New insight into the mechanism of methyl transfer during the biosynthesis of fosfomycin

Article abstract of DOI:10.1039/b614678c

Hydroxyethylphosphonate is a required intermediate in fosfomycin biosynthesis. The Royal Society of Chemistry.

Full text of DOI:10.1039/b614678c

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New insight into the mechanism of methyl transfer during the
biosynthesis of fosfomycin{
Ryan D. Woodyer,a Gongyong Li,a Huimin Zhao*ab and Wilfred A. van der Donk*a
Received (in Cambridge, MA, USA) 9th October 2006, Accepted 8th November 2006
First published as an Advance Article on the web 23rd November 2006
DOI: 10.1039/b614678c
required for heterologous production. These results in combina-
Hydroxyethylphosphonate is a required intermediate in fosfo-
mycin biosynthesis.
tion with bioinformatics analysis lead to a radically different
mechanism for the methyltransferase encoded by fom3.
(1R,2S)-Epoxypropylphosphonic acid, also known as fosfomycin,
is a clinically utilized, highly effective antibiotic that has low
toxicity and is effective against methicillin and vancomycin
resistant pathogens.1–3 Seminal pioneering studies by Seto and
Hammerschmidt using genetic techniques complemented by
feeding studies with isotopically labeled precursors suggested the
biosynthetic pathway for fosfomycin shown in Scheme 1A.4–11
Yet, the mechanism of an unprecedented methyl transfer step in
the biosynthesis of fosfomycin has remained unresolved.5,11 We
recently identified the complete biosynthetic cluster for this natural
product from Streptomyces fradiae and achieved heterologous
production in S. lividans.12 Here we use genetic and chemical
complementation studies to show that fomC, a gene with a
previously unknown role in fosfomycin biosynthesis, is absolutely
In the currently accepted biosynthetic pathway for fosfomycin
(Scheme 1A), PEP is converted to phosphonopyruvate (PnPy) by
PEP mutase (Fom1),13 followed by decarboxylation by Fom2 to
produce phosphonoacetaldehyde (PnAA). In the subsequent step,
Fom3 has been proposed to catalyze transfer of a methyl anion
from methylcobalamin (MeCbl) to PnAA to form 2-hydroxypro-
pylphosphonate (HPP) (Scheme 2).5,11,14 In the final step, HPP is
oxidized by Fom4 to form the epoxide of fosfomycin.15–23 The
proposed mechanism of the methyl transfer reaction catalyzed by
Fom3 is fundamentally different from the mechanisms for all
known MeCbl dependent methyltransferases in which methylation
takes place via nucleophilic attack by the substrate at the methyl
group of MeCbl.24
Intrigued by the apparent mechanistic paradox in how Fom3
would utilize B12 for methyl transfer, we initiated a reinvestigation
of the pathway. Several genes besides fom1–4 have been suggested
to be part of the biosynthetic cluster and were labeled fomA–F.4
FomA and FomB provide resistance to fosfomycin via phosphor-
ylation.25,26 We recently proposed a function for fomD and
determined that fomE,F are not involved directly in fosfomycin
production.12 No function has yet been proposed for fomC.
Seto and coworkers showed that a mutant strain with a block in
the vitamin B12 biosynthetic pathway could not produce
fosfomycin, but did convert HPP to fosfomycin.15 In addition
14C-labeled MeCbl fed to this blocked mutant resulted in 14C-
labeled fosfomycin.14 Based on these results, it was proposed that
MeCbl was the methyl donor. However, no intermediates could be
isolated from the mutants in which either the B12 pathway15 or the
putative methyltransferase Fom34 was disrupted and neither
PnAA nor aminoethylphosphonate (AEP) could be converted to
fosfomycin in S. lividans expressing Fom3 and Fom4, whereas
HPP was successfully converted.4 Thus, although the pathway in
Scheme 1 Comparison of previously proposed biosynthetic pathway (A)
and revised pathway (B).
aDepartment of Chemistry, University of Illinois at Urbana-Champaign,
600 S Mathews Ave, Urbana, IL 61801, USA. E-mail: vddonk@uiuc.edu;
Fax: (217) 244 8533; Tel: (217) 244 5360
bDepartment of Biomolecular and Chemical Engineering, University of
Illinois at Urbana-Champaign, 600 S Mathews Ave, Urbana, IL 61801,
USA. E-mail: zhao5@uiuc.edu; Fax: +1 (217) 333-5052;
Tel: +1 (217) 333-2631
{ Electronic supplementary information (ESI) available: Procedures for
transposon gene disruption, complementation, and chemical synthesis of
HEP. See DOI: 10.1039/b614678c
Scheme 2 Proposed mechanism of B12-dependent methyl transfer to
PnAA.5,11 The corrin ligand ring system is schematically represented by a
square. L may be the benzimidazole ligand of B12 or an amino acid ligand
from the protein.
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Chem. Commun., 2007, 359–361 | 359

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Scheme 1A is consistent with these findings, they do not provide
insights into whether PnAA is indeed transformed directly into
HPP by Fom3. With our heterologous production system, we set
out to study the genes involved in conversion of PnAA to HPP
with the goal of providing new insights into the methylation step.
A conserved domain search27 of the Fom3 sequence showed it
has two conserved domains. The N-terminal domain was identified
as a B12-like binding domain, whereas the C-terminal domain
shows homology to the radical-SAM protein family,28 containing
three conserved Cys residues that serve as ligands to a [4Fe–4S]
cluster. The FomC sequence has conserved domains similar to the
class III Fe2+-dependent ADH family.29 A sequence alignment of
FomC from S. fradiae with structurally characterized family
members reveals several conserved residues, including His257,
His271, and Asp189 (FomC numbering), which are ligands to the
required Fe2+ in these homologs.30 Typically, another His serves as
the final enzyme ligand to the metal, however, in this place Gln193
is found in FomC.
importance of FomC was suggested by our recent determination
of the minimal gene cluster for fosfomycin production.12 The
requirement of FomC is puzzling since it is not involved in the
biosynthetic pathway in Scheme 1A. Its gene fomC appears to be
transcriptionally coupled to fom1 and fom2 (Fig. 1A), and
therefore its activity is likely linked to these two genes. Thus,
PnAA, the product of the enzymes Fom1 and Fom2, was
hypothesized to be the substrate for the dehydrogenase FomC,
reducing it to hydroxyethylphosphonate (HEP).
The S. lividans clones containing the gene-disrupted clusters
were subsequently grown in the presence of 0.5 mM AEP (Fig. 1C).
Addition of AEP did not successfully complement the gene
disruptions of either fomC or fom3, whereas it has been shown to
complement fom2 loss of function mutants in S. wedmorensis4
(Scheme 1A). Previous studies have also shown that fom3 loss of
function mutations can be complemented by HPP.4 Therefore, it
can be concluded that both fom3 and fomC are involved in the
conversion of PnAA into HPP. Importantly, production of
fosfomycin was successfully complemented in the fomC disrupted
clones by the addition of 0.5 mM HEP to the media (Fig. 1D),
whereas this did not complement fosfomycin production in the
fom3 disrupted clone. These data strongly support the hypothesis
that HEP is a productive intermediate in the fosfomycin pathway
as the product of FomC. This result would also imply that HEP is
the substrate of fom3, resulting in a revised route for fosfomycin
biosynthesis (Scheme 1B).
In order to discern the order and nature of events between the
formation of PnAA and the formation of HPP, four gene
disrupted fosmids containing the fosfomycin biosynthetic cluster12
were obtained by transposon insertion (Fig. 1A). Two of these
gene disruptions, fomC1::mini-MuAE5 and fomC2::mini-MuAE5,
were in fomC, a positive control disruption (orf12::mini-MuAE5)
was y 12 kb downstream of the cluster in orf12 (known not to be
involved in fosfomycin production),12 and the final gene disruption
(fom3::mini-MuAE5) was in the putative methyltransferase fom3.
These gene disrupted fosmids were introduced into S. lividans and
checked for bioactivity (Fig. 1B). As expected, a large growth
inhibition zone of a sensitive E. coli strain was observed for the
positive control. This inhibition has been shown to be due to
fosfomycin production.12 The lack of inhibition zones for the other
three strains suggests that fomC and fom3 are both absolutely
required for fosfomycin production. The requirement of Fom3 is
consistent with previous genetic complementation studies,4 and the
The revised pathway is consistent with all previously reported
studies. In fact, this pathway explains why neither PnAA nor AEP
could be converted to fosfomycin by S. lividans expressing fom3
and fom4 alone, whereas HPP was converted;4 HEP complemen-
tation was not reported in this context. Interestingly, it has been
shown that HEP can be converted to fosfomycin by wild type
producing organisms,11,15 but without compelling evidence HEP
was believed to be an off-pathway intermediate. The revised
pathway is also consistent with the elegant labeling studies by
Hammerschmidt and coworkers.8,10,11 When [2,2-2H2]-HEP was
fed to S. fradiae, 34% of the fosfomycin produced contained label.8
Feeding (S)-[2-2H1]-HEP and (R)-[2-2H1]-HEP resulted in 32%
labeled fosfomycin from the (S)-labeled compound and no labeled
fosfomycin from the (R)-labeled compound.10 Moreover, when
18O-HEP was fed to S. fradiae, 50% of the produced fosfomycin
was 18O-labeled.11 These studies revealed for the first time the
unusual nature of the epoxidation step, but as pointed out by
Hammerschmidt,11 the retention of the label requires remarkably
fast utilization of labeled PnAA since the half-life for exchange of
oxygen in acetaldehyde is about 1 min at pH 7.31 The pathway in
Scheme 1B, however, does not require any kinetic limitations since
the label would never be in an exchangeable position.
The revised pathway argues against the methyl anion mechan-
ism proposed5,11 for Fom3 in Scheme 2. Considering the
bioinformatics data and the observed complementation of fomC
disrupted mutants with HEP, it is likely that HEP is the substrate
for Fom3. Then a chemically more pleasing mechanism can be
drawn (Scheme 3) in which SAM and a reduced [4Fe–4S] cluster
form a 59-deoxyadenosyl radical, as would be typical of radical-
SAM protein family members.32–34 The adenosyl radical then
abstracts the pro-R hydrogen (directly or through an enzyme
radical intermediate35) from the C2 position of HEP. Such
hydrogen atom abstractions by a 59-deoxyadenosyl radical are well
Fig. 1 Chemical complementation. A) Transposon disruptions in the
fosmid of orf12 (positive control), fom3 (negative control), and fomC were
reintroduced into S. lividans. B) Gene disruption of fom3 and fomC
abolishes fosfomycin heterologous production as evidenced by a bioassay
against a sensitive E. coli strain.12 C) Feeding S. lividans gene disrupted
strains 0.5 mM AEP did not complement fosfomycin production. D)
Feeding S. lividans gene disrupted strains 0.5mM HEP resulted in small
inhibition zones for fomC disrupted clones, but no complementation for
fom3 was observed. I = orf12::mini-MuAE5, II = fom3::mini-MuAE5,
III = fomC1::mini-MuAE5, IV = fomC2::mini-MuAE5.
360 | Chem. Commun., 2007, 359–361
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in part by grants from the National Institutes of Health (GM63003
to WAV) and a grant from the Office of Naval Research (N00014-
02-1-0725 to HZ).
Notes and references
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Scheme 3 Proposed methyltransferase mechanism. One electron is
transferred from the reduced iron–sulfur cluster to SAM to form an
adenosyl radical and methionine. The adenosyl radical abstracts the pro-R
hydrogen atom from C2 of HEP, and the resulting substrate radical reacts
with MeCbl yielding HPP and cob(II)alamin. The enzyme is then returned
to the active state by reduction of the 4Fe4S cluster back to the +1 state
and binding of SAM and MeCbl. Alternatively, cob(II)alamin might be
reduced to cob(I)alamin and methylated while bound to the enzyme
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precedented in adenosylcobalamin dependent enzymes.24 The
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without major modification in each case. The results presented
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methylation reactions in secondary metabolism. This strategy
requires stoichiometric use of both SAM and MeCbl to achieve
methylation of non-activated carbon centers.
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The authors thank Bill Metcalf for contribution of plasmids and
bacterial strains. We also thank Andrew Eliot for development of
the transposon disruption method and Josh Blodgett for help with
Streptomycete microbiology techniques. This work was supported
This journal is ß The Royal Society of Chemistry 2007
Chem. Commun., 2007, 359–361 | 361