mixture was stirred
until it became clear. Although clarity is an indication of reaction completeness, it was nonetheless
analyzed by thin layer chromatography (TLC) using iPrOH:NH4OH:H2O = 6:3:1 as solvent. Additional
portions of CDI were added if CMP was not completely consumed. After completion, the solvent was
removed under nitrogen flow, and the resulting syrup was reꢀdissolved in methanol and water (1:1)
containing 5% triethylamine. The solvent was dried in vacuo completely, and the resulting gum was reꢀ
suspended in DMF (10 mL) followed by addition of compound 3 (62.0 mg, 0.5 mmol, 1 equiv). The
resulting reaction mixture was stirred vigorously at room temperature for 2 days. After completion, the
reaction was quenched with water (30 mL), and the resulting mixture was extracted with EtOAc (20 mL)
ten times to remove the DMF from the aqueous phase. The aqueous phase containing the product was
purified over a MacroꢀPrep DEAE column with a linear elution of aqueous ammonium bicarbonate
solution (from 10 mM to 300 mM). Fractions containing product were combined and concentrated in
vacuo. Repeated evaporation on a rotary evaporator in a water bath at 50 oC was performed to remove the
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residual ammonium bicarbonate to give 110.0 mg of HEPꢀCMP as a white powder in 51% yield. 1H NMR
(500 MHz, D2O) δ 7.99 (d, J = 7.7 Hz, 1H), 6.14 (br s, 1H), 5.90 (d, J = 3.8 Hz, 1H), 4.31 – 4.23 (m, 2H),
4.23 – 4.16 (m, 2H), 4.14 – 4.08 (m, 1H), 3.76 (q, J = 16.1, 8.5 Hz, 2H), 2.08 – 1.96 (m, 2H) (Figure S7);
13C NMR (126 MHz, D2O) δ = 163.41, 154.06, 142.45, 96.17, 89.40, 82.90, 82.83, 74.25, 69.12, 64.33,
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64.30, 57.07, 32.07, 31.01; 31P NMR (202 MHz, D2O) δ = 14.39, ꢀ11.34 (Figure S8); P NMR (202
MHz, CDCl3) δ = 28.62 (Figure S9).
Synthesis of HPPꢀCMP.
Scheme 2. Steps involved in the synthesis of HPPꢀCMP.
o
Reactions and conditions: a) chloroacetone, NaI, acetonitrile, rt, overnight; b) NaBH4, EtOH, 0 C, 1 h;
then 6 M HCl, 110 oC, overnight; c) cytidine monophosphate, CDI, TEA, DMF, then 6, DMF, rt, 2 d.
step a:
To a stirred solution of triethylphosphite (1.7 g, 10.2 mmol, 1 equiv) and sodium iodide (152.9 mg, 1.0
mmol, 0.1 equiv) in acetonitrile (100 mL) was added chloroacetone (4.1 mL, 51.0 mmol, 5 equiv), and the
resulting brown reaction mixture was stirred at room temperature overnight. After quenching by addition
of water (100 mL), the reaction mixture was extracted with EtOAc (200 mL), and the organic phase was
washed thoroughly with saturated sodium thiosulfate, water and brine. The organic phase was
concentrated in vacuo, and the resulting residue was purified by silica gel flash chromatography (hexanes
: ethyl acetate = 1:1) to give 1.0 g of compound 5 as a yellowish syrup in 51% yield. 1H NMR (500 MHz,
CDCl3) δ 4.17 – 4.05 (m, 4H), 3.05 (d, J = 22.8 Hz, 2H), 2.28 (s, 3H), 1.30 (t, J = 7.1 Hz, 6H) (Figure
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S10); C NMR (126 MHz, CDCl3) δ = 199.89, 199.84, 62.54, 62.49, 43.81, 42.80, 31.31, 16.27, 16.22
(Figure S11); 31P NMR (202 MHz, CDCl3) δ = 19.64 (Figure S12).
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To an iceꢀwater cooled solution of compound 5 (971.0 mg, 5.0 mmol, 1 equiv) in methanol (25 mL) was
added sodium borohydride (189.2 mg, 5.0 mmol, 1 equiv). The reaction was stirred for 1 h and then
quenched by addition of water (50 mL). The resulting mixture was extracted with EtOAc (100 mL), and
the organic layer was washed thoroughly with water and brine. The organic phase was concentrated in
vacuo, and the resulting residue was used for the next step without further purification.
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The product from the previous step was suspended in HCl (6 M, 30 mL), and the resulting mixture was
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refluxed at 110 C overnight. After cooling to room temperature, the reaction mixture was washed with
diethyl ether (30 mL), and the aqueous phase was dried in vacuo. The resulting residue was reꢀsuspended
in water (10 mL), and the pH was adjusted to 8 with triethylamine. The resulting solution was lyophilized
to give 420 mg of compound 6 as white wax in 61% yield. Compound 6 was pure enough for further
reaction, and no further purification was performed. 1H NMR (500 MHz, D2O) δ 4.09 – 3.93 (m, 1H),
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3.07 (q, J = 7.3 Hz, 4H), 1.95 – 1.68 (m, 2H), 1.18 – 1.13 (m, 9H) (Figure S13); C NMR (126 MHz,
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D2O) δ = 63.82, 46.60, 37.28, 36.23, 23.23, 23.15, 8.20 (Figure S14); P NMR (202 MHz, D2O) δ =
24.08 (Figure S15).
step c:
To a stirred suspension of cytidine 5’ꢀmonophosphate (CMP, 161.6 mg, 0.5 mmol, 1 equiv) in the coꢀ
solvent of DMF (10 mL) and triethylamine (TEA, 10 mL) was added carbonyldiimidazole (CDI, 243.2
mg, 1.5 mmol, 3 equiv) in one portion, and the resulting mixture was stirred at room temperature for 2 h.
Another portion of CDI (81.1 mg, 0.5 mmol, 1 equiv) was added, and the reaction mixture was stirred
until it became clear. Although clarity is an indication of reaction completeness, the reaction was
analyzed by thin layer chromatography (TLC) using iPrOH:NH4OH:H2O = 6:3:1 as solvent. Another
potion of CDI was added if CMP was not consumed completely. After completion, the solvent was
removed under nitrogen flow, and the resulting syrup was reꢀdissolved in methanol and water (1:1)
containing 5% triethylamine. The solvent was dried in vacuo completely, and the resulting gum was reꢀ
suspended in DMF (10 mL) followed by addition of compound 6 (69.0 mg, 0.5 mmol, 1 equiv). The
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resulting reaction mixture was stirred vigorously at room temperature for 2 days. After completion, the
reaction was quenched by water (30 mL), and the resulting mixture was extracted with EtOAc (20 mL)
ten times to remove the DMF from the aqueous phase. The aqueous phase containing the product was
purified over a MacroꢀPrep DEAE column with a linear elution of aqueous ammonium bicarbonate
solution (from 10 mM to 300 mM). Fractions containing product were combined and concentrated in
vacuo. Repeated evaporation on a rotary evaporator in a water bath at 50 oC was performed to remove the
residual ammonium bicarbonate to give 88.0 mg of HPPꢀCMP as a white powder in 40% yield. 1H NMR
(500 MHz, D2O) δ 8.14 (d, J = 8.0 Hz, 1H), 6.23 (d, J = 8.0 Hz, 1H), 5.86 (d, J = 2.8 Hz, 1H), 4.31 – 4.18
(m, 4H), 4.15 – 4.04 (m, 2H), 2.07 – 1.87 (m, 2H), 1.21 (d, J = 6.2 Hz, 3H) (Figure S16); 13C NMR (126
MHz, D2O) δ = 159.08, 148.41, 144.12, 95.21, 89.57, 83.25, 83.18, 74.31, 68.94, 64.16, 63.89, 37.90,
36.83, 23.10, 23.03 (Figure S17); 31P NMR (202 MHz, D2O) δ = 15.50, ꢀ11.47 (Figure S18).
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RESULTS
Strategy for Overproduction of Class B RS Methylases in Soluble Form. In our previous studies of
TsrM, the tsrM gene, cloned into a pSUMO vector, was expressed alongside genes on plasmid pDB1282,
which encode proteins involved in FeꢀS cluster biosynthesis and trafficking.44 The chosen pSUMO vector
confers kanamycin resistance, and expression of the tsrM gene is controlled by an IPTGꢀinducible T7
promoter. By contrast, plasmid pDB1282 confers ampicillin resistance, and expression of the isc gene
cluster on this plasmid is controlled by an arabinoseꢀinducible (PBAD) promoter. During gene expression,
E. coli was cultured in a specialized medium supplemented with ethanolamine as the sole carbon and
nitrogen source, as well as OHCbl. Ethanolamine is metabolized in E. coli to acetaldehyde and ammonia
by ethanolamine ammonia lyase (EAL), an AdoCblꢀdependent enzyme.47 Although E. coli cannot
synthesize AdoCbl de novo, it contains the genes necessary to generate it from cobinamide, an incomplete
corrinoid that lacks the lower axial nucleotide loop (Figure 2d). When cultured on ethanolamine as the
sole source of carbon and nitrogen, E. coli upregulates the expression of EAL, which has been suggested
to enhance the intracellular levels of cobalamin.33 When TsrM is overproduced in this fashion, pure
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protein is obtained in reasonable (0.5 mg Lꢀ1)—but still challenging—yields. Whereas, when TsrM is
overproduced in media lacking ethanolamine, only meager amounts of protein are obtained, and its
homogeneity is 50% or less. Consistent with cobalamin accessibility being one of the limiting factors in
obtaining soluble TsrM, the asꢀisolated protein contains a stoichiometric amount of bound cobalamin.34
In an effort to improve upon the low yields of soluble TsrM, we devised a strategy to increase the
cellular concentrations of cobalamin, with the expectation that the immediate availability of the cofactor
might facilitate proper intracellular folding during and/or after protein synthesis. The E. coli genome
encodes two other cobalaminꢀdependent proteins in addition to EAL, but the organism does not require
the cofactor for growth. E. coli preferentially uses a MeCblꢀdependent methionine synthase (MetH) to
generate methionine from homocysteine,48 but can also catalyze the reaction in the absence of cobalamin
using a cobalaminꢀindependent methionine synthase.49 It also contains QueG, which uses cobalamin to
catalyze the final step in the biosynthesis of queuosine, a modified nucleoside found in the anticodon stem
loop of some tRNAs.50, 51
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Cobalamin uptake in E. coli is a regulated process that minimally involves four dedicated gene
products: BtuC, BtuD, BtuF, and BtuB (Figure 3).52 BtuB is a highꢀaffinity cobalamin transporter located
in the outer membrane of E. coli, which acts in concert with TonB to transport various cobalamin
derivatives into the periplasmic space of the bacterium in an energyꢀdependent process.53ꢀ55 Transport
across the cytoplasmic membrane is mediated by BtuC and BtuD, which form a membraneꢀbound α2β2
complex that functions as an ABC transporter.56ꢀ59 Lastly, BtuF is a periplasmic cobalaminꢀbinding
protein that delivers cobalamin to the BtuCD complex.60 Regulation of the cobalamin uptake system is
mediated by AdoCblꢀdependent feedback inhibition of btuB gene expression. When intracellular
concentrations of AdoCbl are sufficiently high, AdoCbl binds to btuB messenger RNA (mRNA),
including a 5′ꢀleader sequence, inhibiting translation by preventing mRNA binding to the ribosome.61
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Figure 3. Cobalamin uptake system in E. coli. Cobalamin is transported across the outer membrane by
the TonBꢀdependent outer membrane protein BtuB. BtuF, a periplasmic membrane transporter, then
delivers the cobalamin to BtuC and BtuD, which subsequently transport cobalamin across the inner
membrane into the cytosol.
We designed a plasmid that incorporates the btuC, btuE, btuD, btuF and btuB genes to allow for their
overexpression. The btuC and btuD genes are part of a btuCED operon in E. coli, although the btuE gene
product does not play a role in cobalamin uptake and trafficking. In fact, it is predicted to be a peroxidase
based on its primary structure.62 The btuB and btuF genes are found elsewhere in the E. coli chromosome.
To facilitate cloning, the btuE gene was not excised from the btuCED operon. The pBAD42 plasmid was
chosen as a compatible vector for use with the expression system that we typically employ to overproduce
RS enzymes. This plasmid encodes resistance to spectinomycin, which allows it to be maintained in the
same host with the kanamycin resistanceꢀencoding pETꢀbased vectors that we use for gene expression of
RS enzymes, the ampicillin resistanceꢀencoding pDB1282 vector for expression of the isc operon, and the
chloramphenicol resistanceꢀencoding plasmids that are present in some expression strains for the
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expression of lowꢀcopy tRNAs. The multiple cloning site of pBAD42 is downstream of the PBAD
promoter, which is also the relevant promoter in the pDB1282 plasmid. This strategy allows for temporal
control of expression of the cobalamin uptake genes with respect to expression of the RS gene of interest.
Typically, expression of the isc operon is induced at an OD600 of 0.3 by addition of arabinose, while
expression of the RS gene is induced by addition of IPTG at an OD600 of approximately 0.6, such that the
FeꢀS cluster assembly proteins are present prior to expression of the target RS gene.44 Likewise, the
cobalamin uptake genes are under the control of the same PBAD promoter and are induced simultaneously
with the genes in the isc operon.
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The btuCED, btuF, and btuB genes were cloned from E. coli genomic DNA and ligated into the
pBAD42 vector sequentially using semiꢀnested restriction sites (Figure 4). To improve translation of the
btuCEDFB mRNAs, the spacing between genes and their respective ribosome binding sites (RBS) was
further optimized. The interstitial sequences between btuD and btuF and between btuF and btuB were
removed by siteꢀdirected mutagenesis such that the stop codon of the upstream gene and the start codon
of the downstream gene overlap by one base pair. Because the btuCED fragment was cloned as an
operon, the spacing between these genes was not altered. The btuE and btuD genes overlap by one base
pair in the E. coli genome; however, there is a >60 baseꢀpair intergenic region between btuC and btuE,
necessitating an additional RBS. Neither the RBS encoded by the vector nor the RBS encoded in the
intergenic region between btuC and btuE were optimal for translation in E. coli. Therefore, the sequence
of each RBS was changed by siteꢀdirected mutagenesis to 5’ꢀ TAAGGAGGT –X5ꢀATGꢀ3’, where the last
three bases are the start codon of the gene. The final optimized plasmid was termed pBAD42ꢀBtuCEDFB.
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Figure 4. The pBAD42ꢀBtuCEDFB vector. (a) A plasmid map of the vector. Expression of btuCEDFB is
driven by an arabinose inducible araBAD promoter. (b) The cloning regions of the btuCEDFB operon.
The optimized ribosome binding sites are shown in red, while the start (bold) and stop (underlined)
codons are also displayed.
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Analysis of Cobalamin Content in E. coli by LCꢀMS/MS. LCꢀMS/MS analysis was used to compare
the concentration of cobalamin in E. coli harboring the pBAD42ꢀBtuCEDFB plasmid to the concentration
of cobalamin in E. coli containing an empty pBAD42 vector. Gene expression was carried out in E. coli
cultured in M9 minimal media supplemented with OHCbl, LB media supplemented with OHCbl, or the
M9ꢀethanolamine media previously used to improve cobalamin uptake. Because OHCbl is converted into
MeCbl or AdoCbl in vivo,63 it was necessary to determine not only if induction of the btuCEDFB operon
increases the cobalamin content of E. coli cells, but also to determine what forms of cobalamin are
present in the cell. In Figure 5, relative intracellular concentrations of OHCbl, AdoCbl, and MeCbl are
displayed for E. coli cultured in different growth media and in the absence or presence of pBAD42ꢀ
BtuCEDFB. In M9 minimal media and in LB media, induction of the btuCEDFB operon results in a 13ꢀ
and 19ꢀfold increase in total (sum of all forms) cobalamin concentration, respectively, as compared to the
vectorꢀonly control, while a 54ꢀfold increase is observed for E. coli cultured in M9ꢀethanolamine media.
In addition to their increased cobalamin content detected by LCꢀMS/MS, cell pellets of E. coli containing
the pBAD42ꢀBtuCEDFB plasmid are noticeably pinker in color (Figure S19). The substantial
enhancement in cobalamin uptake indicates that overexpression of the operon elicits the intended
outcome, increasing the intracellular cobalamin concentration for enhanced cobalamin incorporation into
TsrM or other cobalaminꢀcontaining proteins.
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All three forms of cobalamin are detected in E. coli extracts (Figure 5); however, OHCbl and AdoCbl
predominate. By contrast, relatively small amounts of MeCbl are observed under each of the conditions.
When the expression of the btuCEDFB operon is induced in E. coli cultured in M9 minimal media,
approximately 52% of the cobalamin is found as OHCbl, while <1% and 47% is found as MeCbl and
AdoCbl, respectively. However, when expression is induced in E. coli cultured in M9ꢀethanolamine
media, 63% of the cobalamin is found as AdoCbl, and <1% and 37% are found as MeCbl and OHCbl,
respectively. Similarly, 19% of total cobalamin is found as OHCbl, 81% as AdoCbl, and <1% as MeCbl
when the btuCEDFB operon is induced in E. coli cultured in LB media (Figure 5). These results clearly
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indicate that E. coli cells harboring the pBADꢀBtuCEDFB plasmid uptake significantly more cobalamin
than those that do not have the plasmid and that OHCbl and AdoCbl are the major forms of cobalamin
found in the cell.
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Figure 5. Relative concentrations of MeCbl (orange), OHCbl (blue), and AdoCbl (red) in E. coli cultured
in LB, M9, and M9ꢀethanolamine (Eth) media with and without pBAD42ꢀBtuCEDFB normalized to the
amount of AdoCbl in E. coli cultured in M9ꢀethanolamine media with the pBAD42ꢀBtuCEDFB plasmid
.
pBAD42ꢀBtuCEDFBꢀDependent Enhancement of TsrM Solubility. TsrM was purified to homogeneity
from soluble crude lysate in a previous study, but the strategy employed yielded meager amounts of
protein (~0.5 mg/L) that was only soluble when overproduced in the RosettaBlue (DE3) pLysS strain of
E. coli and when cultured in M9ꢀethanolamine media supplemented with cobalamin.34, 41 When a similar
strategy was applied to TsrM overproduced in the E. coli BL21 (DE3) strain rather than the RosettaBlue
(DE3) strain, only ~0.5 mg/L soluble protein that was approximately 25% pure was obtained (Figure
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S20). To determine if additional cobalamin in the cell could increase the amount of soluble TsrM, TsrM
was overproduced as a fusion protein with SUMO as previously described,34 but under conditions in
which proteins encoded by the pBAD42ꢀBtuCEDFB plasmid are present. TsrM was subsequently purified
as previously described, and a yield of ~3.7 mg/L of >95% homogenous protein was collected upon
removal of the SUMO tag using Ulp1 protease (Figure S21). Thus, coꢀexpression of tsrM with the genes
on pBAD42ꢀBtuCEDFB in BL21 (DE3) yields a 30ꢀfold increase in soluble protein as compared to
expression in the same strain without induction of the btuCEDFB operon, and more than a 7ꢀfold increase
in yield as compared to expression in the RosettaBlue (DE3) strain.
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pBAD42ꢀBtuCEDFBꢀDependent Enhancement of Fom3 Solubility. To further test the efficacy of the
pBAD42ꢀBtuCEDFB construct in enhancing class B RS methylase solubility, we set out to purify Fom3
from Streptomyces wedmorensis, which has previously been reported to be insoluble upon overproduction
in E. coli.64 In a previous study, this protein was purified from inclusion bodies and resolubilized and
refolded before being reconstituted with its FeꢀS and cobalamin cofactors.64 As a control, a SUMOꢀtagged
Fom3 was first overproduced in the absence of the pBAD42ꢀBtuCEDFB plasmid; however, as has been
previously reported, no soluble protein was obtained from the crude lysate. However, when Fom3 was
overproduced in the presence of pBAD42ꢀBtuCEDFB, it could be purified to ≥95% homogeneity using
the same protocol as that described for TsrM (Figure S22).34 This strategy yields 3.0 mg/L of soluble
Fom3 as determined by Bradford analysis.
Forms of Cobalamin Bound to TsrM and Fom3. After purification of Fom3 and TsrM, each protein
was treated with potassium cyanide to form dicyanocobalamin to determine the molar ratio of bound
cobalamin to protein. Using the extinction coefficient of dicyanocobalamin (ɛ367 = 30,800 Mꢀ1 cmꢀ1),40 it
was determined that TsrM and Fom3 contain 1.03 and 0.86 equivalents of cobalamin per polypeptide,
respectively (Figure S23). Additionally, the relative ratios of the different forms of cobalamin present in
TsrM and in Fom3 were determined by LCꢀMS/MS analysis. Fom3 contains 0.85 mol of MeCbl, 0.15
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mol of OHCbl, and 0.00 mol of AdoCbl per polypeptide (Figure 6a). Consistent with previous studies,37
TsrM contains 0.00 mol of MeCbl, 0.63 mol of OHCbl, and 0.40 mol of AdoCbl per polypeptide (Figure
6b). The lack of MeCbl in TsrM may derive from the ready availability of tryptophan, its substrate,
during gene expression, while the substrate for Fom3 is not expected to be present in E. coli. In line with
this argument, when TsrM is overproduced in E. coli, 2ꢀmethyltryptophan (MeTrp) is observed in cell
extracts.
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Figure 6. LCꢀMS/MS analysis of the cobalamin content of (a) asꢀisolated Fom3 (20 µM) and (b) asꢀ
isolated TsrM. (a) Asꢀisolated Fom3 contains 18% OHCbl (blue line), 0% AdoCbl (black line) and 82%
MeCbl (red line). (b) TsrM contains 61% OHCbl (blue line), 39% AdoCbl (black line) and <1% MeCbl
(red line).
Activity of Fom3. Fom3 is one of the enzymes in a microbial biosynthetic pathway for production of
fosfomycin from phosphoenolpyruvate. It was originally predicted to catalyze formation of 2ꢀ
hydroxypropylphosphonate (2ꢀHPP) from 2ꢀhydroxyethylphosphonate (2ꢀHEP), and indeed, one
laboratory reported this activity with Fom3 purified from inclusion bodies.9, 11, 64 However, when we
incubate Fom3 (purified from the soluble fraction of E. coli lysate) with 2ꢀHEP, SAM, reductant
(flavodoxin reducing system, reduced methyl viologen, or dithionite), formation of neither 2ꢀHPP nor Sꢀ
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adenosylhomocysteine (SAH) is detected by LCꢀMS/MS. Recently, Sato et al. found that Fom3 actually
catalyzes the Cꢀmethylation of cytidylylꢀ2ꢀhydroxyethylphosphonate (HEPꢀCMP) to give cytidylylꢀ2ꢀ
hydroxypropylphosphonate (HPPꢀCMP) (Figure S24).65 When HEPꢀCMP is used in our Fom3 activity
determinations, formation of HPPꢀCMP, SAH, and 5′ꢀdeoxyadenosine (5′ꢀdA) are readily observed
(Figure 7). Sato et al. also reported that the steadyꢀstate kinetic analysis of Fom3 required the addition of
MeCbl to their reaction mixtures and suggested that MeCbl is a coꢀsubstrate in the reaction rather than a
cofactor, meaning that it is regenerated outside of the Fom3 active site. As shown in Figure 7, Fom3,
overproduced and isolated as described in this study, exhibits a kcat of 1.81 hꢀ1 in the absence of added
MeCbl, which is 2.03 times greater than the turnover number reported by Sato et al (0.89 hꢀ1).65
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Figure 7. Timeꢀdependent formation of HPPꢀCMP (black), SAH (red), and 5'ꢀdA (blue) by Fom3 (9 ꢁM)
in the presence of 1.5 mM HEPꢀCMP, 1.5 mM SAM, 1 mM methyl viologen and 2 mM NADPH.
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