Stuffed Methyltransferase Catalyzes the Penultimate Step of Pyochelin Biosynthesis

Article abstract of DOI:10.1021/acs.biochem.8b00716

Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into the final natural product. A structurally unique set of tailoring domains are found to be stuffed within adenylation domains and have only recently begun to be characterized. PchF is the NRPS termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain responsible for S-adenosylmethionine (AdoMet)-dependent N-methylation. Recent studies of stuffed methyltransferase domains propose a model in which methylation occurs on amino acids after adenylation and thiolation rather than after condensation to the nascent peptide chain. Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through the synthesis and use of substrate analogues, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an S_N2 reaction after thiolation, condensation, cyclization, and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.

Full text of DOI:10.1021/acs.biochem.8b00716

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Article
Stuffed Methyltransferase Catalyzes
Penultimate Step of Pyochelin Biosynthesis
Trey A. Ronnebaum, Jeffrey S McFarlane, Thomas E. Prisinzano, Squire J. Booker, and Audrey L Lamb
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00716 • Publication Date (Web): 10 Dec 2018
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Stuffed Methyltransferase Catalyzes Penultimate Step of
Pyochelin Biosynthesis
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Trey A. Ronnebaum,1 Jeffrey S. McFarlane,2 Thomas E. Prisinzano,3 Squire J. Booker,4 Audrey
L. Lamb1, 2
1Department of Chemistry, 2Department Molecular Biosciences, 3Department of Medicinal
Chemistry, The University of Kansas, Lawrence, Kansas 66045, United States. 4 Department of
Chemistry, Department of Biochemistry and Molecular Biology, and the Howard Hughes
Medical Institute, The Pennsylvania State University, University Park, Pennsylvania, 16802.
Corresponding Author: Audrey L. Lamb, email: lamb@ku.edu, phone: (785) 864-5075
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Abstract
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Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into
the final natural product. A structurally unique set of tailoring domains are found to be stuffed
within adenylation domains and have only recently begun to be characterized. PchF is the NRPS
termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain
responsible for S-adenosylmethionine (AdoMet) dependent N-methylation. Recent studies of
stuffed methyltransferase domains propose a model in which methylation occurs on amino acids
after adenylation and thiolation rather than after condensation to the nascent peptide chain.
Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through
the synthesis and use of substrate analogs, steady-state kinetics, and onium chalcogen effects.
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We provide evidence that methylation occurs through an S 2 reaction after thiolation,
N
condensation, cyclization and reduction of the module substrate cysteine and is the penultimate
step in pyochelin biosynthesis.
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Abbreviations
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AdoMet, S-adenosylmethionine; AdoHCys, S-adenosylhomocysteine; CFA, cyclopropane fatty
acid synthase; COMT, Catechol O-methyltransferase; Cys, cysteine; HP, hydroxyphenyl;
HRMS, high-resolution mass spectrometry; IPTG, isopropyl β-D-thiogalactopyranoside; KIEs,
kinetic isotope effects; IPP, inorganic pyrophosphatase; MesG, 2-amino-6-mercapto-7-
methylpurine; MesGR, 2-amino-6-mercapto-7-methylpurine ribonucleoside; Mj, Methanococcus
jannaschii; MLP, MbtH-like protein; MTA, 5’-methylthioadenosine; NNACs, N-
acetylethylenediamine amides; NRPS, nonribosomal peptide synthetase; ONACs, N-
acetylethanolamine esters; PchF, nonribosomal peptide synthetase from P. aeruginosa for the
production of pyochelin; PchF-AMT, PchF construct consisting of stuffed adenylation
methyltransferase didomains and thiolation domain, and without the condensation and
thioesterase domains; PchF-FL, full-length construct of PchF; PNP, purine nucleoside
phosphorylase; Ppant, 4’phosphopantetheinyl; SeAdoMet, Se-Adenosyl selenomethionine;
SeMTA, 5’-methylselenoadenosine; SNACs, N-acetylcysteamine thioester; T, thiazoline; Tox,
thiazole; Tred, thiazolidine; Tred-M, N-methylated thiazolidine; TeAdoMet, Te-Adenosyl
telluromethionine; TeMTA, 5’-methyltelluroadenosine.
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Introduction
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Bacteria, fungi, and plants generate siderophores, small molecules with high affinity for
ferric iron, to scavenge the surrounding environment during times of iron starvation. Indeed,
many siderophores have been identified as virulence factors in pathogenic bacteria due to the
metal-limiting environment of the host species. Siderophores are classified by the chemical
nature of the oxygen involved in ferric iron coordination.1 Pyochelin is a phenolate siderophore
from Pseudomonas aeruginosa, a commensal and opportunistic gram-negative pathogen,
notorious for causing infections in immunocompromised patients, burn-wound victims, and lung
infections of cystic fibrosis patients.2-5
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The precursors of pyochelin are chorismate and two cysteine molecules (Figure 1A).6-8
Chorismate, from the shikimate pathway, is converted to salicylate in two steps, and is the
initiating hydroxy acid. The cysteine molecules are added in succession by nonribosomal
peptide synthetases (NRPSs), forming peptide bonds.6 Encoded within the pch operon are
tailoring domains, which perform the epimerization, reduction and methyltransferase activities
required to make the final bioactive compound.6, 9-11 Pyochelin is a tricyclic acid: the 2’-
hydroxyphenyl (HP) ring derived from salicylate attached to a thiazoline (T) ring from one
cyclized and epimerized cysteine, followed by a methylated 4’-thiazolidine (Tred-M) heterocycle
from the second cyclized cysteine.
NRPSs are multi-domain, multi-catalytic proteins arranged in modules to activate and
add one substituent each to the growing chain, with each module containing a condensation or
cyclization domain, an adenylation domain and a thiolation domain. The NRPS assembly line
uses a 4’phosphopantetheinyl (Ppant) thiotemplate tethering system reminiscent of polyketide
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and fatty acid biosynthesis. This means that the peptide bond forming and tailoring chemistries
are
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Figure 1: Synthesis of pyochelin. A) Biosynthetic pathway for pyochelin production. PchA, an
isochorismate synthase, and PchB, an isochorismate pyruvate lyase, convert chorismate to
salicylate. PchD-G consists of 12 different domains constituting three NRPS modules with three
tailoring domains. Together, they assemble pyochelin from salicylate and two L-cysteines, with
co-substrates AdoMet, ATP, and NADPH. AdoMet = S-adenosylmethionine, AdoHcys = S-
adenosylhomocysteine. PchD is a stand-alone adenylation domain (A; blue). PchE has N- and C-
terminal thiolation domains (T; yellow), a cyclization domain (Cy; grey), an adenylation domain
(A; blue) and a stuffed epimerase domain (E; purple). PchG is a stand-alone NADPH-dependent
reductase. PchF is a full-NRPS module consisting of 5 different domains: a cyclization domain
(Cy; grey), adenylation domain (A; blue), stuffed methyltransferase tailoring domain (M; red),
thiolation domain (T; yellow), and thioesterase domain (TE; green). B) PchF-FL refers to the
“full-length” protein, whereas PchF-AMT only includes the stuffed adenylation
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methyltransferase didomain and the thiolation domain. G667I-PchF-AMT has a mutation to the
proposed AdoMet binding domain of the variant PchF-AMT. C) The proposed order of
chemistries performed by PchF. PchF incorporates L-cysteine to the growing peptide chain. The
Cy-domain of PchF continues chain elongation by generating a peptide bond between L-cysteine
and the upstream hydroxyphenyl-D-thiazoline moiety of PchE (an intermediate attached to the
Ppant of the T-domain of PchE). The Cy-domain then cyclizes the cysteine to form a
hydroxyphenyl-(D)-thiazoline-(L)-thiazoline intermediate. PchG (green) reduces the terminal
thiazoline of the hydroxyphenyl-bis-heterocycle to thiazolidine using NADPH. The stuffed
methyltransferase domain of PchF catalyzes the AdoMet-dependent methyl transfer (highlighted
in red) onto the nitrogen of the newly reduced thiazolidine ring before transferring the complete
heterocyclic siderophore to the TE-domain releasing the fully mature pyochelin by hydrolysis in
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the TE-domain. The alternate synthetic schemes where methyl transfer occurs at a different
position in the sequence of biosynthesis are shown in Figure S1. D) During reconstitution
assays, premature hydrolysis releases peptidyl intermediates during biosynthesis. Intermediates
previously recovered by Walsh and colleagues are noted with an asterisk. Intermediate analogs
used as potential substrates in this study are surrounded by a square. Nomenclature of
intermediates is as follows: HP, hydroxyphenyl; Tox, thiazole; T, thiazoline; Tred, thiazolidine;
CO2- carboxyl; Et, ethyl; Me/M, methyl.
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performed while the intermediates are covalently attached to a thiolation domain. The NRPS
tailoring domains are affiliated with particular modules in several geometries. The most studied
geometry has the tailoring domain included at the end of a module following the thiolation
domain, as an independent domain that works in cis with the peptide bond forming domains of
the module. Pyochelin biosynthesis lacks this type of tailoring domain. Some tailoring domains
are stand-alone: independently expressed proteins acting in trans, such as the reductase of
pyochelin biosynthesis (PchG).11 Finally, there are tailoring domains that are incorporated
within the adenylation domain.12 Adenylation domains activate the substrate and attach it to the
thiotemplate tethering system. The “stuffed” tailoring domains replace a loop within an
adenylation domain, making a true di-domain (Figure 1B).13 Pyochelin biosynthesis uses two
stuffed tailoring domains. The first is an adenylation-epimerase didomain in the protein named
PchE, and the second is an adenylation-methyltransferase didomain in PchF.10, 12-14
Examples of stuffed tailoring domains, also known as “interrupted adenylation
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domains”,12 participating in natural product biosynthesis include oxygenases (i.e. epothilone,15
myxothiazol,16 thuggacins)17, ketoreductases (i.e. cereulide)18, methyltransferases (i.e.
thiocoraline,1, 19 cyclosporine,20 echinomycin,21 enniatins,22 micacocidin,23 microcystin,24
pyochelin,9, 10 yersiniabactin9), and epimerases (i.e. pyochelin,9, 10 yersiniabactin9). Importantly,
some of the natural products synthesized using stuffed tailoring domains have been isolated and
experimentally show anticancer (i.e. epothilone, thiocoraline, echinomycin), antifungal (i.e.
myxothiazol), antibacterial (i.e. myxothiazol, thuggacin, micacocidin), or immunosuppressive
(i.e. cyclosporine) activity. However, the mechanistic and structural characterization of stuffed
tailoring domains has only just begun.
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Recently, mechanistic and structural studies of the stuffed methyltransferase domains
from the biosynthesis of the microbial depsipeptides thiocoraline and kutzneride have been
reported.19, 25-27 A main goal of these studies was to determine when in the biosynthetic pathway
methylation occurs, with four possibilities: methylation of 1) the free amino acid, 2) the
aminoacyl-AMP intermediate (after the amino acid is activated by ATP in the adenylation
domain), 3) after thiolation (after the amino acid is attached to the Ppant tether of the thiolation
domain), or 4) after condensation to the upstream peptide. Evidence suggests that the stuffed
methyltransferase does not use the free amino acid as a substrate, but rather the substrate is either
the aminoacyl-AMP intermediate or the amino acid attached to the thiolation domain (after
thiolation).19, 25-27 The possibility remains that methyl transfer occurs after elongation with the
upstream peptide; however, thiocoraline and kutzneride are complex natural products, making
intermediate analog synthesis to test the hypothesis of late stage methylation difficult.
Contradictory to the proposed model, isolates observed during reconstitution assays of
epothilone biosynthesis and mechanistic studies of EpoB indicate that tailoring occurs after
condensation to the upstream peptide chain.14, 15 Indeed, this is logical. Epothilone biosynthesis
employs a hybrid NRPS/PKS assembly line in which EpoB, a protein encoding a single NRPS
module, has a stuffed oxidase domain, which oxidizes the condensed thiazoline. The EpoB
stuffed oxidase domain substrate is simply a 2-methylthiazoline attached to the Ppant of the
thiolation domain.
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Here, using the termination module of pyochelin biosynthesis found in the protein PchF,
we test the opposing hypotheses for the order of chemistry. An order of catalytic events has been
proposed in the literature (Figure 1C). The PchF adenylation domain activates the cysteine
substrate and attaches the cysteine to the thiotemplate of the thiolation domain.6 The cyclization
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domain of PchF links the nascent chain donated from the PchE thiolation domain (HPT) to the
cysteine on the thiolation domain of PchF (HPT-Cys), and subsequently cyclizes this cysteine
generating a thiazoline ring (HPTT). While still tethered to the thiolation domain of PchF, PchG
reduces the second thiazoline ring generating a thiazolidine (HPTTred). The N-methyltransferase
stuffed domain in PchF modifies the ring to generate an N-methylated thiazolidine (HPTTred-M),
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the final proposed synthetic step before release of the mature pyochelin (HPTTred-MCO2 ) by the
thioesterase. Two intermediates have been hydrolyzed from the thiotemplate tether, isolated and
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characterized during pyochelin reconstitution assays: HPT-Cys and HPTTred-CO2 (Figure 1D).6,
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The chemical release and isolation process also produced a more stable, off-pathway product
− 6, 11
hypothesized to be the result of air oxidation: HPToxT- CO2 .
Since none of these isolated
compounds contain the N-methyl on the thiazolidine, the order of the steps proposed for PchF is:
adenylation, thiolation, condensation, cyclization, reduction, methylation and hydrolysis.
However, there are some contradictory data that suggest that methylation may proceed before
reduction: labeled methyl groups, from either 14C-S-adenosylmethionine (AdoMet) or 3H-
AdoMet were incorporated into the product in the absence of the reductase or NADPH.10
Altogether, the order of catalysis, and in particular when methyl transfer occurs remains elusive,
leaving four reasonable pathways: 1) before condensation, 2) before cyclization, 3) before
reduction, or 4) before hydrolysis (Figure S1). Herein, we synthesized a series of substrate
analogues and developed kinetic analyses for the adenylation and methyltransferase activities of
PchF and PchF variants to establish the order of catalytic steps performed by PchF in pyochelin
biosynthesis. Additionally, onium chalcogen effects were used to define the mechanism of
PchF’s methyltransferase reaction.
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Materials and Methods
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Preparation of full-length PchF (pchf-fl) Overexpression Plasmid. The pchF (Uni-Prot ID
O85740) overexpression construct was a gift from the laboratory of Christopher Walsh.6 The
construct received was the kanamycin resistant, pET28b plasmid (Novagen).
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Preparation of Overexpression Plasmid containing PchF adenylation, methyltransferase, and
thiolation domains (pchf-amt). The variant plasmid was produced by site directed mutagenesis
using Herculase polymerase supplemented with 0-10% DMSO and the genomic DNA from
Pseudomonas aeruginosa PAO1, which was purified using the DNeasy® Blood & Tissue Kit
(Qiagen) as per the manufacturer’s instructions. The forward primer (5’-ATT AGA CAT ATG
GTC GAG GCG CCG CCG CAG G-3’) including an NdeI site (underlined) and reverse primer
(5’-TA ATA CTC GAG TCA GGT TCC GGC GCG CTG CGC AG-3’) including an XhoI site
(underlined) were used to amplify the gene of interest. The amplified sequence was ligated into
the kanamycin resistant pET28b plasmid (Novagen) digested with the same restriction enzymes.
Upon sequencing, it was determined that there were an extra 2 nucleotides inserted before the
stop codon. Therefore, using QuikChange XL (Agilent), a forward primer (5’-GCA GCG CGC
CGG AAT GAC TCG AGC AC-3’) and reverse primer (5’-GTC CTC GAG TCA TTC CGG
CGC GCT GC-3’) were designed to remove the 2-nucleotide insertion before the stop codon
(underlined). The isolated plasmid sequence encodes the entire adenylation domain,
methyltransferase domain, and thiolation domain, residues 502 to 1483 of PchF-FL.
G667i-pchf-amt Overexpression plasmid. The variant plasmid was produced by using the
kanamycin resistant pchF-amt plasmid and the QuikChange XL (Agilent) as per the
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manufacturer’s instructions. The forward primer (5’-
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GCGGCGGCGGTGATGGCGCCAAGCTC-3’) and reverse primer (5’-
GAGCTTGGCGCCATCACCGCCGCCGC-3’) were used to mutate the desired nucleotides
(underlined). The isolated plasmid was sequenced showing that only the desired mutations were
present.
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Pa2412 Overexpression Plasmid. The pa2412 (Uni-Prot ID Q9I169) overexpression plasmid
was a gift from the laboratory of Andrew Gulick.28 PA2412 is an MbtH-like protein (MLP) that
increases P. aeruginosa NRPS protein expression including PchF (data not shown).29-32 The
pa2412 gene is in a modified pET15b plasmid, conferring ampicillin resistance, containing a
5Xhis affinity tag and a TEV protease recognition site in place of the thrombin site.28
Methanococcus jannaschii (Mj) S-Adenosyl Methionine (AdoMet) Synthetase Overexpression
Plasmid was a gift from Doug Markham from the Fox Chase Cancer Institute.33
PchF-FL protein overexpression and purification. BL21 (DE3) E. coli containing the pchf-fl and
pa2412 expression plasmids were grown in LB broth containing 50 μg/mL kanamycin and 200
µg/mL ampicillin at 37 °C with shaking (250 rpm). Protein expression was induced when the
OD600 reached ~0.6 by the addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a final
concentration of 200 μM. The temperature was reduced to 22 °C following induction. The cells
were harvested by centrifugation (4 230 x g, 10 min, 4 °C) after ~21 hours. The cell pellet was
resuspended in 25 mM Tris-HCl pH 8, 500 mM NaCl, 50 mM imidazole (buffer A). Cells were
disrupted by French press (35 000 psi), and cellular debris was removed by centrifugation (23
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430 x g, 45 min, 4 °C). The supernatant was applied to a chelating Sepharose fast-flow column
(Amersham Biosciences) charged with nickel chloride and pre-equilibrated in buffer A. Two
column volumes of buffer A were followed by a 15-column volume linear gradient of 0-500 mM
imidazole in buffer A. PchF-FL and PA2412 proteins eluted together in 1 peak at ~150 mM
imidazole. Fractions containing the proteins of interest were pooled and concentrated using an
Amicon stirred cell with YM30. The concentrated fractions were applied to a Superdex 200 size-
exclusion column (Amersham Biosciences) equilibrated with 50 mM Tris pH 8, 150 mM sodium
citrate, 10% (v/v) glycerol (buffer B). PchF-FL eluted separately from PA2412 and the PchF-FL
fractions were concentrated as above to 1.4 mg/mL as determined by the general Beer-Lambert
law and the predicted A280 of ε = 276 620 M-1 cm -1 (ProtParam).34 A total of 14.7 mg was
obtained from the 2 L of cell growth. The protein was flash cooled and stored at -80 °C.
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PchF-AMT and G667I-PchF-AMT protein overexpression and purification. BL21 (DE3) E. coli
containing the pchf-amt or g667i-pchf-amt and pa2412 expression plasmids were grown in LB
broth containing 50 μg/mL kanamycin and 200 µg/mL ampicillin at 37 °C with shaking (225
rpm). When the OD600 reached ~0.6, protein expression was induced by the addition of IPTG to
a final concentration of 200 μM and the temperature was reduced to 15 °C. The cells were
harvested by centrifugation (4 230 x g, 10 min, 4 °C) after ~26 hours. The cell pellet was
resuspended in 25 mM Tris-HCl pH 8, 500 mM NaCl, 5 mM imidazole, 10 % glycerol (buffer
C). Cells were disrupted by use of a French pressure cell (35 000 psi), and cellular debris was
removed by centrifugation (11 950 x g, 60 min, 4 °C). The supernatant was applied to a
chelating Sepharose fast-flow column (Amersham Biosciences) charged with nickel chloride and
pre-equilibrated in buffer C. After injection, the column was washed with four column volumes
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of buffer C, followed by four column volumes of 50 mM imidazole in buffer C. A linear gradient
from 50-500 mM imidazole in buffer C over six column volumes allowed elution of PchF-AMT
or G667I-PchF-AMT and PA2412 proteins in a single peak around 200 mM imidazole. The
pooled fractions were dialyzed using SnakeSkin Dialysis Tubing (10 kDa cutoff) against 50
mM Tris pH 8, 1 mM dithiothreitol (DTT), 10% (v/v) glycerol (buffer D). The dialysate was
changed for fresh buffer twice: at 1 and 2 hours before exchanging overnight. The protein
solution was loaded onto a Source 30Q (GE Healthcare Life Sciences) anion exchange column
pre-equilibrated in buffer D. After injection, two column volumes of buffer D were followed by
a 0-500 mM NaCl linear gradient over 15 column volumes in buffer D. PchF-AMT or G667I-
PchF-AMT eluted at ~150 mM NaCl. The collected fractions were concentrated and applied to a
Superdex 200 size-exclusion column (Amersham Biosciences) equilibrated with 50 mM Tris pH
8, 100 mM sodium citrate, 1 mM DTT, 10% (v/v) glycerol (buffer C). The fractions containing
PchF-AMT or G667I-PchF-AMT were pooled and concentrated using a 30 kDa cutoff Amicon
ultracentrifugal spin filter. The final concentration of PchF-AMT was 7.8 mg/mL as determined
by the general Beer-Lambert law and the predicted A280 of ε = 149 310 M-1 cm -1 (ProtParam).34
A total of 13.3 mg of PchF-AMT was obtained from the 3 L cell growth. The protein was flash
cooled and stored at -80 °C. G667I-PchF-AMT was concentrated to 1.4 mg/mL, also determine
by Beer-Lambert’s law and the predicted A280 of ε = 149 310 M-1 cm -1 (ProtParam).34 A total of
0.621 mg was obtained from the 3 L cell growth. The protein was flash cooled and stored at -
80˚C.
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Methanococcus jannaschii (Mj) AdoMet Synthetase (Uni-Prot ID Q58605) overexpression and
purification. BL21 (DE3) E. coli containing the pMJ1208-1 expression plasmid were grown in
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LB broth containing 100 µg/mL ampicillin at 37 °C with shaking (225 rpm). When the OD600
reached ~0.6, protein expression was induced by the addition of IPTG to a final concentration of
500 μM. Cultures were incubated for an additional 5 hours. The cells were harvested by
centrifugation (10 820 x g, 10 min, 4 °C). The cell paste (50 g) was resuspended in 50mM
HEPES pH 7.5, 10 mM β-mercaptoethanol, 300 mM NaCl, 5 mM imidazole, supplemented with
1 mg/mL lysozyme, 0.1 mg/mL DNAse I, 1 mM PMSF, and 0.1% Triton X (Buffer E). Cells
were lysed by sonication (6 cycles of 45 seconds with 7 minutes between cycles, 4˚C) and
subsequently heat denatured (50 min, 75˚C). Cell debris and denaturants were removed by
centrifugation (50 000 x g, 60 min, 4˚C). The supernatant was loaded onto a Ni-NTA agarose
column equilibrated with Buffer E. The column was washed with five column volumes of Buffer
E and eluted with Buffer E supplemented with 500 mM imidazole. Fractions containing protein
were concentrated using a 10 kDa cutoff Amicon ultracentrifugal spin filter. The protein was
loaded onto a G-25 column equilibrated in 50 mM Tris-HCl pH 8, 50 mM KCl, 0.1% BME, 10%
(v/v) glycerol (Buffer F). Protein containing fractions determined by Bradford assay and were
collected and concentrated using a 30-kDa cutoff Amicon ultracentrifugal spin filter. The
concentrated protein was determined by the general Beer-Lambert law and the predicted A280 of
ε = 24 870 M-1 cm -1 (ProtParam) before being flash cooled and stored at -80 ˚C.34 A total of 211
mg of Mj AdoMet synthetase was obtained from the 10 L of cell growth.
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−
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Scheme 1: Substrate analog synthesis of HPToxT-CO2 , HPToxT-CO2Et, HPToxTred-CO2
HPToxTred-CO2Et and HPT-Cys-Me. Substrate analogs were generated to assess the
methyltransferase capabilities of PchF. Reagents and conditions: a) Pd(PPh3)4, K2CO3/DME,
H2O, 150 °C, 49%;42 b) MeOH, K2HPO4(aq) (pH 6.5), 60 °C, 70%;42 c) SOCl2/EtOH, 40 °C,
49%;42 d) Pd(PPh3)4, K2CO3/DME, H2O, 100˚C, 70%; e) EtOH/KCO2CH3 (aq), RT, 95%; f)
SOCl2/EtOH, 40˚C, 55%; g) DIEA, COMU/DMF, 0˚C 1hr, RT 3hr, 12%; h) generated in the
presence of 5mM TCEP in vitro.
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Preparation of Substrate Analogues (Scheme 1).
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General experimental procedures. Chemical reagents purchased from commercial suppliers were
not further purified prior to use. Flash column chromatography was performed using silica gel
(4-63 mm) purchased from Sorbent Technologies. Other chemical separations were performed
using a Teledyne Isco CombiFlash Rf. A Biotage microwave reactor was used when
microwaving was necessary. 1H and 13C NMR were recorded using a 500 MHz Bruker AVIII
spectrometer equipped with a cryogenically-cooled carbon observe probe. Tetramethylsilane was
used as an internal standard and chemical shifts (δ) are reported in ppm and coupling constants
(J) are reported in Hz. High-resolution mass spectrometry (HRMS) was performed using an
electrospray ion source in either positive or negative mode on an LCT Premier (Micromass Ltd.,
Manchester UK). Melting points were measured with a Thomas Capillary Melting Point
Apparatus and are uncorrected. Chiral analysis was carried out using HPLC, 1 ml/min
hexane:isopropanol (90:10) on a CHIRALCEL OD-H column, monitored at 320 nm unless
otherwise specified.
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2-(2-hydroxyphenyl)thiazole-4-carbaldehyde
A microwave vial was charged with 2-bromothiazole-4-carbaldehyde (2.08 mmol), 2-
hydroxyphenyl boronic acid (2.71 mmol), and Pd(PPh3)4 (0.104 mmol). The vial was sealed and
flushed with argon for 5 minutes. Dimethoxyethane (16 mL) and a 2 M potassium carbonate
solution (2.0 mL, 4.17 mmol) were added through the septum, the reaction was stirred at room
temperature for an additional 5 minutes and heated to 100˚C for 45 minutes using a microwave
reactor. The reaction was cooled to room temperature and filtered through a pad of celite and
sepharose. The reaction was concentrated under reduced pressure and purified using flash
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chromatography (0:1-1:6 EtOAc/Hexane gradient) to yield a yellow solid (300 mg, 70%). The
purified product was in agreement with literature values for melting point, 1H NMR, and 13C
NMR.35
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(2RS-4R)-2-(2-(2-hydroxyphenyl)thiazol-4-yl)thiazolidine-4-carboxylic acid (HPToxTred -CO2 )
2-(2-Hydroxyphenyl)thiazole-4-carbaldehyde (1.46 mmol) and potassium acetate (9.71 mmol)
were dissolved in a solution of EtOH/H2O (10:3, 104 mL) in a round bottom flask. L-Cysteine-
HCl (5.40 mmol) was added and the reaction was stirred at room temperature for 1 hour. The
reaction was washed with hexanes (50 mL), diluted with H2O and acidified with citric acid to
~pH 2. The aqueous layer was extracted with CH2Cl2 (3 x 75 mL). The organic layers were
combined and dried over Na2SO4. The solvent was removed in vacuo to afford a yellow/white
solid (430 mg, 95%). The product was purified as a mixture of epimers and was in agreement
with literature values for 1H NMR and 13C NMR.36 HRMS: [M + H]+ 309.0289 (calcd),
309.0363 (found).
Ethyl (2RS-4R)-2-(2-(2-hydroxyphenyl)thiazol-4-yl)thiazolidine-4-carboxylate (HPToxTred-
CO2Et).
Thionyl chloride (1.75 mmol) was added dropwise to anhydrous ethanol (3.25 mL) with
vigorous stirring in a -10˚C ice water bath. The mixture was warmed to room temperature.
−
HPToxTred-CO2 (0.649 mmols) was added and the reaction was heated to 40 ˚C and stirred for 16
hours. The mixture was cooled to room temperature and the solvent was removed in vacuo. The
residue was reconstituted in saturated Na2CO3 (30 mL) and extracted with CH2Cl2 (4 x 50 mL).
The organic layers were combined, and the mixture was concentrated under reduced pressure and
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purified using the CombiFlash (0:1-1:2 EtOAc/Hexane gradient) to yield a clear oil (119 mg,
55%). The product was purified as a diastereomer (38:62) as determined by chiral
chromatography (Figure S2). 1H NMR (500 MHz, DMSO-d6) δ 11.50 (d, J = 764.2 Hz, 2H),
8.37 (s, 1H), 8.05 (dd, J = 7.9, 1.7 Hz, 1H), 8.02 (dd, J = 7.9, 1.7 Hz, 1H), 7.69 (s, 1H), 7.57 (s,
1H), 7.34 – 7.21 (m, 2H), 7.00 (ddd, J = 8.3, 5.4, 1.2 Hz, 2H), 6.91 (dddd, J = 8.1, 7.2, 5.7, 1.1
Hz, 2H), 5.84 (d, J = 9.9 Hz, 1H), 5.72 (d, J = 12.1 Hz, 1H), 4.41 (ddd, J = 8.7, 6.9, 5.1 Hz, 1H),
4.25 – 4.10 (m, 4H), 4.03 – (ddd, J = 12.1, 8.8, 7.0 Hz, 1H), 3.94 (t, J = 9.4 Hz, 1H), 3.59 (t, J =
12.2 Hz, 1H), 3.42 – 3.30 (m, 1H), 3.15 – 3.03 (m, 1H), 1.21 (td, J = 7.1, 4.7 Hz, 6H) 13C NMR
126 MHz, DMSO-d6) δ 171.30, 170.84, 164.90, 163.92, 163.59, 155.35, 154.87, 152.37, 131.22
– 131.01 (m), 127.43, 127.34, 119.27, 118.95, 118.82, 116.64, 116.51, 115.14, 67.06, 65.23,
64.96, 61.08, 60.80, 38.03, 37.94, 14.08, 14.04. HRMS: [M + H]+ 337.0675 (calcd), 337.0708
(found).
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(S)-2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid (HPT-CO2 )
Synthesis was performed as previously described in Bergeron et al and Zamri et al except
starting materials differed by using D-cysteine instead of L-cysteine.37, 38 Product analysis agreed
with previously reported 1H NMR and 13C NMR. HRMS: [M + H]+ 224.0303 (calcd), 224.0378
(found). Chiral analysis was performed using HPLC and a CHIRALCEL OD-H column.
Elution was performed using a hexane:isopropanol (90:10) mixture supplemented with 0.1%
trifluoroacetic acid at a flow rate of 0.500 mL/min, Rf = 14.7 minutes.
Dimethyl 3,3’-disulfanediyl(2R,2’R)-bis(2-((S)-2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-
carboxamido)propanoate)
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D-HPT-CO2 , L-cystine dimethyl ester dichloride (Sigma), and 1-cyano-2-ethoxy-2-
oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (Sigma)
were purged under argon in a 25 mL round bottom flask before being dissolved in DMF (10 mL)
at 0˚C. Diisopropylethylamine was then added dropwise and the reaction was stirred vigorously
for 1 hour at 4˚C and 3 hours at room temperature. Ethyl acetate (50 mL) was then added to the
reaction. The organic fraction was washed with 1M HCl (2 x 50 mL), water (2 x 50 mL), and
brine (2 x 50 mL) before being concentrated under reduced pressure. The residue was purified
using the CombiFlash (DCM from 0-5 minutes before a gradient to 5% MeOH from 5-8 minutes
and holding the 5% MeOH in DCM for 5 more minutes) eluting a peak at 10 minutes. An
additional purification step was performed using a Waters Acquity UPLC with a photodiode
array UV detector and an LCT premiere TOF mass spectrometer. The UPCL/HRMS used a
gradient elution increasing 20% CH3CN in water over 5 minutes. Injections of a 1 mL DMSO
stock were made onto a Waters (T3 C18) 19 x 150 mm, 5 µm column with a flow rate of 20
mL/min to yield a white solid (35 mg, 12%). 1H NMR (500 MHz, DMSO-d6) δ 12.09 (s, 1H),
8.86 (d, J = 8.0 Hz, 1H), 7.45 (ddd, J = 15.5, 7.8, 1.7 Hz, 2H), 7.02 – 6.92 (m, 2H), 5.38 (t, J =
9.0 Hz, 1H), 4.68 (td, J = 8.7, 4.8 Hz, 1H), 3.66 (s, 3H), 3.55 (dd, J = 11.1, 8.4 Hz, 1H), 3.23 (dd,
J = 13.9, 4.9 Hz, 1H), 3.07 (dd, J = 13.9, 9.1 Hz, 1H). 13C NMR 126 MHz, DMSO-d6) δ 173.03,
171.05, 169.83, 158.61, 134.75, 131.09, 119.79, 117.35, 116.24, 77.99, 54.07, 52.32, 33.93.
HRMS: [M + H]+ 679.0946 (calcd), 679.1008 (found).
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Preparation of Co-substrate Analogues.
Se-Adenosyl selenomethionine (SeAdoMet).
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SeAdoMet was biosynthesized and purified by a modification of known methods.39 10 mM L-
methionine was added to a reaction mixture containing 100 mM Tris pH 8, 50 mM KCl, 40 mM
MgCl2, 15 mM ATP, 8% 2-mercaptoethanol, 1.3 U inorganic pyrophosphatase (Sigma), 130 µM
Mj AdoMet Synthetase in a final volume of 10 mL. The reaction was gently rocked for 4 hours,
quenched with 1 mL 1 M H2SO4, and neutralized with 1 mL 2.5 M Ammonium Acetate. The
mixture was centrifuged (3 082 x g for 10 minutes at 4˚C), flash cooled, and stored as 800 µL
aliquots at -80˚C for further purification. 400 µL was injected onto a Waters Semi-Prep C18
column (10 x 250 mm, 5µM) previously equilibrated in 95% 83 mM triethyl ammonium amine
pH 5 (solvent A) and 5% methanol running at 5 mL/min. SeAdoMet was isolated at 3.7 minutes.
A linear gradient of 0-50% acetonitrile and 5-50% methanol from 8-18 minutes was used to flush
the column. A gradient back to 95% solvent A and 5% methanol was performed at 19 minutes to
re-equilibrate the column for subsequent runs. Immediately after collection, SeAdoMet was flash
cooled and lyophilized overnight. The lyophilized sample was resuspended in 250 µL of 100
mM H2SO4 and the concentration was determined spectrophotometrically (AdoMet, ε260 = 15
400 M-1cm-1)40 to be 35.3 mM.
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Te-Adenosyl telluromethionine (TeAdoMet).
The TeAdoMet was synthesized as previously described.39
Steady-state Adenylation Assay
Reactions contained 50 mM Tris pH 7.5, 1 mM MgCl2, 200 µM 2-amino-6-mercapto-7-
methylpurine ribonucleoside (MesGR), 0.002 U purine nucleoside phosphorylase (PNP, Sigma),
0.001 U inorganic pyrophosphatase (IPP, Sigma), 2 mM ATP, 150 mM hydroxylamine, 0.600
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µM enzyme, and varying concentrations of L-cysteine. Stock concentrations (200 mM – 12.5
µM) of L-cysteine were prepared by serial dilution in 25 mM Tris pH 8 buffer supplemented
with 5 mM dithiothreitol. ATP, L-cysteine, and hydroxylamine were made fresh for each
triplicate reaction. A parent mixture containing all reaction components except L-cysteine were
incubated for 15 minutes at room temperature. The reaction (final volume, 100µL) was initiated
by the addition of the parent mixture to substrate in a 96-well flat bottom plate (Corning Cat.
#9017) and production of 2-amino-6-mercapto-7-methylpurine (MesG) was monitored at A360 on
a Cary 50 UV-VIS Spectrophotometer with a plate reader attachment. Steady-state turnover was
converted from AU/min to µM/min using the general Beer-Lambert law using 0.29 cm as the
path-length and 11,000 cm-1M-1 as the molar extinction coefficient of MesG.41 The calculated
rate was divided in half because each pyrophosphate released from the adenylation activity
undergoes catalytic hydrolysis by inorganic pyrophosphatase creating two inorganic phosphates,
both used individually by purine nucleoside phosphorylase to convert MesGR to MesG.
Negative controls (without enzyme, without L-cysteine, and without ATP) were carried out with
each triplicate reaction. The largest rate from the multiple negative control reactions was
subtracted as background from all of the calculated rates. Triplicate trials were performed twice,
on separate days for each enzyme. The initial rates were fit to the Michaelis-Menten equation
using the nonlinear regression function of KaleidaGraph (Synergy Software). Kinetic parameters
were calculated for each of the six individual trials per enzyme. The reported values represent the
average and standard deviation of these six sets of kinetic parameters.
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Methyltransferase Activity Assay
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Methylated product formation assay. Methyltransferase activity was observed with HPToxTred-
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CO2Et, but not observed with HPToxTred-CO2 , HPToxT-CO2Et,42 or HPT-Cys-Me at 50 µM
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during a 1-hour incubation. Product formation of HPToxTred-M-CO2Et was monitored at A320 by
HPLC and confirmed by selected ion recording mass spectrometry. Each assay mixture (100 µL)
contained 100 mM Tris-HCl pH 8.0, 45 nM enzyme, 1 mM AdoMet, varying amounts of
HPToxTred-CO2Et (1 µM – 30 µM) in DMSO (final DMSO concentration ≤ 2%), and 1 mM
tryptophan (an internal standard). A parent mixture of all reaction components, excluding
HPToxTred-CO2Et, was incubated at room temperature for 5 minutes before initiating the reaction
with HPToxTred-CO2Et. A time-course assay was performed for both low (1 µM) and high (30
µM) HPToxTred-CO2Et to determine that 15 minutes was within the linear steady-state turnover
for PchF-FL and PchF-AMT. Therefore, an endpoint assay was employed after a 15-minute
incubation at room temperature. Reactions were quenched with 5 µL of 1 M H2SO4, vortexed,
and neutralized with 10 µL of 1M sodium citrate pH 5.5.
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S-adenosylhomocysteine formation assay. Methyltransferase activity by PchF-AMT or PchF-FL
was assessed with the substrates HPToxTred-CO2Et or L-Cys-OEt (Sigma) by measuring the
formation of the coproduct, S-adenosylhomocysteine (AdoHCys), following a 3-hour incubation.
Each assay mixture (100 µL) contained 100 mM Tris-HCl pH 8.0, 1 µM enzyme (PchF-AMT or
PchF-FL), 1 mM AdoMet, and 30 µM HPToxTred-CO2Et or 30 µM or 3 mM L-Cys-OEt.
Reactions were quenched with 5 µL of 1 M H2SO4 and neutralized with 10 µL of 1M sodium
citrate pH 5.5. A Waters Acquity UPLC was used to inject 10 µL of sample onto a Phenomenex
Luna silica C18 column (4.6 X 250 mm, 5 µM) previously equilibrated in 99% solvent B
(0.016% formic acid, 0.02% triethylamine) and 1% acetonitrile. Isocratic flow of 99% Solvent B
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: 1% ACN at 0.8 mL/min for 10 minutes separated AdoMet (3.0 min) and AdoHCys (7.0 min),
as measured by absorbance at 260 nm. To determine the concentration of AdoHCys a standard
curve was generated by plotting the area of the elution peaks at varied AdoHCys concentrations.
Final AdoHCys concentrations are reported in µM after subtracting for AdoHCys formed by
non-enzymatic decomposition as measured in negative controls. Measurements were made for at
least 3 replicates of all reaction and standard curve trials.
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Steady-state kinetics varying onium chalcogen co-substrates using the methylated product
formation assay. Each assay mixture (100 µL) contained 100 mM Tris-HCl pH 8.0, 45 nM
enzyme, 50 µM HPToxTred-CO2Et, varying amounts of AdoMet, SeAdoMet or TeAdoMet (0-2
mM), and 1 mM tryptophan (an internal standard). A parent mixture of all reaction components,
excluding AdoMet, SeAdoMet, or TeAdoMet, was incubated at room temperature for 5 minutes
before initiating the reaction with AdoMet, SeAdoMet or TeAdoMet. Reactions were quenched
with 5 µL of 1 M H2SO4, vortexed, and neutralized with 10 µL of 1 M sodium citrate pH 5.5.
Linear steady-state turnover was assessed for low (5 µM) and high (1 mM) concentrations of
AdoMet and SeAdoMet. The reaction was quenched at 10 minutes, within the linear steady-state
for both AdoMet and SeAdoMet. TeAdoMet reactions, also within steady-state, were incubated
for 4 hours and contained twice the concentration of enzyme. AdoMet was varied in reactions
from 0 – 2000 µM, SeAdoMet was varied from 0 – 650 µM, whereas TeAdoMet was varied
from 0 – 800 µM.
Methyltransferase steady-state kinetic analysis using the methylated product formation assay.
Quenched and neutralized methyltransferase reaction samples were centrifuged (10 600 x g, 10
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mins, 4 ˚C) to remove denatured enzyme, and 100 µL of supernatant was injected onto an IRIS
Technologies LLC IProSIL C18 column (4.6 X 250 mm, 5 µM) previously equilibrated in 92%
solvent B (0.016% formic acid, 0.02% triethylamine) and 8% acetonitrile. A linear gradient of 8-
80% acetonitrile from 0-15 mins, an 80-100% linear gradient from 15-20 minutes, and a 100-8%
gradient from 20-21 minutes at 1 mL/min gave good separation of HPToxTred-CO2Et (19.9 min)
and methylated HPToxTred-CO2Et (21.6 min). A standard curve for product formation was
determined by monitoring assay completion of 0-10 µM HPToxTred-CO2Et reactions with PchF-
AMT performed in triplicate (9 000 ± 100 mV/µM, Figure S5). Due to substrate concentrations
being less than 100 times enzyme concentration, Equation 1 was used to calculate steady-state
parameters.43 The reported kinetic parameters were calculated by finding the average and
standard deviation of each individual assay performed in triplicate.
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ꢉ
�
([ ] [ ]
ꢅ + ꢇ ꢆ + ꢈꢇ
)
([ ] ) ([ ] )
� ꢅ
([ ] )([ ] )
−
+
ꢅ
+ ꢈꢇ� − ꢊ ꢅ
ꢇ
ꢆ
ꢆ
ꢆ
ꢆ
ꢆ
⎛
⎝
⎞
⎠
ꢀ = ꢁꢂꢃꢄ
+ ꢂꢊꢄ
([ ] )
ꢉ ꢅ
ꢆ
Equation 1.
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Cloning, sequencing, and purification of PchF-FL, PchF-AMT, and G667I-PchF-AMT.
Three variants of PchF were generated heterologously in an E. coli expression system. The first
is the full-length protein as produced by P. aeruginosa, called PchF-FL herein (Figure 1B). The
second consists of the adenylation-methyltransferase-didomain with the thiolation domain, and is
thus abbreviated PchF-AMT. This construct removes the N-terminal cyclization domain and the
C-terminal thioesterase domain to eliminate confounding activities. Previously, Patel and
colleagues developed a pyochelin reconstitution assay using recombinant PchD, PchE, PchF,
PchG with substrates and cofactors, monitoring the formation of mature pyochelin by HPLC
analysis.10 Patel et al reported that mutation of the second glycine of a core AdoMet binding
motif (GxG) to an arginine (G1167R) in PchF resulted in undetectable amounts of pyochelin.10
The authors suggested that pyochelin formation was inhibited due to a lack of methyl transfer
from AdoMet to the HPTTred intermediate (Figure 1B-D). We attempted to generate G667R-
PchF-AMT, but overexpression yielded negligible amounts of protein. We therefore proposed
that substitution of the same glycine with a bulky hydrophobic residue would likewise inhibit
AdoMet binding, and designed a glycine to isoleucine mutation, G667I-PchF-AMT, which
proved easier to purify in necessary amounts for kinetic analysis.
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Each of the PchF variants was co-expressed with PA2412, an MbtH-like protein (MLP)
from the pyoverdin biosynthetic cluster of P. aeruginosa, which increases the amount of the
PchF variants purified. This is consistent with other reports that have shown that MLPs greatly
enhance NRPS solubility and are sometimes even necessary for expression.28, 29, 44, 45 Recently,
MLPs were co-crystallized with NRPS modules showing complex formation with the
adenylation domains.28, 45-48 While each PchF variant was co-expressed with PA2412 and the
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proteins co-eluted by nickel chelating chromatography (both are histidine tagged), the proteins
were separated by gel filtration chromatography. Therefore, the final purified PchF variants did
not contain PA2412. The PchF-AMT and G667I-PchF-AMT purification protocols included an
additional anion exchange step to achieve >95% purity, estimated by polyacrylamide gel
electrophoresis.
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Steady-state kinetics of adenylation activity. Aldrich et al previously described a continuous assay
to measure activity of adenylation domains by linking pyrophosphate production to an absorbance
change (Figure S3).49 The coupled assay uses inorganic pyrophosphatase (IPP) to hydrolyze the
pyrophosphate generated by the adenylation reaction to two inorganic phosphate ions.
Subsequently, purine nucleoside phosphorylase (PNP) converts 7-methylthioguanosine (MesGR)
to ribose 1-phosphate and the chromogenic 7-methythioguanine (MesG), with the change
measured as an increase in absorbance at 360 nm. This assay was optimized to measure
adenylation of L-cysteine in the stuffed adenylation-methyltransferase didomain of PchF-FL,
PchF-AMT, and G667I-PchF-AMT. Without hydroxylamine addition, detection of the
downstream chromogenic MesG was within error of negative controls through 30 minutes,
whereas the inclusion of hydroxylamine gave reliable steady state turnover (Figure 2), suggesting
that the adenylation domain remains in a closed conformation and inorganic pyrophosphate is not
released until after attachment of the amino acid to the Ppant tether (mimicked here by the
hydroxylamine surrogate). A representative assay with and without hydroxylamine can be found
in Figure 2, along with Michaelis-Menten curves for the three PchF variants. Steady state kinetic
parameters for adenylation of all three constructs can be found in Table 1. Note that kcat values
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Figure 2: Adenylation activity of PchF variants. PchF catalyzes the adenylation of L-cysteine
by ATP forming an aminoacyl-adenylate bond, releasing pyrophosphate. In the adenylation
assay (Figure S3), pyrophosphate is converted to two inorganic phosphates by inorganic
pyrophosphatase. 7-methylguanosine is then phosphorylated by purine nucleoside phosphorylase
to generate 7-methylguanine, which absorbs at 360 nm. A) PchF variants do not turnover with L-
cysteine and ATP (A), but do with the addition of hydroxylamine as a nucleophilic surrogate (B).
Michaelis-Menten steady-state kinetics (C) were performed to compare the L-cysteine
adenylation activity of PchF-FL, PchF-AMT, and the methyltransferase null mutant of G667I-
PchF-AMT.
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