Biosynthesis of Rhizocticins, Antifungal Phosphonate Oligopeptides Produced by Bacillus subtilis ATCC6633

Article abstract of DOI:10.1016/j.chembiol.2009.11.017

Rhizocticins are phosphonate oligopeptide antibiotics containing the C-terminal nonproteinogenic amino acid (Z)-l-2-amino-5-phosphono-3-pentenoic acid (APPA). Here we report the identification and characterization of the rhizocticin biosynthetic gene cluster (rhi) in Bacillus subtilis ATCC6633. Rhizocticin B was heterologously produced in the nonproducer strain Bacillus subtilis 168. A biosynthetic pathway is proposed on the basis of bioinformatics analysis of the rhi genes. One of the steps during the biosynthesis of APPA is an unusual aldol reaction between phosphonoacetaldehyde and oxaloacetate catalyzed by an aldolase homolog RhiG. Recombinant RhiG was prepared, and the product of an in vitro enzymatic conversion was characterized. Access to this intermediate allows for biochemical characterization of subsequent steps in the pathway.

Full text of DOI:10.1016/j.chembiol.2009.11.017

Chemistry & Biology
Article
Biosynthesis of Rhizocticins, Antifungal
Phosphonate Oligopeptides Produced by
Bacillus subtilis ATCC6633
1,2,
Svetlana A. Borisova,1 Benjamin T. Circello,1,2 Jun Kai Zhang,2 Wilfred A. van der Donk,1,3,4, and William W. Metcalf
*
*
1Institute for Genomic Biology
2Department of Microbiology
3Department of Chemistry
4Howard Hughes Medical Institute
University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
*Correspondence: vddonk@uiuc.edu (W.A.V.), metcalf@uiuc.edu (W.W.M.)
DOI 10.1016/j.chembiol.2009.11.017
SUMMARY
L-threonine (Figure 1C) (Kugler et al., 1990; Laber et al., 1994).
Hence, APPA interferes with the biosynthesis of threonine and
Rhizocticins are phosphonate oligopeptide antibi- related metabolic pathways, ultimately affecting protein synthe-
sis and leading to growth inhibition. The inhibitory activity of
APPA is due to the structural resemblance to phosphohomoser-
otics containing the C-terminal nonproteinogenic
amino acid (Z)-L-2-amino-5-phosphono-3-pentenoic
acid (APPA). Here we report the identification and
characterization of the rhizocticin biosynthetic gene
cluster (rhi) in Bacillus subtilis ATCC6633. Rhizocticin
B was heterologously produced in the nonproducer
strain Bacillus subtilis 168. A biosynthetic pathway
ine, but it possesses a hydrolytically stable C-P bond in place of
the C-O-P moiety of phosphohomoserine.
Rhizocticins exhibit antifungal activity, whereas plumbemy-
cins are antibacterials. It has been demonstrated that plumbe-
mycins also enter Escherichia coli K-12 via the oligopeptide
transport system (Diddens et al., 1979). As in the case of rhizoc-
is proposed on the basis of bioinformatics analysis
of the rhi genes. One of the steps during the biosyn-
thesis of APPA is an unusual aldol reaction between
ticins, L-threonine reverses the growth inhibition by plumbemy-
cins in a concentration-dependent manner (Park et al., 1977b).
Therefore, similarly to rhizocticins, plumbemycins must be
phosphonoacetaldehyde and oxaloacetate cata- cleaved by peptidases of the target cell to release the active
substance, APPA. The selectivity of these tripeptide antibiotics
is thus not due to a difference in mode of action, but rather is
lyzed by an aldolase homolog RhiG. Recombinant
RhiG was prepared, and the product of an in vitro
enzymatic conversion was characterized. Access to
this intermediate allows for biochemical character-
determined by the recognition of proteinogenic amino acids
attached at the N terminus of APPA by a specific oligopeptide
transport system or peptidase. This feature can potentially be
ization of subsequent steps in the pathway.
exploited to create APPA-containing oligopeptides with specific
selectivity for a particular organism. Furthermore, the target of
APPA, threonine synthase, is not present in mammals, reducing
the likelihood of toxicity to humans. Thus, such phosphonate
INTRODUCTION
Rhizocticins are phosphonate-containing oligopeptide antibi- oligopeptides can be promising therapeutics.
otics produced by the gram-positive bacterium B. subtilis
Phosphonate compounds are prevalent among biologically
ATCC6633. They were originally discovered in 1949 on the basis active molecules, mainly because of their ability to function as
of their antifungal activity and were collectively termed ‘‘rhizoc- stable mimics of carboxylate and phosphate-containing metab-
tonia factor’’ (Michener and Snell, 1949). The structures of rhi- olites. At present, biosynthetic pathways of only a handful of
zocticins were determined 40 years later (Rapp et al., 1988). phosphonates have been extensively studied (see Metcalf and
They are di- and tripeptide antibiotics consisting of a variable van der Donk, 2009 for a recent comprehensive review on the
amino acid at the N terminus followed by arginine and the topic), including fosfomycin (Hidaka et al., 1995; Woodyer
nonproteinogenic amino acid (Z)-L-2-amino-5-phosphono-3- et al., 2006), dehydrophos (B. T. Circello and W.W.M., unpub-
pentenoic acid (APPA; Figure 1A). Interestingly, APPA is also the lished data), FR-900098 (Eliot et al., 2008), and phosphinothri-
C-terminal amino acid of the tripeptide antibiotics plumbemycin cin-containing peptides (e.g., PTT) (Blodgett et al., 2007)
A and B produced by Streptomyces plumbeus (Figure 1B) (Park (Figure 1D). These studies have shown that the biosyntheses
et al., 1977a; Park et al., 1977b).
of this class of compounds provide a rich source of novel
Rhizocticins enter the target fungal cell through the oligopep- biochemistry as exemplified by many unique enzymatic transfor-
tide transport system (Kugler et al., 1990). They are then cleaved mations (Cicchillo et al., 2009; Higgins et al., 2005). Therefore,
by host peptidases to release APPA, which inhibits threonine a better understanding of the rhizocticin pathway will be of great
synthase, an enzyme catalyzing the pyridoxal 50-phosphate interest. Furthermore, biosynthetic access to the APPA warhead
(PLP)–dependent conversion of phosphohomoserine to of the rhizocticins would be advantageous because organic
28 Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Biosynthesis of Rhizocticins
rhiA
B
C
D
E F
G
H
I
N
J
K L
M
Sensor His kinase
orf 2 345 6 7 8
Ser/Thr kinase
orf9 10 11 12
rhi cluster
skfH
B. subꢀlis 6633
skfA
ArsR regulator
B. subꢀlis 168
Figure 2. Organization of the Rhizocticin Gene Cluster and
Surrounding Genes on the B. subtilis ATCC6633 Chromosome
The same locus of B. subtilis 168 genome is also shown for comparison. Genes
with a high degree of homology between the two strains (>90% identity) are
shown in yellow. The biosynthetic clusters for rhizocticin and sporulation killing
factor are shown in blue and green, respectively. The corresponding location
of these loci in the other genome is denoted with a star of the same color.
the clones produced the desired PCR fragment, possibly
because of a highly divergent sequence of the PEP mutase
gene, which prevented annealing of the primers. Alternatively, a
PEP mutase–independent pathway might be used by B. subtilis
ATCC6633 for production of rhizocticin.
To investigate these alternatives, the genome of B. subtilis
ATCC6633 was sequenced using the 454 sequencing platform.
The details of the genome sequencing will be reported else-
where. Briefly, sequencing data were assembled into 37 contigs
spanning approximately 4.0 Mb. A total of 3769 open reading
frames (ORFs) were determined and annotated using the RAST
Server (Rapid Annotations using Subsystems Technology)
(Aziz et al., 2008). For comparison, the closely related B. subtilis
168 strain has a genome of 4.2 Mb comprising 4114 coding
sequences (Kunst et al., 1997). When searched for the PEP
mutase gene, the sequence of the genome of B. subtilis
ATCC6633 produced a single hit. This gene appears to be
a part of an operon consisting of 13 ORFs and is preceded by
a differentially transcribed additional ORF encoding a transcrip-
tional regulator. Careful analysis of the ORFs comprising this
operon led to the conclusion that these genes are likely to consti-
tute a rhizocticin biosynthetic gene cluster (Figure 2).
Figure 1. Chemical Structures of the Phosphonate Antibiotics
(A) Chemical structure of rhizocticins.
(B) Chemical structure of plumbemycins.
(C) The threonine synthase reaction inhibited by APPA.
(D) Chemical structures of representative phosphonate antibiotics whose
biosynthetic pathways have been studied.
synthesis of this molecule is very challenging. For instance,
biosynthetic preparation of APPA would provide an avenue to
combinatorially vary the N-terminal amino acids to create
analogs with desired specificity.
To this end, we identified the rhizocticin biosynthetic cluster in
the genome of B. subtilis ATCC6633 and confirmed its identity by
heterologous expression in B. subtilis 168. Although the first two
steps of the biosynthetic pathway appear to be the same as for
other phosphonate metabolites, the third step—an aldol reaction
between phosphonoacetaldehyde (PnAA) and oxaloacetic acid
(OAA)—is unprecedented. Biochemical characterization of this
step is also reported here. On the basis of genetic and biochem-
ical information, a pathway for the biosynthesis of rhizocticins is
proposed.
With the genome sequence available, the fosmid library of
B. subtilis ATCC6633 was screened as described above using
two sets of sequence-specific primers designed to amplify
short sequences upstream of the putative rhizocticin cluster
(within orf6) and within rhiM. Fosmid 2-11E was identified and
sequenced via the Sanger protocol using transposon insertions,
as described elsewhere (Blodgett et al., 2005; Eliot et al., 2008).
The sequence of the insert of 2-11E originating from B. subtilis
ATCC6633 DNA was identical to that of the corresponding frag-
RESULTS AND DISCUSSION
Identification of the Rhizocticin Biosynthetic Gene
Cluster
The first step in the biosynthetic pathways of the majority of ment obtained through 454 sequencing of the genome, with the
phosphonates is the isomerization of phosphoenolpyruvate exception of a single base-pair mismatch located outside of the
(PEP) to phosphonopyruvate (PnPy) catalyzed by phosphoenol- putative rhizocticin gene cluster.
pyruvate phosphomutase (PEP mutase) (Seidel et al., 1988).
B. subtilis ATCC6633 possesses a high degree of nucleotide
Previously, we successfully identified phosphonate biosynthetic sequence homology to B. subtilis 168. The putative rhizocticin
gene clusters by screening fosmid libraries with degenerate PCR gene cluster appears to be a single site insertion of approxi-
primers designed to amplify PEP mutase-encoding genes mately 13 kb into the genome of B. subtilis 168. Although the
(Blodgett et al., 2005; Eliot et al., 2008). We attempted a similar genes of the rhizocticin cluster have no homologs within the
approach here to identify the rhizocticin gene cluster. A fosmid B. subtilis 168 genome, the nucleotide sequences outside of
library of B. subtilis ATCC6633 was constructed and screened the cluster are approximately 90% identical. Interestingly,
by PCR for a PEP mutase gene fragment as described in the B. subtilis 168 contains a gene cluster (skf) located near the
Supplemental Information available online. However, none of ‘‘insertion site’’ of the rhizocticin gene cluster (blue star in
Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved 29

Chemistry & Biology
Biosynthesis of Rhizocticins
Table 1. Summary of the Open Reading Frames of the Rhizocticin Gene Cluster
Percentage
of aa identitya
ORF
orf6
orf7
orf8
rhiA
No. of aa Protein homology (NCBI No.)
325
223
322
296
B. subtilis 168 putative hydrolase/transferase (CAB11993) (325 aa)
94
95
87
65
18
46
28
27
62
B. subtilis 168 two-component response regulator YbdJ (BAA33098) (223 aa)
B. subtilis 168 sensor histidine kinase YbdK (BAA33099) (320 aa)
B. licheniformis transcriptional activator of the cysJI operon (AAU21843) (298 aa)
Salmonella enterica Typhimurium transcriptional regulator CysB (NP_460672) (324 aa)
Sphaerobacter thermophilus threonine synthase (ZP_04494878) (420 aa)
Mycobacterium tuberculosis threonine synthase (2D1F_B) (360 aa)
rhiB
rhiC
433
408
B. subtilis ATCC6633 threonine synthase ThrC (this study) (352 aa)
B. licheniformis hypothetical protein, related to NikS (YP_077482) (405 aa)
Streptomyces ansochromogenes nikkomycin biosynthesis protein SanS, D-Ala-D-Ala ligase homolog
30
(AAK53061) (424 aa)
rhiD
rhiE
rhiF
rhiG
407
167
186
337
B. licheniformis MFS transporter (YP_077483) (408 aa)
68
40
40
34
25
56
42
36
14
35
22
42
38
35
23
32
19
25
Sorangium cellulosum sulfopyruvate decarboxylase a-subunit (YP_001617955) (170 aa)
S. hygroscopicus phosphonopyruvate decarboxylase (Q54271) (401 aa)
Legionella pneumophila 4-hydroxy-2-oxovalerate aldolase (YP_096686) (295 aa)
Pseudomonas sp. bifunctional aldolase-dehydrogenase DmpG (1NVM_A) (345 aa)
Paenibacillus larvae putative PEP phosphomutase (ZP_02329666) (297 aa)
S. viridochromogenes PEP phosphomutase of PTT biosynthesis (AAU00071) (313 aa)
Pseudomonas syringae hypothetical protein (BAF32889) (354 aa)
rhiH
rhiI
296
362
132
393
85
Mycoplasma pneumonia HPr kinase/phosphatase (1KNX_A) (312 aa)
Chloroherpeton thalassium protein of unknown function UPF0047 (YP_001997537) (138 aa)
E. coli conserved hypothetical protein YjbQ (ZP_03048862) (138 aa)
Thermotoga lettingae aminotransferase class V (YP_001471385) (381 aa)
Methanocaldococcus jannaschii broad-specificity class V aspartate aminotransferase (NP_247954) (385 aa)
Natronomonas pharaonis glutaredoxin (CAI48716) (82 aa)
rhiN
rhiJ
rhiK
rhiL
rhiM
orf9
E. coli glutaredoxin 3 (1FOV_A) (82 aa)
215
413
256
Frankia sp. EAN1pec putative metallophosphoesterase (YP_001510901) (243 aa)
E. coli metal-dependent phosphodiesterase YfcE (P67095) (184 aa)
B. licheniformis hypothetical, related to NikS (YP_077482) (405 aa)
S. ansochromogenes nikkomycin biosynthesis protein SanS, D-Ala-D-Ala ligase homolog (AAK53061) (424 aa) 26
B. subtilis 168 putative serine/threonine protein kinase YbdM (O31435) (256 aa)
B. subtilis 168 putative phage protein YbdN (CAB11998) (285 aa)
B. subtilis 168 putative phage protein YbdO (CAB11999) (394 aa)
90
94
89
orf10 284
orf11 394
Note: aa, amino acid.
a The closest homologs were based on NCBI searches conducted October 8, 2009. The homolog whose biochemical function was experimentally
supported is shown for proteins of particular interest.
Figure 2). This gene cluster is absent from B. subtilis ATCC6633 analyzed with the Basic Local Alignment Search Tool (BLAST)
(its corresponding location is shown as a green star). The skf program at NCBI (Altschul et al., 1990) and the Phyre server
gene cluster is responsible for the biosynthesis and export of (Kelley and Sternberg, 2009). The gene annotations, along with
and the immunity to sporulation killing factor. This peptide the closest and functionally confirmed homologs, are shown in
antibiotic produced by sporulating B. subtilis 168 causes lysis Table 1.
of nonsporulating sibling B. subtilis 168 cells (Gonzalez-Pastor
As mentioned above, the genes surrounding the putative rhi-
et al., 2003). Thus, the rhi and skf gene clusters occupy essen- zocticin gene cluster (rhiA-rhiM; e.g., immediately adjacent
tially the same locus on the genomic DNA of related species, orf6-8 and orf9-11) have nearly identical counterparts in B. sub-
as commonly seen for the genes involved in secondary tilis 168. Therefore, they are not likely to be involved in rhizocticin
metabolism.
biosynthesis.
The rhiA gene encodes a putative transcriptional regulator of
the LysR family (Schell, 1993). The helix-turn-helix DNA-binding
Bioinformatic Analysis of the rhi Cluster
The genes of the rhizocticin biosynthetic cluster were first anno- motif, typical of many LysR regulators (Maddocks and Oyston,
tated using the RAST Server (Aziz et al., 2008) and further 2008), is predicted by the Phyre server to be located within the
30 Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Biosynthesis of Rhizocticins
N-terminal residues 30–85. A putative ligand-binding domain is thesis is usually a two-subunit enzyme (e.g., ComD and ComE
also present at the C terminus of RhiA. The rhiA gene is located in Methanococcus jannaschii) (Graupner et al., 2000). ComD
upstream and in the opposite direction of the other genes in the (a-subunit) is homologous to the N-terminal portion of single
rhi operon, as commonly seen for LysR-regulated operons (Mad- chain PnPy decarboxylases, whereas ComE (b-subunit) has
docks and Oyston, 2008).
homology to the C terminus. In a similar manner, RhiE and
The rhiB gene encodes a putative threonine synthase. Inter- RhiF are homologs of the a- and b-subunits of sulfopyruvate
estingly, the genome of B. subtilis ATCC6633 contains another decarboxylase, respectively, and the N- and C-terminal portions
copy of a threonine synthase gene, thrC, located in an operon of single chain PnPy decarboxylases. RhiF (like ComE) contains
with genes involved in the biosynthesis of threonine that is a canonical TPP-binding motif. To the extent of our knowledge,
present at the same site as the threonine synthase gene in the RhiE/RhiF is the first example of a PnPy decarboxylase consist-
B. subtilis 168 genome. Unlike RhiB, ThrC is highly homologous ing of two subunits.
to threonine synthases of gram-positive bacteria, predominantly
A search of the NCBI database for protein sequences homol-
Bacillus species (98% identical to threonine synthase of B. sub- ogous to the translated product of rhiG yielded a number of puta-
tilis 168), suggesting ThrC is a bona fide threonine synthase tive 4-hydroxy-2-oxovalerate aldolases with modest homology
involved in primary metabolism. RhiB and ThrC appear to be to RhiG (identity of 35% and lower). The closest homologs
evolutionary distinct because they share only 27% amino acid of RhiG that have been biochemically characterized are the
identity. It is conceivable that rhiB is involved in rhizocticin self- 4-hydroxy-2-oxovalerate aldolases NahM and DmpG (25%
resistance by encoding a threonine synthase homolog that is identity) of Pseudomonas putida strains (Manjasetty et al.,
not inhibited by APPA.
2003; Platt et al., 1995). They belong to the class II family of aldol-
The translated products of rhiC and rhiM are homologs (26% ases that are dependent on divalent metal ions for catalysis.
identity). The proteins with the closest homology are NikS and DmpG (and NahM) catalyzes the penultimate step of the meta-
SanS, participating in the biosynthesis of the peptidyl nucleoside cleavage pathway from catechol to pyruvate and acetyl-CoA
antibiotic nikkomycin (Lauer et al., 2001; Li et al., 2004). They during the catabolism of aromatic compounds by Pseudomonas
belong to the carboxylate-amine/thiol ligase superfamily of strains. DmpG is a part of a bifunctional enzyme complex
enzymes possessing a signature ATP-grasp structural motif because it physically associates with the enzyme of the following
(Galperin and Koonin, 1997). The enzymes of this superfamily step, acetaldehyde dehydrogenase (acylating) DmpF, to ensure
catalyze the ATP-dependent formation of peptide or thioester efficient transfer of the reactive intermediate acetaldehyde
bonds via a reactive acylphosphate intermediate. Examples (Figure S1).
include D-Ala-D-Ala ligase of peptidoglycan biosynthesis (Fan
The rhiH gene encodes a putative PEP mutase that presum-
et al., 1994; Zawadzke et al., 1991), glutathione synthetase ably would catalyze the first step in the biosynthetic pathway,
(Fan et al., 1995; Yamaguchi et al., 1993), and biotin carboxylase the conversion of PEP to PnPy.
(Artymiuk et al., 1996; Waldrop et al., 1994). Despite a variety of
The translated product of rhiI has no significant end-to-end
reactions catalyzed by the members of the ATP-grasp super- homology to any of the entries in the NCBI database. However,
family, enzymes catalyzing the formation of a ‘‘conventional’’ the C terminus of RhiI (approximately 213 amino acids) shows
proteinogenic a-peptide bond between L-amino acids have low homology to the C-terminal domain of the histidine-contain-
been identified only recently (Kino et al., 2009; Kino et al., ing phospho carrier protein (HPr) kinase/phosphorylase from
2008a; Kino et al., 2008b; Tabata et al., 2005). During the course several species. In low GC gram-positive bacteria, HPr is
of our study, the identification and substrate specificity of RhiM involved in the regulation of carbon catabolism (Martin-Ver-
was reported (named RizA by the authors) (Kino et al., 2009). straete et al., 1999). HPr kinase/phosphorylase is a bifunctional
RhiM (RizA) is capable of ligating L-arginine to 19 other amino protein that modifies Ser-46 of HPr and accepts ATP or
acids, including a saturated analog of L-APPA, 2-amino-5- pyrophosphate (PPi) as a phosphate group donor. RhiI contains
phosphonopentanoic acid (Kino et al., 2009). A sequence of an easily identifiable canonical nucleotide binding P loop
RhiC has also been deposited into GenBank (accession number (GSKGKGKS). It is conceivable that RhiI catalyzes an ATP-
BAH56723) by Kino and co-workers; its activity has not been dependent phosphorylation of a small molecule or plays a regu-
reported.
latory role similar to HPr kinase/phosphorylase.
RhiD is a putative transporter of the major facilitator super-
The translated gene product of rhiN shows homology to
family (MFS). Between 8 and 10 transmembrane helixes are pre- a number of hypothetical proteins belonging to an uncharacter-
dicted by different topology prediction tools (http://ca.expasy. ized protein family UPF0047 (ExPASy, Prosite). The members of
org). RhiD is likely responsible for the export of rhizocticins this family are small proteins of 14–16 kDa and are widely distrib-
from the cell.
uted among the three domains of life. Although several crystal
The genes rhiE and rhiF encode two subunits of a putative structures were solved (submitted to the Protein Data Bank but
PnPy decarboxylase. PnPy decarboxylases catalyze the irre- not published) for the UPF0047 family, no function has been
versible thiamin pyrophosphate (TPP)–dependent decarboxyl- establishedforanyofthehomologs.OneoftheUPF0047proteins,
ation of PnPy to PnAA. This step is present in the majority of YjbQ of E. coli, has a low promiscuous activity as thiamine phos-
biosynthetic pathways of known phosphonates, including fosfo- phate synthase (Morett et al., 2008), catalyzing the coupling of the
mycin and phosphinothricin tripeptide (Metcalf and van der thiazole and pyrimidine portions of thiamine phosphate via
Donk, 2009). Unlike RhiE/RhiF, these enzymes usually consist a nucleophilic substitution reaction proceeding through a carbo-
of a single polypeptide chain. On the other hand, the functionally cation intermediate (Peapus et al., 2001). However, the biological
similar sulfopyruvate decarboxylase of coenzyme M biosyn- function of YjbQ and its homologs remains to be elucidated.
Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved 31

Chemistry & Biology
Biosynthesis of Rhizocticins
The gene rhiJ encodes a putative aminotransferase belonging mass spectrometry (LC-MS) further supported the presence of
to a family of Fold Type I PLP-dependent enzymes (Eliot and rhizocticin B (see Figure 3B and Experimental Procedures for
Kirsch, 2004). It can be further classified into phylogenetic class details). No phosphonates were produced in a control experi-
V of aminotransferases (also referred to as subgroup IV) (Mehta ment with the parent B. subtilis 168 strain (data not shown).
et al., 1993). One of the closest homologs (38% identical) of Taken together, these results confirm that B. subtilis MMG272
RhiJ, MJ0959 of Methanocaldococcus jannaschii, displayed produces rhizocticin B and that the rhi gene cluster is respon-
the highest specific activity for transamination of aspartate and sible for its biosynthesis.
a-ketoglutarate when assayed in vitro (Helgadottir et al., 2007).
However, MJ0959 is thought to be a part of L-phosphoserine Proposed Rhizocticin Biosynthetic Pathway
biosynthesis, converting phosphohydroxypyruvate to L-phos- On the basis of the amino acid sequence homology of Rhi
phoserine (Helgadottir et al., 2007).
proteins to enzymes with known activities and previous knowl-
BLAST analysis revealed that RhiK is a homolog of glutaredox- edge of phosphonate biosynthetic pathways, a proposed
ins, small proteins related to thioredoxins and involved in the biosynthetic pathway for rhizocticins is shown in Figure 4. First,
maintenance of the reducing environment of the cytoplasm. PEP is converted to PnPy by the action of the PEP mutase
RhiK contains a CPYC motif conserved among glutaredoxins RhiH. PnPy then undergoes decarboxylation catalyzed by
and is predicted to have a typical bababba thioredoxin fold PnPy decarboxylase RhiE/RhiF to yield PnAA. These steps
(Pan and Bardwell, 2006). RhiK also shows homology to the are common to the vast majority of known phosphonate
N-terminal domain of glutathione S-transferase, another antibiotic biosyntheses (Metcalf and van der Donk, 2009).
member of the thioredoxin-like superfamily.
The subsequent step is a novel transformation, an aldol
The translated sequence of rhiL belongs to the calcineurin-like reaction between PnAA and pyruvate (Py) catalyzed by the
superfamily (PF00149) that includes metal-dependent phospho- aldolase homolog RhiG. An alternative possibility is that the
monoesterases and phosphodiesterases catalyzing the hydro- enolate of pyruvate is generated by decarboxylation of OAA
lysis of diverse substrates, from phosphorylated proteins to (see below).
nucleic acids (Koonin, 1994). Several conserved amino acid resi-
A minimum of two steps, dehydration and aminotransfer, are
dues are present in RhiL, most notably all those comprising the required to convert the putative RhiG product I to APPA. The
binuclear metal center, suggesting it may have a phosphodies- aminotransferase RhiJ is likely responsible for the introduction
terase activity as well.
of the amino group at C-2. It is less obvious how dehydration
happens. It is possible that the aminotransferase RhiJ is also
capable of catalyzing a PLP-dependent g-elimination of water
Heterologous Production of Rhizocticin B
To confirm that the identified gene cluster is responsible for the in tandem with aminotransfer, single-handedly converting I to
biosynthesis of rhizocticins in B. subtilis ATCC6633, we intro- APPA. Another possibility is activation of the hydroxyl leaving
duced the rhi cluster in the B. subtilis 168 genome through group via phosphorylation by the action of the kinase homolog
homologous recombination (see the Experimental Procedures RhiI. Elimination could then be achieved by a yet unknown
and Figure S2 for details). To do this, a spectinomycin resistance activity of RhiI (e.g., via acid-base catalysis) or by the action of
cassette (Spec) was introduced into fosmid 2-11E downstream RhiJ. Alternatively, RhiG could be responsible for aldol addition
of the rhi cluster using l Red recombinase-mediated recombina- followed by dehydration. Regardless of the order in which dehy-
tion (Datsenko and Wanner, 2000). The resulting fosmid dration and aminotransfer happen (path ‘‘a’’ versus ‘‘b’’), the
2-11E+Spec was linearized by restriction digestion and used APPA product can then be decorated at its N terminus with
for the transformation of B. subtilis 168. We anticipated that Arg and Val (or Leu/Ile) by the action of carboxylate-amine
the level of homology between the DNA sequence immediately ligases. Two of these proteins, RhiC and RhiM, are encoded by
outside of the rhi cluster in B. subtilis ATCC6633 and the corre- the rhi cluster, suggesting that each of the peptide bonds is
sponding sequence of B. subtilis 168 (over 90% identity on the formed by the action of a dedicated ligase.
nucleotide level) was sufficiently high for the homologous recom-
The timing of dehydration could also be later in the pathway.
bination to occur. This proved correct, because the recombinant Namely, once intermediate I is converted by RhiJ to amino
B. subtilis 168 colonies selected on spectinomycin-containing acid III, III may be incorporated by RhiC and RhiM into di- or
medium contained the rhi cluster, as verified by PCR amplifica- tripeptide precursors IV of rhizocticins. In this case, the dehydra-
tion of rhiC and rhiM genes.
tion would commence on a peptide intermediate IV. In this
One of the recombinant strains, B. subtilis MMG272, was scenario, no a-amino group would be available for RhiJ-
grown for the production of rhizocticins, and its clarified spent catalyzed PLP-dependent chemistry and at least one another
medium was partially purified and fractionated as described in enzyme must be involved. This path (‘‘c’’) is particularly
Experimental Procedures. Samples were analyzed by phos- appealing because it avoids the presence of toxic APPA as an
phorus (31P) NMR spectroscopy for the presence of phospho- intermediate.
nates. One of the fractions produced a major phosphonate
The functions of the proteins RhiK, RhiL, and RhiN are unclear.
peak with a characteristic chemical shift (d) of 20.7 ppm in the Although unusual for secondary metabolite biosynthesis, the
31P NMR spectrum (Figure 3A). Addition of purified rhizocticin glutaredoxin homolog RhiK may be involved in maintaining
B to the sample resulted in an increase in intensity of the a reduced active state for specific proteins of the pathway, and
20.7 ppm peak and no new peaks in the 31P NMR spectrum RhiL or RhiN or both may be involved in a dehydration sequence.
(Figure 3A), suggesting that the major phosphonate product is Further studies are needed to clarify the functions of these
rhizocticin B. Analysis of the sample by liquid chromatography- proteins.
32 Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Biosynthesis of Rhizocticins
Figure 3. Analysis of rhizocticin B Production
by B. subtilis MMG272
A
(A) 31P NMR spectra of partially purified spent medium
of B. subtilis MMG272 and of the same sample supple-
mented with rhizocticin B authentic standard. The
concentrations of components in the sample B. subtilis
MMG272 + rhizocticin B are the same as in the indi-
vidual sample of B. subtilis MMG272. Both spectra
were collected for 400 transients and adjusted to the
same absolute vertical scale.
B. subꢀlis MMG272
(B) LC-MS analysis of partially purified spent medium
of B. subtilis MMG272. The fragmentation of the rhi-
zocticin B parent ion is shown, and the peaks corre-
sponding to the characteristic fragments are labeled.
30 29 28 27 26 25 24 23 22 21 20 19 18 17 16
, ppm
with RhiH-N-His and Ppd-Bf-His. The ex-
tent of the reaction was analyzed using 31P
NMR spectroscopy. Upon incubation, PEP
(ꢀ0.2 ppm) was converted to PnAA, as dem-
onstrated by the appearance of a new peak
at 9.9 ppm in the 31P NMR spectrum, consis-
tent with the reported value (Blodgett et al.,
2007). Upon prolonged storage, PnAA
undergoes a nonenzymatic degradation, as
attested by the appearance of a broad peak
at 15.4 ppm in the 31P NMR spectrum con-
sistent with previously reported behavior
(Zhang et al., 2003a).
B. subꢀlis MMG272 + Rhizocꢀcin B
30 29 28 27 26 25 24 23 22 21 20 19 18 17 16
, ppm
B
Intens.
RhiH-N-His, together with Ppd-Bf-His,
were used for the enzymatic preparation of
PnAA. Because of the labile nature of PnAA,
the enzyme-free reaction mixture was
used as a source of PnAA without further
purification.
5
x10
2.5
2.0
1.5
1.0
b2+H2O
274.0
b2
256.0
Investigation of RhiG Catalytic Activity
To obtain additional support for the biosyn-
thetic proposal depicted in Figure 4, we set
out to experimentally confirm the function
of RhiG. Obtaining the product of RhiG via
an enzymatic reaction would also provide
M+H−H2O
M+H
0.5
0.0
433.1
200
250
300
350
400
450
m/z
Catalytic Activity of the PEP Mutase RhiH
the substrate for biochemical investigation of subsequent
The rhiH gene encoding putative PEP mutase was expressed in biosynthetic steps. RhiG was purified as a C-terminal fusion with
E. coli as a fusion protein with an N-terminal hexahistidine tag. a hexahistidine tag, RhiG-C-His (molecular weight, 38.7 kDa),
Recombinant RhiH-N-His was purified to near homogeneity using metal affinity chromatography. The purified protein con-
using metal affinity chromatography. The reversible reaction tained no chromophore as attested by a UV-vis spectrum trans-
catalyzed by PEP mutase favors the formation of PEP (Seidel parent above 300 nm. Native RhiG-C-His appears to be a
et al., 1988; Bowman et al., 1988). Subsequent decarboxylation homodimer (native molecular weight, 75 kDa) as determined by
of PnPy to PnAA catalyzed by PnPy decarboxylase provides the size-exclusion chromatography.
necessary driving force in many phosphonate pathways. There-
As seen for other class II aldolases, we expected that the
fore, the enzymatic activity of RhiH-N-His was tested using activity of RhiG is dependent on a divalent metal cation, most
a coupled assay with PnPy decarboxylase from Bacteroides fra- likely Mg2+ or Mn2+. Because the PnAA solution prepared with
gilis (Zhang et al., 2003b) prepared as a C-terminally His-tagged RhiH-N-His and Ppd-Bf-His already contains Mg2+, no additional
protein (Ppd-Bf-His).
metals were supplied to the reaction. Incubation of a PnAA
Assay conditions were based on published procedures (Zhang solution with pyruvate and RhiG-C-His did not produce new
et al., 2003a; Blodgett et al., 2007) and are described in detail in phosphonate compounds when examined by 31P NMR spec-
the Supplemental Information. Briefly, the assay mixture con- troscopy. We then evaluated OAA as substrate for the aldol
taining PEP, catalytic TPP cofactor, and Mg2+ was incubated reaction with PnAA. Indeed, incubation of PnAA with OAA and
Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved 33

Chemistry & Biology
Biosynthesis of Rhizocticins
O
P
O
S4) allowed for the unequivocal assignment of the structures
Ia and Ib (Figure 5A). The structure of Ia was further supported
by LC-MS analysis (Supplemental Information and Figure S5).
The trans configuration of the double bond in Ib, and not a cis
double bond as seen in APPA, suggests nonenzymatic formation
of Ib with anti-elimination of water from Ia resulting in a trans-
isomer. Therefore, the involvement of a specific enzyme (other
than RhiG) is necessary for the dehydration during rhizocticin
biosynthesis.
O
P
O
P
O
-O
RhiH
O-
RhiEF
CO2
RhiG
O-
-O
O-
O
O-
O-
H
O-
O
O
O-
O
O-
Py
PEP
PnPy
PnAA
O
a
O
Dehydratase
(RhiI?)
P
O-
O
P
O
OH
RhiG
O-
-O
O-
-O
O-
O
O
H2O
O
I
II
O-
-O
O
b/c
a
RhiJ
RhiJ
OAA
O
RhiG-C-His also catalyzes the formation of pyruvate from OAA
in the absence of PnAA. This conversion was complete after
incubation with RhiG-C-His at room temperature for 15 min,
whereas only 13% of OAA was converted to pyruvate in the
absence of enzyme (Supplemental Information).
b
O
O
P
OH
P
O-
O-
Dehydratase
(RhiI?)
O-
O-
O-
O-
+H3N
+H3N
APPA
III
H2O
O
O
Peptide bond
formation:
a/b
c
RhiC + RhiM
RhiC + RhiM
Summary of the RhiG Reaction
O
P
OH
c
O
P
O-
RhiG catalyzes the formation of 2-keto-4-hydroxy-5-phospho-
nopentanoic acid from PnAA and OAA. Carbon dioxide is
presumably a second product of this reaction. The requirement
for divalent metal cation was not investigated at this point but
is assumed on the basis of the studies of known class II aldol-
ases. Clearly, if required, Mg2+ present in the assay was suffi-
cient for successful conversion to I. OAA serves as a surrogate
Dehydratase
(RhiI?)
O-
O-
IV
O-
O-
O-
Val-Arg-NH
Val-Arg-NH
H2O
O
Rhizocticin B
O
Figure 4. Proposed Pathways for the Biosynthesis of Rhizocticins
RhiG-C-His resulted in the formation of a new compound, of pyruvate, and the corresponding three-carbon moiety is incor-
denoted Ia, as demonstrated by the appearance of a new peak porated into the final product. We propose that OAA coordinates
(19.8 ppm) in the 31P NMR spectrum (Figure 5B). Approximately to a divalent metal cation (Mg2+ in our assay) via its 1-carboxylate
80% of the PnAA was converted to Ia, as estimated by integra- and 2-ketone moieties and undergoes decarboxylation to
tion of the 31P NMR signals. The product Ia was observed only produce the enolate form of pyruvate. The enolate is stabilized
when OAA and RhiG-C-His were both added to the assay. No by the divalent cation acting as an electron sink. Subsequent
new phosphonates were detected when 2-ketoglutaric acid attack of the enolate on the electrophilic carbonyl moiety of
was used in place of OAA. Interestingly, upon storage of the PnAA furnishes the carbon-carbon bond of I (Figure 6A).
enzyme-free assay mixture, a slow conversion occurred of the
The proposed RhiG mechanism draws from the homology of
phosphonate Ia to another phosphonate-containing compound RhiG to 4-hydroxy-2-oxovalerate aldolase DmpG. Particularly,
(15.8 ppm), denoted Ib. Degradation of Ia and its highly polar the residues comprising Mn2+ ligands in DmpG are also
nature complicated its purification by HPLC. Therefore, the conserved in RhiG (Figure 6B) (Manjasetty et al., 2003). A crystal
structures of compounds Ia and Ib were determined using spec- structure of DmpG contains either pyruvate (a product) or
troscopic analyses of a crude assay mixture.
oxalate (a structural analog of pyruvate enolate) as an equatorial
A comprehensive NMR analysis of the RhiG-C-His reaction bidentate ligand to Mn2+ (Manjasetty et al., 2003). An analogous
mixtures prepared with unlabeled and 13C-labeled PEP and position could be occupied by OAA in RhiG as depicted in
OAA substrates (Supplemental Information and Figures S3 and Figure 6A.
Figure 5. Reaction Catalyzed by RhiG-C-His
(A) Chemical reaction equation for the RhiG-C-His catalyzed process.
A
O
P
O
P
O-
O
P
O-
O
O
2
OH
O
2
(B) 31P NMR spectrum of the RhiG-C-His assay with unlabeled
O-
O
O-
O-
Non-enzymatic
-O
-O
RhiG-C-His
3
1
1
3
5
5
4
4
H
O-
substrates.
PnAA
O
O
Ia
Ib
H2O
CO2
O
O-
HO
OAA
O
B
Ia
PnAA
by-product
Ib
PnAA
22
20
18
16
14
12
10
8
, ppm
34 Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Biosynthesis of Rhizocticins
Figure 6. Proposed Mechanism for the RhiG-Catalyzed
Transformation
A
Amino acid residues coordinating the divalent metal cation
(panel A, RhiG numbering, W denotes water) are based on the
alignment with the homolog DmpG shown in panel B (con-
served residues are in bold, ligands to M2+ are labeled with
arrows).
B
RhiG (7) DCTLRDGGN
DmpG (13) DVTLRDGSH
(189) VGFHGHNNLGL
(197) VGMHAHHNLSL
without further purification. 13C-labeled phosphoenolpyruvic acid potassium
salt and aspartate were from Isotec of Sigma-Aldrich group. Media
components were purchased from Thermo Fisher Scientific or VWR (West
Chester, PA). Bacterial strains and PCR primers are listed in Tables S1 and
S2, respectively.
The first step in this mechanistic proposal, decarboxylation of
OAA, is analogous to that catalyzed by macrophomate synthase
from the fungus Macrophoma commelinae (Watanabe et al.,
2000), (Ose et al., 2003). The pyruvate enolate generated by
macrophomate synthase is stabilized by coordination to Mg2+
and carries out either a Diels-Alder or Michael-type aldol reac-
tion, the specifics still being a matter of debate. Macrophomate
synthase was recently reported to also possess promiscuous
aldolase activity catalyzing an aldol reaction of OAA with alde-
hydes, analogous to the RhiG transformation (Serafimov et al.,
2008). Interestingly, the amino acid sequence of RhiG has no
homology to that of macrophomate synthase.
Preparation of Rhizocticin Heterologous Producer B. subtilis
MMG272
The spectinomycin cassette was incorporated into 2-11E fosmid (Supple-
mental Information) using l Red mediated recombination (Datsenko and
Wanner, 2000) with modifications as described below. A spectinomycin resis-
tance cassette was amplified by PCR with primers Spec-red-fwd2 and Spec-
red-rev2 using pAIN750 as a template. The primers were designed to contain
51 bp regions of homology to the sequences flanking the site of Spec insertion
in fosmid 2-11E. The PCR product (Spec fragment, 1247 nt) was digested with
DpnI and purified from an agarose gel. Electrocompetent E. coli MMG194 was
transformed with pKD46, plated on LB agar containing 12 mg/mL chloram-
phenicol (Cm) and 100 mg/mL ampicillin (Amp), and grown at 30ꢁC overnight.
One of the transformants was picked and grown overnight at 30ꢁC in LB-Cm,
Amp. The culture was then diluted 100-fold into SOB medium containing Cm,
Amp, and 2 mM arabinose (to induce l recombinase) and grown to OD600 ꢂ0.6
at 30ꢁC. The cells were made electrocompetent by extensive washing with ice-
cold 10% glycerol and were concentrated 100-fold. These cells (50 ml aliquot)
were transformed with the PCR fragment (35 ng) via electroporation, recov-
ered in SOC medium at 37ꢁC for 2 hr, and plated on LB agar containing
7 mg/mL Cm and 100 mg/mL spectinomycin (Spec). Several colonies were
inoculated into LB-Cm, Spec and grown overnight at 37ꢁC. The fosmid DNA
was isolated using QIAprep kit and analyzed by PCR amplification of the
rhiC, rhiM genes and Spec fragment. The amplification of the DNA fragments
of the desired size confirmed the incorporation of Spec into 2-11E and forma-
tion of fosmid 2-11E+Spec.
SIGNIFICANCE
Despite the demonstrated utility of phosphonates as thera-
peutics and agricultural chemicals, our knowledge of phos-
phonate biosynthetic pathways remains limited to a handful
of examples (Metcalf and van der Donk, 2009). We report
here the identification of the gene cluster responsible for
the biosynthesis of rhizocticins in B. subtilis ATCC6633
and biochemical characterization of early transformations
in the biosynthetic pathway. In addition to gaining valuable
insight into the details of phosphonate biosynthesis, access
to the pathway intermediate allows further investigation of
the subsequent steps in the pathway and ultimately may
provide a source of biologically active APPA or APPA-
containing peptides.
The fosmid DNA 2-11E+Spec was used to transform E. coli WM4489 to yield
E. coli MMG273 strain. This strain was grown in the presence of 10 mM rham-
nose to induce a high copy number for the fosmid 2-11E+Spec, and the fosmid
DNA was reisolated. The 2-11E+Spec DNA was digested by restriction endo-
nuclease NotI, purified by ethanol precipitation, and used to transform B. sub-
tilis 168 following a published protocol (Henner, 1990). Recombinants were
selected on LB agar plates containing 100 mg/mL Spec. The recombination
was confirmed by culture PCR of selected recombinant strains as described
above for verification of the fosmid 2-11E+Spec. One of the strains, B. subtilis
MMG272, was chosen for rhizocticin production analysis as described below.
The majority of currently known phosphonate biosyn-
thetic pathways share PnAA as a common intermediate
(Metcalf and van der Donk, 2009). The metabolic fate of
PnAA is either transamination or reduction. Transamination
produces 2-aminoethylphosphonate (AEP), which is incor-
porated into a variety of structural macromolecules, such
as lipids and polysaccharides. Reduction, on the other
hand, generates 2-hydroxyethylphosphonate (HEP) and
leads to antibiotics such as fosfomycin, PTT, and dehydro-
phos. In this work, we discovered a third route by which
phosphonate natural products are made from PnAA—
through an aldol reaction with oxaloacetate. This type of
transformation may prove to be a gateway to greater diver-
sity of natural phosphonate compounds.
Rhizocticin B Production in B. subtilis MMG272 and Analysis
by 31P NMR Spectroscopy and LC-MS
The heterologous producer B. subtilis MMG272 was grown for metabolite
production, as described for B. subtilis ATCC6633 (Supplemental Information),
with several exceptions. Spectinomycin was added to all of the media at
100 mg/mL. Additionally, PL medium was supplemented with tryptophan at
50 mg/mL, and the fermentation culture volume was 2 L. The cell-free superna-
tant was taken through the same purification steps through Biogel P2 fraction-
ation as described for rhizocoticin purification (Supplemental Information). The
P2 fractions corresponding to the rhizocticin B elution volume were analyzed
by 31P NMR spectroscopy and compared to an authentic standard. Several
phosphonates with chemical shifts in the range 17–27 ppm were detected;
EXPERIMENTAL PROCEDURES
Materials
Chemical reagents used in this study were the products of Sigma-Aldrich
(St. Louis, MO) or Thermo Fisher Scientific (Pittsburgh, PA) and were used
Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved 35

Chemistry & Biology
Biosynthesis of Rhizocticins
fractions eluted from the column with 90–100 ml of water (B7-8) contained
a major phosphonate with a chemical shift of 20.7 ppm. The NMR sample of
B7-8 was supplemented with 8 mM rhizocticin B and reanalyzed by 31P
NMR spectroscopy. Sample B7-8 was analyzed by LC-MS as described for
rhizocticin B analysis (Supplemental Information), and its retention time and
fragmentation pattern were consistent with the presence of rhizocticin B
(Figure 3B).
Blodgett, J.A., Thomas, P.M., Li, G., Velasquez, J.E., van der Donk, W.A., Kel-
leher, N.L., and Metcalf, W.W. (2007). Unusual transformations in the biosyn-
thesis of the antibiotic phosphinothricin tripeptide. Nat. Chem. Biol. 3,
480–485.
Bowman, E., McQueney, M., Barry, R.J., and Dunaway-Mariano, D. (1988).
Catalysis and thermodynamics of the phosphoenolpyruvate/phosphonopyru-
vate rearrangement: entry into the phosphonate class of naturally occurring
organophosphorus compounds. J. Am. Chem. Soc. 110, 5575–5576.
RhiG Activity Assays
Cicchillo, R.M., Zhang, H., Blodgett, J.A., Whitteck, J.T., Li, G., Nair, S.K., van
der Donk, W.A., and Metcalf, W.W. (2009). An unusual carbon-carbon bond
cleavage reaction during phosphinothricin biosynthesis. Nature 459, 871–874.
A stock of 100 mM oxaloacetic acid was freshly prepared in 100 mM sodium
cacodylate buffer (pH 7.5). It was added to the PnAA sample (Supplemental
Information) to a final OAA concentration of 12 mM. The reaction was initiated
by the addition of RhiG-C-His (45 mM, see the Supplemental Information for the
details of the protein purification) and the assay mixture was incubated at 30ꢁC
for 1 hr. A precipitate that formed during incubation was removed by centrifu-
gation, and soluble proteins were removed by filtration through a Microcon
YM-30 unit. We found that adding OAA and RhiG without prior removal of
RhiH-N-His and Ppd-Bf-His, or even simultaneously with PnAA formation,
reduced the amount of the PnAA degradation product formed. Therefore,
the samples intended for extensive NMR characterization were prepared in
this manner to reduce the processing time. The Microcon units were sequen-
tially rinsed with 0.1 M sodium hydroxide, water, and finally reaction buffer
prior to use to eliminate trace amounts of glycerol because it produced 1H
NMR signals in the region of interest. The enzymatic preparation of 13C-labeled
compounds and the spectroscopic characterization of compounds Ia, Ib,
Ia0,Ia00 and Ib0 are described in the Supplemental Information.
Datsenko, K.A., and Wanner, B.L. (2000). One-step inactivation of chromo-
somal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad.
Sci. USA 97, 6640–6645.
Diddens, H., Dorgerloh, M., and Zahner, H. (1979). Metabolic products of
microorganisms. 176. On the transport of small peptide antibiotics in bacteria.
J. Antibiot. (Tokyo) 32, 87–90.
Eliot, A.C., and Kirsch, J.F. (2004). Pyridoxal phosphate enzymes: mecha-
nistic, structural, and evolutionary considerations. Annu. Rev. Biochem. 73,
383–415.
Eliot, A.C., Griffin, B.M., Thomas, P.M., Johannes, T.W., Kelleher, N.L., Zhao,
H., and Metcalf, W.W. (2008). Cloning, expression, and biochemical character-
ization of Streptomyces rubellomurinus genes required for biosynthesis of anti-
malarial compound FR900098. Chem. Biol. 15, 765–770.
Fan, C., Moews, P.C., Walsh, C.T., and Knox, J.R. (1994). Vancomycin resis-
tance: structure of D-alanine:D-alanine ligase at 2.3 A resolution. Science
266, 439–443.
ACCESSION NUMBER
Fan, C., Moews, P.C., Shi, Y., Walsh, C.T., and Knox, J.R. (1995). A common
fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and
D-alanine:D-alanine ligase of Escherichia coli. Proc. Natl. Acad. Sci. USA 92,
1172–1176.
The sequence of the insert in fosmid 2-11E containing rhizocticin biosynthetic
gene cluster has been deposited in GenBank under accession number
FJ935779.
Galperin, M.Y., and Koonin, E.V. (1997). A diverse superfamily of enzymes with
ATP-dependent carboxylate-amine/thiol ligase activity. Protein Sci. 6, 2639–
2643.
SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures, two tables, and Experimental
Procedures. It can be found with this article online at doi:10.1016/
j.chembiol.2009.11.017.
Gonzalez-Pastor, J.E., Hobbs, E.C., and Losick, R. (2003). Cannibalism by
sporulating bacteria. Science 301, 510–513.
Graupner, M., Xu, H., and White, R.H. (2000). Identification of the gene encod-
ing sulfopyruvate decarboxylase, an enzyme involved in biosynthesis of coen-
zyme M. J. Bacteriol. 182, 4862–4867.
ACKNOWLEDGMENTS
We thank Feng Lin (Keck NMR laboratory, UIUC) for assistance with advanced
NMR experiments, Lucas Li (Biotechnology Center, UIUC) for performing LC-
MS analysis, and Nurul Zulkepli for help in preparation of pRhiG-C-His. This
work was supported by the National Institutes of Health (GM PO1
GM077596). Its contents are solely the responsibility of the authors and do
not necessarily represent the official views of the NIGMS or NIH.
Helgadottir, S., Rosas-Sandoval, G., Soll, D., and Graham, D.E. (2007).
Biosynthesis of phosphoserine in the Methanococcales. J. Bacteriol. 189,
575–582.
Henner, D.J. (1990). Inducible expression of regulatory genes in Bacillus sub-
tilis. Methods Enzymol. 185, 223–228.
Hidaka, T., Goda, M., Kuzuyama, T., Takei, N., Hidaka, M., and Seto, H. (1995).
Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Strep-
tomyces wedmorensis. Mol. Gen. Genet. 249, 274–280.
Received: October 19, 2009
Revised: November 23, 2009
Accepted: November 30, 2009
Published: January 28, 2010
Higgins, L.J., Yan, F., Liu, P., Liu, H.W., and Drennan, C.L. (2005). Structural
insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme.
Nature 437, 838–844.
REFERENCES
Kelley, L.A., and Sternberg, M.J. (2009). Protein structure prediction on the
Web: a case study using the Phyre server. Nat. Protoc. 4, 363–371.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990). Basic
Kino, K., Nakazawa, Y., and Yagasaki, M. (2008a). Dipeptide synthesis by
L-amino acid ligase from Ralstonia solanacearum. Biochem. Biophys. Res.
Commun. 371, 536–540.
local alignment search tool. J. Mol. Biol. 215, 403–410.
Artymiuk, P.J., Poirrette, A.R., Rice, D.W., and Willett, P. (1996). Biotin carbox-
ylase comes into the fold. Nat. Struct. Biol. 3, 128–132.
Kino, K., Noguchi, A., Nakazawa, Y., and Yagasaki, M. (2008b). A novel L-
Aziz, R.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., Edwards, R.A., For-
msma, K., Gerdes, S., Glass, E.M., Kubal, M., et al. (2008). The RAST Server:
rapid annotations using subsystems technology. BMC Genomics 9, 75.
amino acid ligase from Bacillus licheniformis. J. Biosci. Bioeng. 106, 313–315.
Kino, K., Kotanaka, Y., Arai, T., and Yagasaki, M. (2009). A novel L-amino acid
ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-
antibiotic rhizocticin. Biosci. Biotechnol. Biochem. 73, 901–907.
Blodgett, J.A., Zhang, J.K., and Metcalf, W.W. (2005). Molecular cloning,
sequence analysis, and heterologous expression of the phosphinothricin tri-
peptide biosynthetic gene cluster from Streptomyces viridochromogenes
DSM 40736. Antimicrob. Agents Chemother. 49, 230–240.
Koonin, E.V. (1994). Conserved sequence pattern in a wide variety of phos-
phoesterases. Protein Sci. 3, 356–358.
36 Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved

Chemistry & Biology
Biosynthesis of Rhizocticins
Kugler, M., Loeffler, W., Rapp, C., Kern, A., and Jung, G. (1990). Rhizocticin A,
an antifungal phosphono-oligopeptide of Bacillus subtilis ATCC6633: biolog-
ical properties. Arch. Microbiol. 153, 276–281.
Park, B.K., Hirota, A., and Sakai, H. (1977b). Studies on new antimetabolite
N-1409. Agric. Biol. Chem. 41, 161–167.
Peapus, D.H., Chiu, H.J., Campobasso, N., Reddick, J.J., Begley, T.P., and
Ealick, S.E. (2001). Structural characterization of the enzyme-substrate,
enzyme-intermediate, and enzyme-product complexes of thiamin phosphate
synthase. Biochemistry 40, 10103–10114.
Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V.,
Bertero, M.G., Bessieres, P., Bolotin, A., Borchert, S., et al. (1997). The
complete genome sequence of the gram-positive bacterium Bacillus subtilis.
Nature 390, 249–256.
Platt, A., Shingler, V., Taylor, S.C., and Williams, P.A. (1995). The 4-hydroxy-2-
oxovalerate aldolase and acetaldehyde dehydrogenase (acylating) encoded
by the nahM and nahO genes of the naphthalene catabolic plasmid pWW60-
22 provide further evidence of conservation of meta-cleavage pathway gene
sequences. Microbiology 141, 2223–2233.
Laber, B., Lindell, S.D., and Pohlenz, H.D. (1994). Inactivation of Escherichia
coli threonine synthase by DL-Z-2-amino-5-phosphono-3-pentenoic acid.
Arch. Microbiol. 161, 400–403.
Lauer, B., Russwurm, R., Schwarz, W., Kalmanczhelyi, A., Bruntner, C.,
Rosemeier, A., and Bormann, C. (2001). Molecular characterization of co-
transcribed genes from Streptomyces tendae Tu901 involved in the biosyn-
thesis of the peptidyl moiety and assembly of the peptidyl nucleoside antibiotic
nikkomycin. Mol. Gen. Genet. 264, 662–673.
Rapp, C., Jung, G., Kugler, M., and Loeffler, W. (1988). Rhizocticins—new
phosphono-oligopeptides with antifungal activity. Liebigs Ann. Chem.,
655–661.
Schell, M.A. (1993). Molecular biology of the LysR family of transcriptional
Li, Y., Zeng, H., and Tan, H. (2004). Cloning, function, and expression of sanS:
a gene essential for nikkomycin biosynthesis of Streptomyces ansochromo-
genes. Curr. Microbiol. 49, 128–132.
regulators. Annu. Rev. Microbiol. 47, 597–626.
Seidel, H.M., Freeman, S., Seto, H., and Knowles, J.R. (1988). Phosphonate
biosynthesis: isolation of the enzyme responsible for the formation of
a carbon-phosphorus bond. Nature 335, 457–458.
Maddocks, S.E., and Oyston, P.C. (2008). Structure and function of the LysR-
type transcriptional regulator (LTTR) family proteins. Microbiology 154, 3609–
3623.
Serafimov, J.M., Gillingham, D., Kuster, S., and Hilvert, D. (2008). The putative
Diels-Alderase macrophomate synthase is an efficient aldolase. J. Am. Chem.
Soc. 130, 7798–7799.
Manjasetty, B.A., Powlowski, J., and Vrielink, A. (2003). Crystal structure of
a bifunctional aldolase-dehydrogenase: sequestering a reactive and volatile
intermediate. Proc. Natl. Acad. Sci. USA 100, 6992–6997.
Tabata, K., Ikeda, H., and Hashimoto, S. (2005). ywfE in Bacillus subtilis codes
for a novel enzyme, L-amino acid ligase. J. Bacteriol. 187, 5195–5202.
Martin-Verstraete, I., Deutscher, J., and Galinier, A. (1999). Phosphorylation of
HPr and Crh by HprK, early steps in the catabolite repression signalling
pathway for the Bacillus subtilis levanase operon. J. Bacteriol. 181, 2966–
2969.
Waldrop, G.L., Rayment, I., and Holden, H.M. (1994). Three-dimensional struc-
ture of the biotin carboxylase subunit of acetyl-CoA carboxylase. Biochemistry
33, 10249–10256.
Watanabe, K., Mie, T., Ichihara, A., Oikawa, H., and Honma, M. (2000).
Detailed reaction mechanism of macrophomate synthase: extraordinary
enzyme catalyzing five-step transformation from 2-pyrones to benzoates.
J. Biol. Chem. 275, 38393–38401.
Mehta, P.K., Hale, T.I., and Christen, P. (1993). Aminotransferases: demon-
stration of homology and division into evolutionary subgroups. Eur. J. Bio-
chem. 214, 549–561.
Metcalf, W.W., and van der Donk, W.A. (2009). Biosynthesis of phosphonic
Woodyer, R.D., Shao, Z., Thomas, P.M., Kelleher, N.L., Blodgett, J.A., Metcalf,
W.W., van der Donk, W.A., and Zhao, H. (2006). Heterologous production of
fosfomycin and identification of the minimal biosynthetic gene cluster.
Chem. Biol. 13, 1171–1182.
and phosphinic acid natural products. Annu. Rev. Biochem. 78, 65–94.
Michener, H.D., and Snell, N. (1949). Two antifungal substances from Bacillus
subtilis cultures. Arch. Biochem. 22, 208–214.
Morett, E., Saab-Rincon, G., Olvera, L., Olvera, M., Flores, H., and Grande, R.
(2008). Sensitive genome-wide screen for low secondary enzymatic activities:
the YjbQ family shows thiamin phosphate synthase activity. J. Mol. Biol. 376,
839–853.
Yamaguchi, H., Kato, H., Hata, Y., Nishioka, T., Kimura, A., Oda, J., and Kat-
sube, Y. (1993). Three-dimensional structure of the glutathione synthetase
from Escherichia coli B at 2.0 A resolution. J. Mol. Biol. 229, 1083–1100.
Zawadzke, L.E., Bugg, T.D., and Walsh, C.T. (1991). Existence of two D-ala-
nine:D-alanine ligases in Escherichia coli: cloning and sequencing of the
ddlA gene and purification and characterization of the DdlA and DdlB
enzymes. Biochemistry 30, 1673–1682.
Ose, T., Watanabe, K., Mie, T., Honma, M., Watanabe, H., Yao, M., Oikawa, H.,
and Tanaka, I. (2003). Insight into a natural Diels-Alder reaction from the struc-
ture of macrophomate synthase. Nature 422, 185–189.
Pan, J.L., and Bardwell, J.C. (2006). The origami of thioredoxin-like folds.
Zhang, G., Allen, K.N., and Dunaway-Mariano, D. (2003a). Enzymatic synthesis
Protein Sci. 15, 2217–2227.
of radiolabeled phosphonoacetaldehyde. Anal. Biochem. 322, 233–237.
Park, B.K., Hirota, A., and Sakai, H. (1977a). Structure of plumbemycin-A and
plumbemycin-B, antagonists of L-threonine from Streptomyces plumbeus.
Agric. Biol. Chem. 41, 573–579.
Zhang, G., Dai, J., Lu, Z., and Dunaway-Mariano, D. (2003b). The phosphono-
pyruvate decarboxylase from Bacteroides fragilis. J. Biol. Chem. 278, 41302–
41308.
Chemistry & Biology 17, 28–37, January 29, 2010 ª2010 Elsevier Ltd All rights reserved 37