Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway

Article abstract of DOI:10.1073/pnas.1303568110

Bioactive natural products containing a C-P bond act as mimics of phosphate esters and carboxylic acids, thereby competing with these compounds for active sites of enzymes. Dehydrophos (DHP), a broad-spectrum antibiotic, is a phosphonotripeptide produced by Streptomyces luridus, in which glycine and leucine are linked to an aminophosphonate analog of dehydroalanine, ΔAla(P). This unique feature, in combination with the monomethylation of the phosphonic acid, renders DHP a Trojan horse type antibiotic because peptidase-mediated hydrolysis will release methyl acetylphosphonate, a potent inhibitor of pyruvate dehydrogenase. Bioinformatic analysis of the biosynthetic gene cluster suggested that ΔAla(P) would be generated from Ser(P), the phosphonate analog of Ser, by phosphorylation and subsequent elimination, and that ΔAla(P) would be condensed with Leu-tRNA^Leu. DhpH was anticipated to carry out this elimination/ligation cascade. DhpH is a multidomain protein, in which a pyridoxal phosphate binding domain is fused to an N-acetyltransferase domain related to the general control non-derepressible- 5 (GCN5) family. In thiswork, the activity of DhpH was reconstituted in vitro. The enzyme was able to catalyze the β-elimination reaction of pSer(P) to generate ΔAla(P), but it was unable to condense ΔAla(P) with Leu. Instead, ΔAla(P) is hydrolyzed to acetyl phosphonate, which is converted to Ala(P) by a second pyridoxal phosphate-dependent enzyme, DhpD. Ala(P) is the substrate for the condensation with Leu-tRNA^Leu catalyzed by the C-terminal domain of DhpH. DhpJ, a 2-oxoglutarate/Fe(II)-dependent enzyme, introduces the vinyl functionality into Leu-Ala(P) acting as a desaturase, and addition of Gly by DhpK in a Gly-tRNA^Gly-dependent manner completes the in vitro biosynthesis of dehydrophos.

Full text of DOI:10.1073/pnas.1303568110

Revisiting the biosynthesis of dehydrophos reveals
a tRNA-dependent pathway
a,b
a,b
a,b,1
Despina J. Bougioukou , Subha Mukherjee , and Wilfred A. van der Donk
a
b
Institute for Genomic Biology, Department of Chemistry, and Howard Hughes Medical Institute, University of Illinois at Urbana–Champaign, Urbana,
IL 61801
Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved May 23, 2013 (received for review February 23, 2013)
Bioactive natural products containing a C-P bond act as mimics of
phosphate esters and carboxylic acids, thereby competing with
these compounds for active sites of enzymes. Dehydrophos (DHP),
a broad-spectrum antibiotic, is a phosphonotripeptide produced by
Streptomyces luridus, in which glycine and leucine are linked to an
aminophosphonate analog of dehydroalanine, ΔAla(P). This unique
feature, in combination with the monomethylation of the phos-
phonic acid, renders DHP a Trojan horse type antibiotic because
peptidase-mediated hydrolysis will release methyl acetylphospho-
nate, a potent inhibitor of pyruvate dehydrogenase. Bioinformatic
analysis of the biosynthetic gene cluster suggested that ΔAla(P)
would be generated from Ser(P), the phosphonate analog of Ser,
by phosphorylation and subsequent elimination, and that ΔAla(P)
inhibitor for alanine racemase (14), enzymatic hydrolysis of
dehydrophos will unmask 1-aminovinylphosphonate, ΔAla(P), in
its monomethylated form (Fig. 1B). The enamine of ΔAla(P) will
tautomerize to the corresponding imine and hydrolyze to afford
methyl acetylphosphonate (MAP), a 125 times stronger inhibitor
of pyruvate dehydrogenase compared with nonesterified acetyl-
phosphonate (15) and a potent inhibitor of bacterial 1-deoxy-D-
xylulose 5-phosphate synthase (16, 17).
As part of a program focused on elucidating phosphonate bio-
synthetic pathways, the dehydrophos gene cluster was integrated
into the chromosome of S. lividans and heterologous production of
dehydrophos (DHP) was accomplished (18). Bioinformatic anal-
ysis of the proteins involved revealed the apparent duplication of
several enzymatic functions (Fig. 1C) including two putative 2-
oxoglutarate/Fe(II)-dependent oxygenases, DhpA and DhpJ, two
putative alcohol dehydrogenases, DhpC and DhpG, two putative
pyridoxal 5′-phosphate (PLP)-dependent enzymes, DhpD and
DhpH (N-terminal domain), and two putative nonribosomal
peptidyl transferases, DhpH (C-terminal domain) and DhpK (18).
Single-gene deletions and characterization of the accumulated
Leu
would be condensed with Leu-tRNA . DhpH was anticipated to
carry out this elimination/ligation cascade. DhpH is a multidomain
protein, in which a pyridoxal phosphate binding domain is fused to
an N-acetyltransferase domain related to the general control non-
derepressible-5 (GCN5) family. In this work, the activity of DhpH was
reconstituted in vitro. The enzyme was able to catalyze the β-elim-
ination reaction of pSer(P) to generate ΔAla(P), but it was unable to
condense ΔAla(P) with Leu. Instead, ΔAla(P) is hydrolyzed to acetyl
phosphonate, which is converted to Ala(P) by a second pyridoxal
phosphate-dependent enzyme, DhpD. Ala(P) is the substrate
3
1
intermediates by P-NMR spectroscopy delineated the order of
the first four biosynthetic steps (Fig. 1D). With the exception of the
DhpA-catalyzed reaction, these steps are all similar to reactions
described for other phosphonate natural products (19–21). Fur-
thermore, the accumulation of intermediates such as 1, 2-dihy-
droxyethyl phosphonate (DHEP), 1-hydroxy-2-phosphorylethyl
phosphonate (HP-EP), and 1-amino-2-phosphorylethyl phospho-
nate [pSer(P)] suggested a pathway that resembles that of serine
biosynthesis in Escherichia coli (Fig. 1E and SI Appendix, Fig. S2).
Based on that scheme, the two PLP-dependent enzymes (DhpD
and DphH) would act in tandem and the transient product of the N-
terminal domain of DhpH, ΔAla(P), would be the substrate of the
factors essential for methicillin (Fem) XAB-like C-terminal do-
main of DhpH for amide bond formation (Fig. 1F). The fact that
the two functional domains are fused and encoded by one gene
Leu
for the condensation with Leu-tRNA catalyzed by the C-terminal
domain of DhpH. DhpJ, a 2-oxoglutarate/Fe(II)-dependent enzyme,
introduces the vinyl functionality into Leu-Ala(P) acting as a desatur-
Gly
ase, and addition of Gly by DhpK in a Gly-tRNA -dependent man-
ner completes the in vitro biosynthesis of dehydrophos.
hosphonopeptides containing a P-terminal aminophosphonate
P
moiety are well-documented bioactive agents. In 1971, in-
corporation of L-1-aminoethylphosphonic acid, L-Ala(P), in the
dipeptide mimetic L-Ala-L-Ala(P) lead Roche researchers to the
discovery of alaphosphin (Fig. 1A), a potent inhibitor of cell-wall
biosynthesis (1). Since then, numerous synthetic phosphonopep-
tides have been screened for their antimicrobial action (2), and
several naturally occurring oligopeptides harboring a C-P bond
have been isolated (3) (SI Appendix, Fig. S1). Two unique modes of
action have been recognized: (i) the intact phosphonopeptide may
directly inhibit an enzymatic activity, such as K-26 (SI Appendix,
Fig. S1), which exhibits antihypertensive properties by inhibiting
angiotensin converting enzyme (4), or (ii) the bioactive phospho-
nate warhead is released after cellular uptake of the peptide mimic
by the target organism and subsequent peptidase-mediated hy-
drolysis, as in the case of alaphosphin (5), bialaphos (6), and rhi-
zocticin (7) (SI Appendix, Fig. S1). The affinity of the C-P
compounds for their targets is usually high owing to their structural
similarity to analogous phosphate esters or carboxylic acids that
are the substrates of the target enzymes (8, 9).
(
dhpH) implies a physical proximity for the two active sites and
could compensate for the expected very short life of the reactive
enamine in ΔAla(P). Thus, it was anticipated that both the carbon-
carbon double bond and the amide bond to Leu would be formed
by the dual action of DhpH. Formation of the second amide bond
was assigned to DphK, whereas DhpI, a phosphonate O-methyl-
transferase, would furnish dehydrophos (Fig. 1F) (22).
In this study, we reconstituted the activity of DhpH in vitro and
discovered that it is not responsible for the formation of the vinyl
group in dehydrophos. Instead, we propose that DhpD and the
PLP domain of DhpH convert the phosphorylated intermediate
1
-oxo-2-phosphorylethylphosphonate (OP-EP) into L-Ala(P), and
that the C-terminal domain of DhpH subsequently forms the
Leu
dipeptide L-Leu-L-Ala(P) in a Leu-tRNA –dependent manner.
Dehydrophos (formerly A53868 factor A), a broad-spectrum
antibiotic isolated originally from Streptomyces luridus (10), has
been of interest because the reassignment of its structure revealed
a unique O-methylated dehydroaminophosphonate connected to
a glycine-L-leucine dipeptide by an amide bond (11) (Fig. 1B).
Structure–activity relationship studies and screening of Salmonella
mutants for dehydrophos sensitivity have provided strong evidence
for a Trojan horse type mechanism (12, 13). Thus, whereas hy-
drolysis of alaphosphin releases L-Ala(P) (Fig. 1A), a competitive
Author contributions: D.J.B. and W.A.v.d.D. designed research; D.J.B. and S.M. performed
research; D.J.B., S.M., and W.A.v.d.D. analyzed data; and D.J.B., S.M., and W.A.v.d.D.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: vddonk@illinois.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1303568110/-/DCSupplemental.
1
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Fig. 1. Proposed biosynthesis of dehydrophos. (A) Alaphosphin: a man-made, Trojan horse type, phosphonopeptide. (B) Conversion of DHP into the active
compound MAP. (C) Organization of the DHP biosynthetic genes and functional assignment of putative proteins. The dhpLMNOP genes that encode putative
transcriptional regulatory proteins and/or secondary transporters are not shown. (D) Confirmed early steps in DHP biosynthesis. (E) Proposed biosynthesis of
pSer(P) based on single-gene deletion experiments. (F) Proposed late steps in DHP biosynthesis. Abbreviations not defined in the text: PEP, phosphoe-
nolpyruvic acid; PnAA, phosphonoacetaldehyde; PnPy: phosphonopyruvic acid; SAHC, S-adenosylhomocysteine; SAM, S-adenosylmethionine; 2-HEP,
2
-hydroxyethylphosphonate. Dashed reaction arrows are hypothetical.
Subsequent methylation by DhpI affords the substrate for the the lack of transaminase activity with pSer(P) suggests that
2
-oxoglutarate/Fe(II)-dependent enzyme DhpJ, which is shown to pSer(P) is not the physiological substrate of DphD.
Gly
be a desaturase. DhpK, a Gly-tRNA -dependent peptidyl trans-
We determined the apparent steady-state kinetic parameters
for the conversion of AP and L-Ala to L-Ala(P) and pyruvate
by coupling the formation of pyruvate with the oxidation of
β-NADH, in the presence of lactate dehydrogenase (LDH) and
ferase, is shown to furnish the final tripeptide.
Results
L-Ala (kcat = 1.9 s− , K
1
= 0.02 mM, kcat/K = 1.0 × 10 M ·s )
m m
5
−1 −1
In Vitro Reconstitution of PLP-Dependent Activity of DhpD and DhpH.
The dhpD and dhpH genes were PCR amplified from the fosmid (SI Appendix, Fig. S7A). The high catalytic efficiency observed
1
7E11-4 harboring the dehydrophos gene cluster (18) and ligated suggested that this transformation could be the physiological re-
into the expression vector pET-15b. Both proteins were expressed action catalyzed by DhpD. The enzyme also converted MAP to
in E. coli as N-terminal hexahistidine-tagged constructs and purified
by immobilized metal affinity chromatography (IMAC). DhpH,
a multidomain protein (SI Appendix, Fig. S3) with an expected
the corresponding aminophosphonate in the presence of alanine,
1
albeit with 100-fold lower catalytic efficiency (i.e., kcat = 2.1 s−
,
3
−1 −1
m m
K = 2.1 mM; kcat/K =1.0 × 10 M ·s ) (SI Appendix, Fig. S7B),
molecular mass of 75 kDa, was copurified with a second protein of suggesting that MAP is not the physiological substrate of DhpD.
approximately 45 kDa after IMAC, based on SDS/PAGE analysis.
6 6
When His -DhpH or His -DhpH-N were incubated with rac-
When the polyacrylamide protein gel was stained with a poly- pSer(P) in the absence or presence of typical amino-recipient keto
histidine sequence-specific dye, the lower molecular mass band was acids such as pyruvate, oxaloacetate, or α-ketoglutarate (α-KG),
also stained (SI Appendix, Fig. S4). We suspected therefore that the
second band was the product of partial proteolytic degradation of
DhpH. Therefore, we generated a second His-tagged construct
(
His -DhpH-N) in which only the first 356 amino acid residues of
6
DhpH were included. His
6
-DhpH-N was also successfully expressed
in E. coli in soluble form.
Based on the previously proposed biosynthetic scheme, DhpD
was expected to reversibly convert the phosphonate analog of
phosphoserine, pSer(P), and an amino-acceptor molecule to OP-
EP (Fig. 1E). In addition, pSer(P) was expected to be the sub-
strate of the PLP domain of DhpH and undergo a β-elimination
reaction (Fig. 1F). To test these hypotheses, we prepared pSer(P)
3
(SI Appendix, Fig. S5) by using phosphorylchloride (POCl ) as
a phosphorylating reagent under conditions similar to those
reported for the phosphorylation of serine (23). Unexpectedly,
when DhpD was incubated with pSer(P) in the presence of
pyruvate, oxaloacetate, or α-ketoglutarate(α-KG) as amino acceptors,
only the starting material was recovered. However, L-Ala(P) and
Ser(P) were converted to acetylphosphonate (AP) and 1-oxo-2-
hydroxyethylphosphonate (OH-EP), respectively, in the presence
of pyruvate (Fig. 2 A and B and SI Appendix, Fig. S6), dem-
onstrating that DhpD has aminotransferase activity. Therefore,
31
Fig. 2.
P-NMR analysis of DhpD activity converting L-Ala(P) to AP and vice
31
versa and DhpH activity with rac-pSer(P). (A) P-NMR spectrum after con-
version of L-Ala(P) to AP by DhpD. Reaction mixture contained 10 mM L-Ala(P),
0 mM pyruvate, and 40 μM DhpD in 50 mM Na-Hepes at pH 8.0. (B) P-NMR
spectrum after conversion of AP to L-Ala(P) by DhpD. Reaction mixture con-
31
1
tained 10 mM L-Ala, 10 mM AP, and 40 μM DhpD in 50 mM Na-Hepes at pH
31
8.0. (C) P NMR spectrum after conversion of rac-pSer(P) to AP by DhpH. (D)
31
P-NMR spectrum of C after spiking with authentic standard of AP.
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AP was observed as the only product by 31P-NMR spectroscopy
Fig. 2 C and D and SI Appendix, Fig. S8). Thus, like DhpD, DhpH
(SI Appendix, Fig. S11A). To identify the nature of the nucleic acids
bound to DhpH, we treated samples of DhpH-C separately with
DNase, RNase, or Proteinase K and analyzed them on a 1% aga-
rose gel. In the RNase-treated sample, nucleic acids were no longer
visible under UV light when the gel was stained with ethidium bro-
mide, whereas nucleic acids were still visible in all other samples,
suggesting that it was RNA that copurified with DhpH-C. Sub-
sequently, we isolated the RNA by phenol extraction and ethanol
precipitation. The isolated RNA sample was incubated either with L-
(
did not convert pSer(P) to OP-EP. AP is the expected product for
β-elimination of the phosphate group followed by tautomeriza-
tion (Fig. 3A). Interestingly, the (S)-enantiomer of the carboxylic
acid analog of pSer(P), phosphoserine (L-pSer), was also a sub-
strate for DhpH-catalyzed β-elimination. By coupling the ob-
served formation of pyruvate with the oxidation of β-NADH in
the presence of LDH, we determined the apparent steady-state
kinetic parameters for both DhpH (kcat = 0.06 s− , K
1
= 0.22 mM,
[ C(U)]-leucine in the presence of ATP and purified leucyl-tRNA
14
m
3
−1 −1
−1
14
kcat/K
m
= 0.3 × 10 M ·s ) and DhpH-N (kcat = 1.10 s , K
m
=
synthetase from E. coli (LeuRS) or with [ C(U)]-glycine in the
4
−1 −1
0
.28 mM, kcat/K
m
= 0.4 × 10 M ·s ) (SI Appendix, Fig. S9). We
presence of ATP and purified glycyl-tRNA synthetase from E. coli
note that DhpH exhibited transamination activity with unphos-
phorylated compounds such as SerP and L-Ala(P) in the presence
of pyruvate as amino acceptor (SI Appendix, Fig. S10). In sum-
mary, the in vitro studies on the PLP-dependent enzymes DhpD
and DhpH-N did not provide confirmation of the proposed
pathway in Fig. 1 E and F, but DhpH generated AP from pSer(P)
and DhpD converted AP to L-Ala(P) (Fig. 3A), prompting further
evaluation whether these reactions could be consecutive steps in
the DHP biosynthetic pathway.
(GlyRS) following a standard aminoacylation assay protocol (30).
14
Only the pair L-[ C(U)]-leucine/LeuRS was able to radioactively
label the RNA sample with carbon-14 (SI Appendix, Fig. S11B).
To evaluate whether the C-terminal domain of DhpH could
generate an amide bond, the expected product L-Leu-ΔAla(P) was
chemically synthesized (SI Appendix, Figs. S12A and S13). This
synthetic standard was first used to assess whether pSer(P) could
be converted to L-Leu-ΔAla(P) by the coordinated action of the
Leu
two domains of DhpH in the presence of Leu-tRNA
as
expected by the pathway in Fig. 1F. We set up a sensitive TLC assay
Leu
In Vitro Reconstitution of DhpH tRNA-Dependent Activity. A PSI-
BLAST search using the last 346 amino acids of DhpH as query
against the UniProtKB database at the PredictProtein server (24)
in which aminoacylated tRNA
was (re)generated in situ by
14
LeuRS in the presence of total tRNA from E. coli, ATP, L-[ C
(U)]-leucine, and thermostable inorganic pyrophosphatase (TIPP).
revealed that this part of the protein shares similarity with numerous Incubation of this regeneration system with DhpH and rac-pSer(P)
uncharacterized proteins that belong to the domain of unknown
function 482 (25). The latter is a member of the acetyltransferase
clan, which consist of 30 families including the Fem resistance
did not result in formation of the desired product (Fig. 3). Thus,
once again, the model in Fig. 1F could not be confirmed.
The results described thus far showed that the PLP domain of
family members FemXAB (26, 27) and the leucyl/phenylalanyl- DhpH converts pSer(P) to AP and that DhpD can generate Ala(P)
tRNA protein transferase family (28). Indeed, a BLAST search of from AP. We thus decided to investigate whether the general
the Protein Data Bank database (29) pinpointed FemX from control nonderepressible-5 (GCN5)-related N-acetyltransferase
Weissella viridescens (FemX ) as the closest 3D structural match to
domain of DhpH could form L-Leu-Ala(P), which was also
Wv
the C-terminal domain of DhpH (sequence identity: 17%; similar- chemically synthesized (SI Appendix, Figs. S12B and S14 A and B).
ity: 29%). When we purified the full-length DhpH protein, we ob-
served a high A260/A280 ratio (approximately 1.3) after IMAC
and desalting purification steps, a strong indicator of nucleic acid
Indeed, when DhpH and DhpD were incubated with rac-pSer(P)
Leu
and L-Ala in the presence of the tRNA regeneration system, an
additional radioactive spot appeared on the TLC (Fig. 3). The R
f
copurification. We also cloned and overexpressed in E. coli the C- value of the product was identical to that of synthetic L-Leu-L-
terminal domain of DhpH as a histidine-tagged fusion protein
His -DhpH-C) and observed the same high A260/A280 ratio. In
contrast, purified His
Ala(P) (SI Appendix, Fig. S15). To confirm the chemical structure
of the radioactive product, L-Ala(P) was incubated on larger scale
(
6
6
-DhpH-N exhibited a A260/A280 ratio of 0.7 with DhpH-C or DhpH in the presence of leucine and the Leu-
14
Fig. 3. Radioactive TLC analysis of the conversion of rac-pSer(P) to L-[ C(U)]-Leu-Ala(P) by DhpH and DhpD in a one-pot reaction. (A) Reaction scheme. (B)
14
Reaction progress and scanned phosphorimaging plate of silica TLC sheets spotted with: lane 1, aliquot of a reaction containing DhpH, rac-pSer(P), L-[ C(U)]-
Leu
Leu, tRNA and (re)generation components of Leu-tRNA ; lane 2, aliquot of a reaction containing L-Ala in addition to the components in lane 1; lane 3, aliquot
of a reaction containing L-Ala and DhpD in addition to the components in lane 1.
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Bougioukou et al.

tRNALeu regeneration components. We purified the product by
high performance liquid chromatography (HPLC) and showed it
exhibited spectral data identical to that of synthetic L-Leu-L-
Ala(P) (SI Appendix, Fig. S14 C and D). Combined, these results
suggest that Ala(P) is the physiological substrate for the C-termi-
nal domain of DhpH, but it raises the question of how the alkene is
then installed in dehydrophos because phosphate elimination is no
longer possible. We address this question in the next section.
A series of control experiments confirmed that amide bond
formation absolutely depends on tRNA (SI Appendix, Figs. S16
and S17). When using L-Ala(P) as substrate, only partial conver-
sion to the corresponding dipeptide was observed in the absence of
externally added nucleic acids. Furthermore, the dipeptide L-Leu-
L-Ala(P) was not formed when DhpH was preincubated with
RNase (SI Appendix, Fig. S18). Thus, the observed partial activity
in the absence of exogenous tRNA was the result of the RNA
content of the DhpH preparation as described above. Lastly, by
coupling the continuous formation of AMP, owing to the com-
bined action of LeuRS and DhpH, with NADH oxidation in the
presence of phosphoenolpyruvate (PEP), myokinase, PEP kinase,
and LDH (31), we were able to demonstrate the dependence of
DhpH and DhpH-C activity on L-Ala(P) concentration (SI Ap-
pendix, Fig. S19).
2
In Vitro Reconstitution of the Fe(II)/α-KG/O –Dependent DhpJ Activity.
3
1
As mentioned above, the formation of L-Leu-L-Ala(P) raises the
question how the alkene is formed in dehydrophos. Based on bio-
informatic analysis, DhpJ exhibits up to 40% sequence similarity
with previously characterized aspartyl/asparaginyl β-hydroxylases
Fig. 4.
P-NMR analysis of DhpJ activity with a 1:1.2 mixture of mono-
methylated and unmethylated L- Leu-L-Ala(P). (A) Reaction scheme of DhpJ
activity. (B) P-NMR spectrum of the a solution of L-Leu-L-Ala(POMe) and
L-Leu-L-Ala(P). (C) P-NMR spectrum taken 6 h after addition of Fe(II)-recon-
stituted DhpJ, α-KG, O , and L-ascorbic acid. MAP was presumably the side
product of L-Leu-ΔAla(POMe) hydrolysis. The phosphate peak (between
and 4 ppm) was omitted for clarity.
31
31
(32). The prediction that DhpJ might modify peptides prompted us
2
to overexpress the protein in E. coli. His-tagged DhpJ possessed
limited solubility and, thus, we elected to work with a maltose
binding protein (MBP) fusion protein (MBP-DhpJ). After ex-
cluding the possibility that MBP-DhpJ acted on other inter-
mediates of dehydrophos biosynthesis, such as 2-HEP, L-Ala(P),
and DHEP, we tested its activity against a panel of dipeptides and
tripeptides listed in SI Appendix, Fig. S20. Interestingly, L-Leu-L-
Ala(P) was converted to L-Leu-ΔAla(P), whereas for all other
substrates, only the starting material was recovered. The formation
of the unsaturated dipeptide was accompanied by the formation of
a second major product (Fig. 4C and SI Appendix, Figs. S21 and
2
MBP-DhpK catalyzed the addition of Gly to the N terminus of
synthetic L-Leu-Ala(P), L-Leu-ΔAla(P), and methylated L-Leu-
ΔAla(P) in assays similar to those described for DhpH-C (SI
Appendix, Figs. S33–S35). DhpK could not ligate Gly to L-Ala(P).
Discussion
Based on nature’s strategy to introduce dehydroalanine residues
into peptides (33) and the small natural product valanimycin (34),
the biosynthesis of the phosphonate analog of dehydroalanine in
dehydrophos could have featured generation of the tripeptide Gly-
1
S22). The H-NMR spectrum of the HPLC-purified side product
suggested that the dipeptide had been hydroxylated at the α-carbon
of L-Ala(P) (Fig. 4A and SI Appendix, Figs. S22 and S24); attempts
to confirm this assignment by mass spectrometry were unsuccessful Leu-Ser(P) and phosphorylation of the alcohol of Ser(P) by a ki-
(
SI Appendix, Fig. S24). When the isolated side product was in- nase. Elimination across the Cα-Cβ bond would then generate the
cubated with a fresh solution of DhpJ in the presence of Fe(II)/ ΔAla(P) group (Fig. 5A). However, although a gene encoding a pu-
α-KG/O
2
and L-ascorbic acid, no conversion to L-Leu-ΔAla(P) was
tative kinase, dhpB, is present in the dehydrophos biosynthetic gene
cluster, a gene encoding a pSer lyase-like enzyme is not. pSer(P)
observed (SI Appendix, Figs. S25–S27). Together, these observa-
tions suggest that the putative hydroxylated dipeptide is not an was the Ariadne’s thread in our previous efforts to resolve the puzzle
intermediate in L-Leu-ΔAla(P) formation and imply that perhaps of dehydrophos biosynthesis. The appearance of phosphorylated
the L-Leu-L-Ala(P) is not the true physiological substrate of DhpJ.
intermediates such as HP-EP and pSer(P) in spent supernatant of
Indeed, incubation of DhpJ with the monomethylated version of the blocked mutants ΔdhpC and/or ΔdhpH (18, 35) suggested that
L-Leu-L-Ala(P) (SI Appendix, Fig. S28) afforded the corresponding
unsaturated dipeptide as the only product (Fig. 4C and SI Appendix,
Figs. S29–S32), suggesting that methylation of L-Leu-L-Ala(P) by
the phosphorylation event occurs early in DHP biosynthesis, and
that the phosphate would be eliminated subsequently to install the
vinyl group. However, DhpH was unable to incorporate pSer(P)
DhpI occurs before desaturation. In support of this hypothesis, into a peptide in our in vitro studies. Instead, DphH eliminated the
when nearly equimolar amounts of L-Leu-L-Ala(POMe) and
phosphate from pSer(P) to generate AP, DhpD converted AP to
L-Leu-L-Ala(P) were allowed to compete for MBP-DhpJ in the Ala(P), and Ala(P) was efficiently condensed with Leu by the C-
same reaction mixture, the methylated substrate was cleanly desa- terminal domain of DhpH. DhpJ then installed the olefin in
turated, whereas the nonmethylated substrate once again was a desaturation reaction (Fig. 5B). This pathway is attractive be-
partially hydroxylated (Fig. 4 B and C).
cause it provides a role for DhpJ, which had no function in the
previous proposal. It also provides further evidence that methyl-
ation occurs late in the biosynthetic pathway, because otherwise
the active compound MAP would be formed inside the producing
In Vitro Reconstitution of DhpK Activity. The observed selectivity of
DhpJ for L-Leu-L-Ala(P)-OMe as substrate suggested that addition
of the N-terminal Gly would be the last step of DHP biosynthesis. organism. The 100-fold lower activity of DhpD on MAP than on
DhpK possesses only 16% identity to the C-terminal domain of AP also agrees with methylation in a subsequent step. However, by
DhpH, yet both amino acid sequences have the FemX peptidyl using DhpD to convert AP to Ala(P), a PLP-dependent enzyme
Wv
transferase as closest 3D structural match. His-tagged DhpK was appears to be missing from the dehydrophos gene cluster to convert
soluble in E. coli only when coexpressed with the chaperones GroEL/
OP-EP to pSer(P). Indeed, when DhpD or DhpH was incubated
S; therefore, we decided to work with a MBP-fused construct. with pSer(P) to look at the reverse reaction (conversion to OP-EP),
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Fig. 5. Revised biosynthesis of dehydrophos. (A) Proposed model for the biosynthesis of dehydrophos based on precedent in dehydroalanine formation. (B)
Proposed model for the biosynthesis of dehydrophos based on the in vitro biochemical analysis of DhpD, DhpH, DhpK, and DhpJ. (C) Alternative mechanism
for the PLP domain of DhpH. A single capital letter denotes proteins participating in the DHP biosynthesis. Capital letters X and Y denote hypothetical
proteins that are not present in the DHP biosynthetic gene cluster.
no activity was observed. We cannot rule out that an aminotrans- this emerging class of ligases is unexplored and most likely distinct
ferase that is not encoded in the biosynthetic gene cluster may from a second recently introduced class of tRNA-dependent cyclo-
generate pSer(P) from OP-EP (e.g., protein Y in Fig. 5B); in fact,
the formation of pSer(P) by the dhpH blocked mutant in combi-
nation with our in vitro characterization of DhpD/DhpH support
this notion. However, an additional aminotransferase is not abso-
lutely necessary if OP-EP encounters DhpH in the pyridoxamine
phosphate (PMP) form (SI Appendix, Fig S36). If so, the N-terminal
domain of DhpH could catalyze phosphate elimination to AP,
followed by the regeneration of the PMP form with an appropriate
amine donor such as Ala (Fig. 5 B and C); indeed, we show that
DhpH has aminotransferase activity. In this explanation, pSer(P)
would never appear as an intermediate during the in vivo bio-
dipeptide synthetases (38, 39). Our results strengthen the notion
that the role of amino acylated-tRNAs goes well beyond their use in
protein and cell wall biosynthesis (40, 41).
The introduction of the vinyl moiety into dehydrophos is ac-
complished in an unusual way: A Fe(II)/α-KG/O
hydroxylase evolved into a desaturase. The apparent dilemma
hydroxylation vs. desaturation) is well-documented for this class
2
-dependent
(
of enzymes (42–44), but the ability of an α-KG–dependent oxy-
genase to desaturate linear peptides has been demonstrated only
very recently (45) and only as a promiscuous activity. It remains to
synthesis of DHP; it would have accumulated in the dhpH mutant be seen how general this function is.
35) because of a fortuitous action of a nonspecific aminotrans-
In conclusion, the biosynthetic pathway to dehydrophos can be
ferase on OP-EP. Indeed, a similar phenomenon has been observed divided into three parts. The first part consists of three steps in-
(
in the case of a dhpG blocked mutant, which accumulates 2-ami-
noethylphosphonate instead of phosphonoacetaldehyde (18) and
in the biosynthesis of phosphinothricin (19). We intended to test
this possibility by accessing OP-EP enzymatically from DHEP by
the consecutive actions of DhpB and DhpC (or vice versa), but
attempts to obtain the kinase DhpB by expression in E. coli
were unsuccessful.
volved in the biosynthesis of many of the natural phosphonates
investigated thus far: installation of the C-P bond, decarboxylation,
and reduction of phosphonoacetaldehyde to 2-HEP (Figs. 1D and
5
). From 2-HEP onwards, the reactions are at present unique to
dehydrophos biosynthesis. The middle part of the pathway in-
volves the introduction of an amine functionality at the α-carbon
and complete removal of functional groups at the β-carbon of
The evolutionary advantage of the fused domains of DhpH (PLP
domain and GCN5-related N-acetyltransferase domain) is un- 2-HEP to generate Ala(P), which heretofore was considered to be
known but, based on our data, is unrelated to channeling of a re- only a manmade compound (Fig. 5B). Hence, although late
active intermediate from one active site to another. DhpH and methylation prevents formation of the active molecule MAP
DhpK are two examples of a small but growing list of peptide ligases during DHP biosynthyesis, the biosynthetic pathway delineated
that are not directly ATP-dependent but instead rely on the cell’s
primary metabolism and use aminoacyl-tRNAs, normally dedicated
to protein biosynthesis, to accomplish their functions in natural
product biosynthesis. Parry and coworkers initially reported the
herein requires the producing organism to protect itself from in-
hibition of Ala racemase by L-Ala(P) in a manner that is unknown.
The final part includes four steps: ligation of the proteinogenic
amino acid leucine to Ala(P) by the C-terminal domain of DhpH,
monomethylation of the phosphonic acid by DhpI, introduction of
the C-C double bond by DhpJ, and attachment of glycine to the
unsaturated dipeptide by DhpK. These four steps are executed by
Ser
involvement of Ser-tRNA in the biosynthesis of a natural product
(
valanimycin) (36), and more recently Walsh and coworkers showed
that PacB, which also has similarity to the FemXWv peptidyl-
Ala
transferase, transfers L-Ala from L-Ala-tRNA to the N terminus
of a tetrapeptide intermediate during biosynthesis of pacidamycin a set of quite unusual enzymes in the context of biosynthesis of
(37). The molecular and structural basis of peptide formation by
natural products (Fig. 5B).
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Bougioukou et al.

Materials and Methods
All experimental techniques are described in detail in SI Appendix.
incubation at ambient temperature, the enzymes were removed by passing
the reaction mixture through an Amicon spin column (10-kDa molecular
weight cutoff; MWCO). The conversion of the reaction (60%) was calculated
by integrating the signals of starting material and product by P-NMR
spectroscopy. To this solution, L-Leu-L-Ala(P) was added such that the final
ratio of monomethylated/unmethylated dipeptide was 1:1.2. After lyophili-
zation, the material was dissolved in 0.5 mL of a solution containing 6 mM
31
Radioactive-TLC Assay. All three reaction mixtures (50 μL) contained 0.3 mg of
14
tRNA, 10 mM rac-pSer(P), 5 mM L-Leu, 0.85 μCi L-[ C(U)]-Leu, 5 mM ATP,
μM DhpH, 5 μM LeuRS and 2 U TIPP, 100 mM Hepes, 10 mM KCl, and 20 mM
MgCl at pH 7.5. The reaction mixture corresponding to TLC lane 2 was
5
2
4 2 4 2
α-KG, 3 mM L-ascorbic acid, 0.2 mM (NH ) Fe(SO ) , and 40 μM MBP-DhpJ
supplemented with 10 mM L-Ala. The reaction mixture corresponding to TLC
lane 3 was supplemented with 10 mM L-Ala and 10 μM DhpD. The reactions
were incubated at room temperature and 0.5-μL aliquots were removed
periodically and spotted on a 5 × 10 cm TLC aluminum sheet Si 60. The TLC
plates were developed by using butanol/water/acetic acid at a ratio of 48/12/
reconstituted on ice in an anaerobic chamber with 1.2 equivalent of Fe(II). The
reaction mixture was incubated for 5 h at room temperature. Then, approx-
imately 100 μL of Chelex 100 resin was added to the reaction tube to remove
iron ions, and the solution was passed through an Amicon spin column (30
2
kDa MWCO). Before NMR analysis, 150 μL of D O was added into the sample.
2
0 as developing solvent and were dried before they were placed in the
Based on integration of the signals of Fig. 4C, about 21% was side-product,
about 37% L-Leu-ΔAla(POMe), about 37% L-Leu-ΔAla(P), and about 5%
belonged to MAP.
phosphorimaging cassette. R values of radioactive spots were compared
f
with those obtained from a TLC plate spotted with standards of leucine,
L-Leu-L-Ala(P), and L-Leu-L-ΔAla(P) developed identically and visualized with
ninhydrin solution (SI Appendix, Fig. S15).
ACKNOWLEDGMENTS. We thank Susan A. Martinis (Department of Bio-
chemistry, University of Illinois at Urbana–Champaign) for providing the
E. coli strain that expressed the leucyl-tRNA synthetase. This work was sup-
ported by National Institutes of Health (NIH) Grant P01 GM077596. NMR
spectra were recorded on a 600-MHz instrument purchased with support
from NIH Grant S10 RR028833.
Competition Experiment Between Monomethylated and Unmethylated L-Leu-L-
Ala(P) in the Presence of MBP-DhpJ. L-Leu-L-Ala(POMe) was prepared by
reacting 3 mM L-Leu-LAla(P) with 5 mM SAM in the presence of 50 μM DhpI
and 20 μM SAHC nucleosidase in 50 mM Na-Hepes at pH 7.5. After 6-h
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