A three enzyme pathway for 2-amino-3-hydroxycyclopent-2-enone formation and incorporation in natural product biosynthesis

Article abstract of DOI:10.1021/ja1002845

A number of natural products contain a 2-amino-3-hydroxycyclopent-2-enone five membered ring, termed C₅N, which is condensed via an amide linkage to a variety of polyketide-derived polyenoic acid scaffolds. Bacterial genome mining indicates three tandem ORFs that may be involved in C₅N formation and subsequent installation in amide linkages. We show that the protein products of three tandem ORFs (ORF33-35) from the ECO-02301 biosynthetic gene cluster in Streptomyces aizunenesis NRRL-B-11277, when purified from Escherichia coli, demonstrate the requisite enzyme activities for C₅N formation and amide ligation. First, succinyl-CoA and glycine are condensed to generate 5-aminolevulinate (ALA) by a dedicated PLP-dependent ALA synthase (ORF34). Then ALA is converted to ALA-CoA through an ALA-AMP intermediate by an acyl-CoA ligase (ORF35). ALA-CoA is unstable and has a half-life of ～10 min under incubation conditions for off-pathway cyclization to 2,5-piperidinedione. The ALA synthase can compete with the nonenzymatic decomposition route and act in a novel second transformation, cyclizing ALA-CoA to C₅N. C ₅N is then a substrate for the third enzyme, an ATP-dependent amide synthetase (ORF33). Using octatrienoic acid as a mimic of the C₅₆ polyenoic acid scaffold of ECO-02301, formation of the octatrienyl-C ₅N product was observed. This three enzyme pathway is likely the general route to the C₅N ring system in other natural products, including the antibiotic moenomycin.

Full text of DOI:10.1021/ja1002845

Published on Web 04/15/2010
A Three Enzyme Pathway for
2-Amino-3-hydroxycyclopent-2-enone Formation and
Incorporation in Natural Product Biosynthesis
Wenjun Zhang, Megan L. Bolla, Daniel Kahne, and Christopher T. Walsh*
Department of Biological Chemistry & Molecular Pharmacology, HarVard Medical School,
Boston, Massachusetts 02115, and Department of Chemistry & Chemical Biology, HarVard
UniVersity, Cambridge, Massachusetts 02138
Received January 12, 2010; E-mail: Christopher_Walsh@hms.harvard.edu
Abstract: A number of natural products contain a 2-amino-3-hydroxycyclopent-2-enone five membered
ring, termed C5N, which is condensed via an amide linkage to a variety of polyketide-derived polyenoic
acid scaffolds. Bacterial genome mining indicates three tandem ORFs that may be involved in C5N formation
and subsequent installation in amide linkages. We show that the protein products of three tandem ORFs
(ORF33-35) from the ECO-02301 biosynthetic gene cluster in Streptomyces aizunenesis NRRL-B-11277,
when purified from Escherichia coli, demonstrate the requisite enzyme activities for C5N formation and
amide ligation. First, succinyl-CoA and glycine are condensed to generate 5-aminolevulinate (ALA) by a
dedicated PLP-dependent ALA synthase (ORF34). Then ALA is converted to ALA-CoA through an ALA-
AMP intermediate by an acyl-CoA ligase (ORF35). ALA-CoA is unstable and has a half-life of ∼10 min
under incubation conditions for off-pathway cyclization to 2,5-piperidinedione. The ALA synthase can
compete with the nonenzymatic decomposition route and act in a novel second transformation, cyclizing
ALA-CoA to C5N. C5N is then a substrate for the third enzyme, an ATP-dependent amide synthetase
(ORF33). Using octatrienoic acid as a mimic of the C56 polyenoic acid scaffold of ECO-02301, formation
of the octatrienyl-C5N product was observed. This three enzyme pathway is likely the general route to the
C5N ring system in other natural products, including the antibiotic moenomycin.
Introduction
hydrogen bonding possibilities for interaction with target
proteins. Examples of these ring systems include pyrrolidine-
A variety of natural products with a vast range of biological
activities have polyketide and nonribosomal peptide fragments
joined together. Frequently, these scaffolds arise from hybrid
nonribosomal peptide synthetase (NRPS)-polyketide synthase
(PKS) assembly lines. In some cases, the polyketide backbone
predominates as in rapamycin and FK506,1,2 where a single
NPRS-derived pipecolate is embedded in a polyketide frame-
work. The reverse can also occur as in bleomycin and its
congeners,3 where a single polyketide fragment interrupts the
nonribosomal peptide backbone. During the biosynthesis of
some NRPS/PKS derived natural products, the non-NRPS/PKS
machinery is enlisted to carry out the condensation between
scaffold fragments. In one example, a polyketide acid, corono-
facic acid, is enzymatically ligated to a nonproteinogenic amino
acid, coronamic acid, by a trans-acting amide synthetase to yield
the phytohormone antagonist coronatine.4,5
2,5-diones as in the methylsuccinamide terminus of andrimid,6
and the pyrrolidine-2,4-dione (tetramic acid) moieties in a wide
range of natural products including equisetin7 and cyclopiaz-
onate.8 In these instances, the pyrrolidine-diones are generated
by the chain termination domains of hybrid PKS-NRPS
assembly lines during product release. Typically, the ketone at
C4 is enolized with the 4-hydroxy-3-ene tautomer predominating.
A distinct type of nitrogen-containing cyclic dione serves as
a hydrogen bond donor/acceptor pharmacophore in more than
30 members of the manumycin family.9 Termed the C5N unit,
this is formally a 2-aminocyclopentanedione unit, but it too
predominates as the enol tautomer, 2-amino-3-hydroxycyclo-
pent-2-enone. In most cases, including the manumycins, limocro-
cin,10 Sch72542411 and ECO-02301,12 the amino group of the
C5N unit is acylated through an amide bond to a polyenoic acid
component of polyketide origin (Figure 1). This suggests an
Of special note are cyclic five-membered nitrogen-containing
ring structures that generate conformational constraints and offer
(6) Jin, M.; Fischbach, M. A.; Clardy, J. J. Am. Chem. Soc. 2006, 128,
10660–10661.
(1) Graziani, E. I. Nat. Prod. Rep. 2009, 26, 602–9.
(7) Sims, J. W.; Fillmore, J. P.; Warner, D. D.; Schmidt, E. W. Chem.
Commun. 2005, 186–188.
(2) Schwecke, T.; Aparicio, J. F.; Molnar, I.; Konig, A.; Khaw, L. E.;
Haydock, S. F.; Oliynyk, M.; Caffrey, P.; Cortes, J.; Lester, J. B.; et
al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7839–43.
(8) Liu, X.; Walsh, C. T. Biochemistry 2009, 48, 8746–8757.
(9) Sattler, I.; Thiericke, R.; Zeeck, A. Nat. Prod. Rep. 1998, 15, 221–
240.
(3) Shen, B.; Du, L.; Sanchez, C.; Edwards, D. J.; Chen, M.; Murrell,
J. M. J. Ind. Microbiol. Biotechnol. 2001, 27, 378–85.
(4) Bender, C.; Palmer, D.; PenalozaVazquez, A.; Rangaswamy, V.;
Ullrich, M. Arch. Microbiol. 1996, 166, 71–75.
(10) Hanajima, S.; Ishimaru, K.; Sakano, K.; Roy, S. K.; Inouye, Y.;
Nakamura, S. J. Antibiot. 1985, 38, 803–805.
(11) Yang, S. W.; Chan, T. M.; Terracciano, J.; Patel, R.; Loebenberg, D.;
Chen, G. D.; Patel, M.; Gullo, V.; Pramanik, B.; Chu, M. J. Antibiot.
2005, 58, 192–195.
(5) Parry, R. J.; Lin, M. T.; Walker, A. E.; Mhaskar, S. J. Am. Chem.
Soc. 1991, 113, 1849–1850.
9
6402 J. AM. CHEM. SOC. 2010, 132, 6402–6411
10.1021/ja1002845  2010 American Chemical Society

Three Enzyme Pathway for 2-Amino-3-hydroxycyclopent-2-enone
A R T I C L E S
Figure 1. Examples of natural products containing C5N (shaded). Candidate ORFs and a putative biosynthetic pathway for C5N formation during ECO-
02301 biosynthesis are boxed. ALAS, 5-aminolevulinate synthase; ACL, acyl-CoA ligase; AMS, amide synthetase.
analogy to the biosynthesis of coronatine and the aminocoumarin
antibiotics, where the acid and amine components are connected
late in the biosynthetic pathway by an amide-forming ligase.13,14
An unusual variant is found in moenomycin biosynthesis where
the C5N unit is in amide linkage to a hexuronic acid ring.15
Initial inspection of the C5N scaffold suggested it could arise
from 5-aminolevulinic acid (ALA) by an unusual cyclization
process (Figure 1).16-18 ALA is a primary metabolite in many
organisms as the immediate precursor to the monopyrrolic
building block, porphobilinogen, for heme and corrin biogen-
esis.19 While ALA can arise from reduction of glutamate on
glutamyl-tRNAglu, ALA can also be generated via the Shemin
pathway in which PLP-dependent condensation of glycine and
succinyl-CoA results in net loss of CO2 and CoASH to form
the five carbon 5-amino-4-keto-levulinic acid.20 In 2006, genetic
studies of the asukamycin producer Streptomyces nodosus subsp.
asukaensis revealed genes for both pathways to ALA.21 The
knockout of gene hemA-asuA abolished asukamycin production.
Furthermore, expression and purification of the encoding PLP-
binding enzyme in Escherichia coli gave low but detectable
activity of ALA synthesis. These results validated that the
glycine and succinyl-CoA condensing enzyme is responsible
for ALA formation and likely involved in the biosynthesis of
C5N.
Two attributes of assembly of C5N-containing amide natural
products intrigued us most: (1) the unusual cyclization of ALA
to C5N and (2) the ability of the amide synthetase to activate
the presumably weak nitrogen nucleophile of C5N as well as
its specificity for the polyenoic or hexuronic acid partner
substrates. To this end, we have examined the biosynthetic gene
cluster of antifungal agent ECO-02301 for possible enzyme
candidates responsible for the formation and ligation of the C5N
unit to the C56 polyenoic acid moiety.12 The production of ECO-
02301 had been predicted by genomic analysis of Streptomyces
aizunensis NRRL B-11277, and then ECO-02301 was success-
fully identified as a 1297 Da linear polyketide product from
the fermentation broth. Notable to our interests is the terminal
C5N moiety and the prediction by bioinformatic analysis that
ORF33-35 in the ECO-02301 gene cluster could be involved
in C5N formation and ligation (Figure 1). ORF33 could be a
putative amide synthetase (AMS, ligation of C5N to polyenoate),
while ORF34 has high sequence similarity to a PLP-dependent
ALA synthase (ALAS) and ORF35 has signature sequences
predicted for an acyl-CoA ligase (ACL). Through in Vitro
biochemical characterization of all three enzymes, we present
experimental validation of the C5N biosynthetic pathway,
including identification of the novel C5N cyclase activity.
Materials and Methods
(12) McAlpine, J. B.; Bachmann, B. O.; Piraee, M.; Tremblay, S.; Alarco,
A. M.; Zazopoulos, E.; Farnet, C. M. J. Nat. Prod. 2005, 68, 493–
496.
Bacterial Strains, Plasmids, Materials, and Instrumentation. S.
aizunensis NRRL B-11277 was obtained from USDA ARS Culture
Collection. Oligonucleotide primers were synthesized by Integrated
DNA Technologies, and PCR was performed with Phusion High-
Fidelity PCR Master Mix (NEB). Cloning was performed using
the Xa/LIC Cloning Kit (Novagen). Recombinant plasmid DNA
was purified with a QIAprep kit (Qiagen). DNA sequencing was
performed at the Molecular Biology Core Facilities of the Dana
Farber Cancer Institute. E. coli BL21 Gold (DE3) Cells (Stratagene)
transformed with pET-derived vectors were used for overexpression
of proteins in LB medium supplemented with kanamycin. Nickel-
nitrilotriacetic acid agarose (Ni-NTA) superflow resin and SDS-
PAGE gels were purchased from Qiagen and Biorad, respectively.
Protein samples were concentrated using 30 kDa MMCO Amicon
Ultra filters (Millipore). DNA and protein concentrations were deter-
mined by Nanodrop 1000 spectrophotometer (Thermo Scientific).
All chemicals were purchased from Sigma-Aldrich unless stated
(13) Liyanage, H.; Penfold, C.; Turner, J.; Bender, C. L. Gene 1995, 153,
17–23.
(14) Galm, U.; Dessoy, M. A.; Schmidt, J.; Wessjohann, L. A.; Heide, L.
Chem. Biol. 2004, 11, 173–183.
(15) Ostash, B.; Saghatelian, A.; Walker, S. Chem. Biol. 2007, 14, 257–
267.
(16) Nakagawa, A.; Wu, T. S.; Keller, P. J.; Lee, J. P.; Omura, S.; Floss,
H. G. J. Chem. Soc., Chem. Commun. 1985, 519–521.
(17) Beale, J. M.; Lee, J. P.; Nakagawa, A.; Omura, S.; Floss, H. G. J. Am.
Chem. Soc. 1986, 108, 331–332.
(18) Endler, K.; Schuricht, U.; Hennig, L.; Welzel, P. Tetrahedron Lett.
1998, 39, 13–16.
(19) Jaffe, E. K. Bioorg. Chem. 2004, 32, 316–325.
(20) Avissar, Y. J.; Ormerod, J. G.; Beale, S. I. Arch. Microbiol. 1989,
151, 513–519.
(21) Petricek, M.; Petrickova, K.; Havlicek, L.; Felsberg, J. J. Bacteriol.
2006, 188, 5113–5123.
9
J. AM. CHEM. SOC. VOL. 132, NO. 18, 2010 6403

A R T I C L E S
Zhang et al.
otherwise. [4-13C]5-Aminolevulinic acid and NMR solvents were
purchased from Cambridge Isotope Laboratories, Inc. 2,5-Pip-
eridinedione was purchased from Small Molecules. Boc-5-ami-
nolevulinic acid was purchased from AnaSpec, Inc.
NMR spectra were recorded on a Varian Inova 500 (500 MHz
1H, 125 MHz 13C) instrument in CDCl3. LC-MS analysis was
performed on an Agilent Technologies 6520 Accurate-Mass Q-TOF
LC-MS instrument or an Agilent Technologies 6210 Accurate-Mass
TOF LC-MS instrument. HPLC analysis was performed on a
Beckman Coulter System Gold with an Inertsil ODS-4 C18 column
(4.6 × 250 mm) from GL Sciences, Inc. A Phenomenex Luna C18
column (21.2 × 250 mm) was used for preparative HPLC. A
SPECTRAmax plus 384 96-well plate reader from Molecular
Devices was used for continuous spectrophotometric assays.
20-50 µM ORF35 was incubated with 2-5 mM acid substrates,
2 mM ATP, 2 mM CoA, and 2 mM MgCl2 for 30 min. The
reactions were stopped by addition of tricholoacetic acid (TCA) to
a final concentration of 5% (v/v), and the supernatant was subjected
to both LC-MS and HPLC analysis. For analysis of cyclase activity,
20-50 µM ORF34 was added to the reaction mixture of ORF35
and incubated for ∼60 min. The reactions were stopped by addition
of 100 µL of CH3CN, and the supernatant was subjected to LC-
MS and HPLC analysis. Alternatively, 20-50 µM ORF34 was
added to buffer containing synthesized ALA-CoA substrate and
reacted for 10 min prior to LC-MS and HPLC analysis. For analysis
of AMS activity, 20-50 µM ORF33 was added to buffer containing
synthesized 2 mM C5N, 2 mM 2,4,6-octatrienoic acid, 5 mM ATP,
and 2 mM MgCl2. The reactions were stopped after 1 h by addition
of CH3CN and the supernatant was subjected to LC-MS and HPLC
analysis. For reconstitution of the entire pathway, reaction mixtures
contained 50 µM ORF33-35, 5 mM glycine, 1 mM succinyl-CoA,
5 mM ATP, 2 mM CoA, 2 mM MgCl2, and 2 mM 2,4,6-octatrienoic
acid. The reactions were stopped after 2-3 h by addition of 100
µL of CH3CN and the supernatant was subjected to LC-MS and
HPLC analysis.
Preparation of ALA-CoA. Boc-ALA [25 µmol, 1 equiv], CoA
sodium salt (25 µmol, 1 equiv), PyBOP (Novobiochem, 50 µmol,
2 equiv) and K2CO3 (100 µmol, 4 equiv) were dissolved in
tetrahydrofuran/H2O [1/1 (v/v), 1 mL] and stirred for 2 h at room
temperature. The reaction mixture was directly purified by prepara-
tive HPLC (two injections, 0.5 mL each) using a linear gradient of
2-60% CH3CN (v/v) over 30 min in H2O supplemented with 0.1%
(v/v) TFA at a flow rate of 10 mL/min. The HPLC-purified mixture
was concentrated on a rotary evaporator and lyophilized to yield
Boc-ALA-CoA (30% yield) as a white solid. The Boc protecting
group was removed by redissolving Boc-ALA-CoA in 1 mL of
5-10% TFA in H2O and stirring for 30 min at room temperature.
The reaction mixture was again purified by preparative HPLC (same
method as above), concentrated, and lyophilized, yielding ALA-
CoA (85% yield) as a white solid.
Preparation of 2,4,6-Octatrienyl-C5N. To a cooled solution (0
°C) of (2E,4E,6E)-octa-2,4,6-trienoic acid23 (97 mg, 0.700 mmol)
in CH2Cl2 (7 mL) and DMF (3 drops) was added a solution of
oxalyl chloride (100 mg, 0.840 mmol, 1.2 equiv) in CHCl2 (0.5
mL) dropwise over 30 min. The resulting solution was stirred at 0
°C for 2 h, then slowly canulated into a cooled solution (0 °C) of
freshly prepared 2-amino-3-hydroxy-2-cyclopenten-1-one hydro-
chloride24 (103 mg, 0.700 mmol), N,N-dimethylaminopyridine (5
mg, 0.041 mmol, 5 mol %) in pyridine (15 mL). The reaction
mixture was slowly warmed to room temperature and maintained
stirring overnight. The reaction was then concentrated (azeotroping
with toluene) and purified by flash column chromatography with
98:2 (CH2Cl2/MeOH) to afford a yellow solid (55 mg, 0.236 mmol,
34%). 1H NMR (500 MHz, CDCl3) δH 13.6 (s, 1H), 7.45 (broad s,
1H), 7.34 (dd, J ) 14.5, 11.5 1H), 6.58 (dd, J ) 15.0, 11.0 1H),
6.22 (dd, J ) 14.5, 12.0 1H), 6.15-6.20 (m, 1 H), 6.00 (dd, J )
14.5, 9.5 1H), 5.95 (d, J ) 15.0 1H), 2.60-2.62 (m, 2H), 2.52-2.55
(m, 2 H), 1.85 (d, J ) 7.0 3 H); 13C NMR (125 MHz, CDCl3) δC
197.3, 177.6, 173.8, 145.0, 142.5, 136.3, 131.3, 127.2, 119.2, 32.3,
25.7, 18.8.
Kinetic Investigations of ORF34. ORF34 activity was measured
at 25 °C in a 100 µL reaction volume containing 100 mM sodium
phosphate (pH 7.8), 0.1 mM 5,5′-dithio-bis(2-nitro-benzoic acid)
(DTNB), 0.2 mM PLP, and 20 µM ORF34. Kinetic parameters for
the succinyl-CoA substrate were determined with the concentration
of glycine maintained at 10 mM and the concentration of succinyl-
CoA varied from 0 to 425 µM; kinetic parameters for the glycine
substrate were determined with the concentration of succinyl-CoA
maintained at 0.5 mM and the concentration of glycine varied from
0 to 5 mM. Kinetic parameters for the ALA-CoA substrate in the
Cloning, Overexpression, and Purification of ORF33-35.
ORF33-35 were PCR amplified from genomic DNA extracted from
S. aizunensis. For ORF33, the forward primer was 5′-GGTAT-
TGAGGGTCGCATGACCCCGCAGGACCATTGGTG-3′ and the
reverse primer was 5′-AGAGGAGAGTTAGAGCCTTAGGAGTC-
GAGCAGCTGCAGCC-3′. For ORF34, the forward primer was
5′-GGTATTGAGGGTCGCATGAACCTGCACCTGGAATCGTA-
3′ and the reverse primer was 5′-AGAGGAGAGTTAGAGCCT-
TACGAAAGCCAGTTCCTGTCGG-3′. For ORF35, the forward
primer was 5′-GGTATTGAGGGTCGCATGACCCGGTCGGTG-
GCGGCCGT-3′ and the reverse primer was 5′- AGAGGAGAGT-
TAGAGCCTTACGCGTAGCGGTGTGCCAGCT-3′. Purified PCR
products were ligated to pET-30 Xa/LIC following the standard
protocol and confirmed by DNA sequencing. The resulting expres-
sion constructs were transformed into E. coli BL21 cells for protein
expression. Expression and purification for all three proteins
followed the same general procedure and is detailed as follows. In
1 L of liquid culture, the cells were grown at 37 °C in LB medium
with 50 µg/mL kanamycin to an OD600 of 0.4. The cells were cooled
on ice for 10 min and then induced with 0.1 mM isopropyl-ꢀ-D-
thiogalactopyranoside (IPTG) for 16 h at 16 °C. The cells were
harvested by centrifugation (6000 rpm, 6 min, 4 °C), resuspended
in 30 mL of lysis buffer (20 mM HEPES, pH 8.0, 0.5 M NaCl,
and 5 mM imidazole), and lysed by sonication on ice. Cellular
debris was removed by ultracentrifugation (35 000 rpm, 30 min, 4
°C). Ni-NTA agarose resin was added to the supernatant (1 mL/L
of culture) and the solution was nutated at 4 °C for 1 h. The protein
resin mixture was loaded into a gravity flow column, and proteins
were eluted with increasing concentrations of imidazole in Buffer
A (50 mM HEPES, pH 8.0, and 2 mM EDTA). Purified proteins
were concentrated and buffer exchanged into Buffer A and 10%
glycerol using Amicon Ultra filters. Gel filtration chromatography
was performed on a Superdex 200 10/300 column connected to an
Amersham automated FPLC system at 4 °C (50 mM phosphate
buffer, and 150 mM NaCl, pH 7.8). The final proteins were flash-
frozen in liquid nitrogen and stored at -80 °C.
HPLC/LC-MS Product Assays. All product assays were
performed in 50 µL of 50 mM HEPES (pH 8.0) at 25 °C. LC-MS
analysis was normally performed with a linear gradient of 0 to 20%
CH3CN (v/v) over 20 min, 20-95% CH3CN (v/v) over 5 min, and
95% CH3CN (v/v) for a further 15 min in H2O supplemented with
0.1% (v/v) formic acid, at a flow rate of 0.5 mL/min. HPLC analysis
was normally performed with a linear gradient of 2-12% CH3CN
(v/v) over 30 min, 12-95% CH3CN (v/v) over 5 min, and 95%
CH3CN (v/v) for a further 10 min in H2O supplemented with 0.1%
(v/v) trifluoroacetic acid (TFA) at a flow rate of 1 mL/min. For
analysis of ALAS activity, 20-50 µM ORF34 was incubated with
5 mM glycine and 1 mM succinyl-CoA for 2 h. The protein was
removed by 5 kDa MMCO filter tubes, and the filtered reaction
mixture was subjected to LC-MS analysis. OPTA derivatization
of amines in the filtered reaction mixture was further performed
following the reported protocol.22 For analysis of ACL activity,
(23) Lensen, N.; Mouelhi, S.; Bellassoued, M. Synth. Commun. 2001, 31,
1007–1011.
(24) Ebenezer, W. J. Synth. Commun. 1991, 21, 351–358.
(22) Morineau, G.; Azoulay, M.; Frappier, F. J. Chromatogr. 1989, 467,
209–216.
9
6404 J. AM. CHEM. SOC. VOL. 132, NO. 18, 2010

Three Enzyme Pathway for 2-Amino-3-hydroxycyclopent-2-enone
A R T I C L E S
Figure 2. Characterization of ORF34 as a 5-aminolevulinate synthase. (A) Schematic of the condensation of glycine and succinyl-CoA to yield 5-aminolevulic
acid catalyzed by ORF34. (B) Extracted ion chromatograms (m/z ) 132 [M + H]+) showing ORF34-catalyzed production of ALA (trace ii) as compared
to the ALA standard (trace iii). ALA was not detected in controls without ORF34 (shown, trace i) or without any of the substrates (not shown). (C) Determination
of ORF34 kinetic parameters by assay with DTNB. Parameters were determined for succinyl-CoA (top panel) by fixing the glycine concentration at 10 mM,
while parameters were determined for glycine (bottom panel) by fixing the succinyl-CoA concentration at 500 µM. Error bars represent standard deviations
from at least three independently performed experiments.
cyclization reaction were determined by varying the concentration
of ALA-CoA from 0 to 400 µM. For each concentration, a control
reaction was carried out without enzyme. Assays were initiated by
the addition of acyl-CoA. Activity was monitored continuously by
following the increase in absorbance at 412 nm resulting from the
reaction between the free thiol of CoASH and DTNB. Initial
velocities (Vini) were calculated using the extinction coefficient of
14150 M-1 cm-1 (Vini ) Vini[with enzyme] - Vini[without enzyme]).
The Vini data were fitted to the Michaelis-Menten equation in
GraphPad Prism to obtain estimates for kcat and Km.
resulting from the production of the ligated product. Kinetic
parameters for the 2,4,6-octatrienoic acid substrate were determined
with the concentration of C5N maintained at 1 mM and the
concentration of acid varied from 0 to 5 mM. Higher acid
concentrations could not be achieved due to poor solubility. Kinetic
parameters for the C5N substrate were determined with the
concentration of 2,4,6-octatrienoic acid maintained at 2 mM and
the concentration of C5N varied from 0 to 1.1 mM. Initial velocities
(Vini) were calculated using a standard curve of synthetically
prepared 2,4,6-octatrienyl-C5N. The Vini data were fitted to the
Michaelis-Menten equation in GraphPad Prism to obtain estimates
for kcat and Km.
ATP-[32P]PPi Exchange Assays for ORF33 and ORF35. A
typical assay contained, in a total volume of 800 µL, 5 mM acid
substrate,
5 mM ATP, 10 mM MgCl2, 1
mM Na[32P]-
pyrophosphate (PPi) (∼2.5 × 106 cpm/mL), and 50 mM HEPES,
pH 8.0. Reactions were initiated by the addition of enzyme
(1 µM of ORF35 or 0.8 µM of ORF33). At regular time intervals,
100 µL aliquots were quenched with 500 µL of a charcoal
suspension (100 mM NaPPi, 350 mM HClO4, and 16 g/L charcoal).
The mixtures were vortexed and then centrifuged at 13 000 rpm
for 6 min. The pellets were washed twice with 500 µL of wash
solution (100 mM NaPPi and 350 mM HClO4). Charcoal-bound
radioactivity was measured on a Beckman LS 6500 scintillation
counter. Turnover was calculated as (% incorporation of [32P]PPi-
)[total PPi]/[Enz].
ATP-PPi Release Assays for ORF35. The inorganic pyrophos-
phate released by enzymatic reaction was measured continuously
using the EnzChek Pyrophosphate Assay Kit (Invitrogen). A typical
assay contained, in a total volume of 100 µL, 0-5 mM acid
substrate, 5 mM ATP, 2 mM MgCl2, and 5 µM ORF35 in 50 mM
HEPES, pH 8.0. MESG substrate, purine nucleoside phosphorylase
and inorganic pyrophosphatase were added according to the
protocol. Reactions were initiated by the addition of acid substrate
and monitored at 360 nm. Initial velocities were calculated using
the standard curve for inorganic pyrophosphate. The data were fitted
to the Michaelis-Menten equation in GraphPad Prism to obtain
estimates for kcat and Km.
Results
Expression and Purification of ORF33-35 in E. coli.
ORF33-35 from S. azuinensis were amplified and cloned into
an expression vector encoding an N-terminal His6-tag. The
corresponding recombinant proteins (ORF34, 49 kDa; ORF35,
59 kDa; ORF33, 60 kDa) were overproduced in E. coli and
purified using Ni-NTA affinity chromatography (Supporting
Information Figure S1). The approximate protein yields were
10 mg/L for ORF34, 26 mg/L for ORF35, and 30 mg/L for
ORF33.
ORF34 Is a PLP-Dependent Aminolevulinate Synthase (ALAS).
Examination of the UV absorption spectra of ORF34 identified
a characteristic absorbance maximum at 420 nm, indicating the
presence of an enzyme-bound PLP Schiff base (Supporting
Information Figure S2).25 Titration with PLP indicated ap-
proximately 60% of the protein was in the holo-form (PLP
bound) upon isolation (Supporting Information Figure S3).
Incubation of purified ORF34 with glycine and succinyl-CoA
led to the formation of a new compound detected by LC-MS,
with retention time and mass (m/z ) 132 [M + H]+) matching
those of the commercial ALA standard (Figure 2A,B). The
identity of the product ALA was further confirmed using OPTA
Kinetic Investigations of ORF33. The amide synthetase activity
of ORF33 was measured at 25 °C in a 100 µL reaction volume
containing 50 mM HEPES, pH 8.0, 5 mM ATP, 2 mM MgCl2, 5
µM ORF33 and acid and amine substrates. Activity was monitored
continuously by following the increase in absorbance at 355 nm
(25) Ferreira, G. C.; Neame, P. J.; Dailey, H. A. Protein Sci. 1993, 2, 1959–
1965.
9
J. AM. CHEM. SOC. VOL. 132, NO. 18, 2010 6405

A R T I C L E S
Zhang et al.
Figure 3. Characterization of ORF35 as an acyl-CoA ligase. (A) Schematic of the ligation of ALA to CoASH catalyzed by ORF35. (B) HPLC traces (260
nm) of ORF35 reaction (trace i) and controls with no ATP (trace ii), no CoA (trace iii), and no enzyme (trace iv). (C) ATP-[32P]PPi exchange assays
monitoring the reversible formation of acyl-AMP as the first half reaction. Calculated rates: k(ALA) ) 6.6 min-1, k(octanoic acid [C8]) ) 17.5 min-1
,
k(levulinic acid [LA]) ) 5 min-1, k(5-aminovaleric acid [AV]) ) 1.7 min-1. (D) Kinetic parameters for the ALA substrate determined by ATP-PPi release
assays. Error bars represent standard deviations from at least three independently performed experiments.
Table 1. ATP-PPi Release Assays for ORF35 on Selected Acidsa
derivatization and LC-MS analysis (Supporting Information
kcat/Km
Figure S4). The product assays confirmed that ORF34 was an
aminolevulinate synthase (ALAS), which is in agreement with
the previous report that ALAS is essential for asukamycin
biosynthesis in S. nodosus subsp. asukaensis. Notably, the gene
encoding the S. nodosus subsp. asukaensis ALAS is also
adjacent to genes encoding AMS and ACL homologues.21
To determine the kinetic parameters of ORF34, the DTNB
assay monitoring the free CoA formation was performed (Figure
2C). The Km values of the enzyme were determined to be
120 µM for succinyl-CoA and 2.5 mM for glycine, which is
within range of the reported values for ALASs originating from
different organisms (17-257 µM for succinyl-CoA and 1.9-9.7
mM for glycine).26,27 The turnover number was determined to
be ∼0.4 min-1, which was ∼10-fold less than those of ALASs
dedicated to heme biosynthesis, but ∼10-fold greater than that
of the ALAS involved in the biosynthesis of asukamycin.21
ORF35 Is an Acyl-CoA Ligase (ACL). ORF35 shows high
sequence similarity to acyl-CoA ligases, and was proposed to
ligate ALA synthesized by ORF34 to CoASH. To probe the
role of ORF35, in Vitro assays containing ALA, CoASH, ATP,
Mg2+ and purified ORF35 were performed in HEPES buffer at
pH 8.0 and monitored by LC-MS. The protein readily precipi-
tated at lower pH values (<7.5). ALA-CoA was detected as a
new product (m/z ) 881.1723 [M + H]+, ∆ ) 2.1 mmu)
(Supporting Information Figure S5), albeit in low yields. Full
sets of control experiments confirmed that ORF35 catalyzed the
formation of ALA-CoA in an ATP-dependent manner (Figure
3A,B). To determine the substrate specificity of ORF35, the
classical ATP-[32P]PPi exchange assay was used to monitor
acids Vmax (mM min-1
)
Km (µM)
[ORF35] (µM)
kcat (min-1
)
(min-1mM-1
)
ALA 6.19 ( 0.14
478 ( 43
5
5
5
5
5
1.24 ( 0.03
1.08 ( 0.02
1.61 ( 0.04
1.31 ( 0.03
1.32 ( 0.05
2.594
0.999
0.217
7.016
7.316
LA
AV
C6
C8
5.43 ( 0.08 1087 ( 46
8.07 ( 0.22 7431 ( 370
6.56 ( 0.15
6.62 ( 0.23
187 ( 15
181 ( 21
a ALA, 5-aminolevulinic acid; LA, levulinic acid; AV, 5-aminovaleric
acid; C6, hexanoic acid; C8, octanoic acid.
the reversible formation of acyl-AMP as the first half reaction.
The enzyme demonstrated specificity toward ALA over substrate
analogs such as levulinic acid and 5-aminovaleric acid (Figure
3C), with a kcat of 6.6 min-1 for ALA. Interestingly, ORF35
recognized octanoic acid (kcat ) 17.5 min-1) better than ALA.
A coupled spectrophotometric assay for PPi release was further
used to kinetically characterize ORF35, and the substrate
specificity was in agreement with the ATP-PPi exchange assays
(for ALA, kcat ) 1.24 min-1, Km ) 478 µM) (Figure 3D, Table
1 and Supporting Information Figure S6). It is notable that the
kcat values obtained from the PPi release assays were relatively
low for all the acids tested, which is likely due to slow, off-
pathway release of the tightly bound acyl-AMP intermediates.
Regardless of assay used, however, ORF35 showed better
specificity toward medium-chain fatty acids, which may indicate
that the ancestor of this enzyme is a fatty acid acyl-CoA ligase.
To confirm the acyl-CoA formation catalyzed by ORF35, in
Vitro reactions followed by LC-MS analyses were performed
with selected acids (Supporting Information Figure S7). As
expected, under the same reaction conditions, hexanoyl-CoA
was quickly formed in good yield and levulinyl-CoA was
formed less efficiently. However, very little ALA-CoA could
be detected as noted above, and no 5-aminovaleryl-CoA could
be detected by UV or mass ion extraction. Instead, 2,5-
(26) Lin, J.; Fu, W.; Cen, P. Bioresour. Technol. 2009, 100, 2293–2297.
(27) Bolt, E. L.; Kryszak, L.; Zeilstra-Ryalls, J.; Shoolingin-Jordan, P. M.;
Warren, M. J. Eur. J. Biochem. 1999, 265, 290–299.
9
6406 J. AM. CHEM. SOC. VOL. 132, NO. 18, 2010

Three Enzyme Pathway for 2-Amino-3-hydroxycyclopent-2-enone
A R T I C L E S
acetonitrile and analyzed by LC-MS. C5N was successfully
detected as a new product with UV absorbance maximum at
250 nm (m/z ) 114.0557 [M + H]+, ∆ ) 0.7 mmu), and its
mass was shifted by +1 using [4-13C]ALA as an alternative
substrate (Supporting Information Figure S8). The product
identity was further confirmed by comparison to authentic C5N
synthesized as a standard by reduction of the corresponding
nitro-cyclopentanedione as described in the Materials and
Methods.24 Control experiments demonstrated that the formation
of C5N from ALA relied on the presence of both enzymes
(ORF34 and ORF35) and all cofactors (CoA, ATP and Mg2+
)
(Figure 5A). Prior reports indicated synthetic C5N is unstable
upon concentration.24 We determined by HPLC analysis that,
under enzymatic reaction conditions, C5N displayed a usable
half-life of 140 min at pH 8.0. HPLC and LC-MS detection of
C5N from enzymatic reactions validated our hypothesis, dem-
onstrating that the PLP-dependent ORF34 can direct the
cyclization of ALA-CoA to yield C5N (Figure 5B).
To further verify ALA-CoA as the substrate for ORF34 and
kinetically characterize the cyclization step, ALA-CoA was
synthesized chemically. The synthesis was undertaken with Boc-
5-ALA and unblocked CoA, with PyBOP as a condensation
reagent to yield Boc-5-ALA-CoA. The Boc group was then
removed by TFA and the ALA-CoA product was purified by
preparative HPLC. DTNB assays indicated that ALA-CoA had
a half-life of ∼10 min at pH 8.0, which is consistent with the
difficulty in detecting the product ALA-CoA in ORF35 LC-
MS assays. C5N was successfully generated after the incubation
of the synthetic ALA-CoA with ORF34, while no C5N was
detectable in the control without enzyme (Figure 5C). These
results unequivocally confirmed the role of ORF34 as an ALA-
CoA cyclase to afford C5N. The enzyme demonstrated substan-
tially higher catalytic efficiency for cyclization (kcat ) 2.6 min-1,
Km ) 36.7 µM, kcat/Km ) 71 min-1 mM-1) than for ALA
synthesis according to the DTNB assays (Figure 5D).
Figure 4. Instability of ALA-CoA. (A) Extracted ion chromatograms (m/z
) 114 [M + H]+) showing the formation of PDO in the reaction mixture
of ORF35 (trace ii). PDO standard is shown in trace iii and the no enzyme
control is shown in trace i. (B) Schematic of the spontaneous cyclization
of ALA-CoA to PDO.
piperidinedione and 2-piperidinone were observed as products,
respectively, which were reasoned to be formed through
nonenzymatic cyclization of the aminoacyl-CoAs. Particularly,
at pH 8.0 the -NH2 group of the product aminoacyl-CoAs is
likely reactive enough to serve as an internal nucleophile and
attack the activated carbonyl of the CoA thioester terminus,
leading to spontaneous formation of the six membered ring
(Figure 4). ALA-CoA was found to be stable in acidic
conditions, which is consistent with our hypothesis. Considering
the instability of ALA-CoA at physiological pH, the mechanism
underlying the cyclization of the activated ALA to C5N instead
of the default 2,5-piperidinedione was intriguing. Because no
C5N was detectable in the ORF35 in Vitro assays, we reasoned
that an additional dedicated cyclase was required to direct the
synthesis of C5N from ALA-CoA.
ORF33 Is a C5N Amide Synthetase. The condensation presum-
ably between a carboxylic acid and the amine of C5N constitutes
the final step for C5N incorporation into the polyketide backbone
as a chain terminator. This is putatively promoted by ORF33,
which shows reasonable sequence similarity (<40%) to amide
synthetases (NovL, CloL, CouL, SimL) in the aminocoumarin
antibiotic family.14 To probe the role of ORF33, the purified
enzyme was incubated with C5N, ATP, MgCl2, and 2,4,6-
octatrienoic acid (OTEA), which was used to mimic the
C-terminal polyolefinic moiety of the polyketide substrate
(Figure 6A,B). LC-MS analysis showed production of a new
PLP-Dependent ORF34 Is a Bifunctional ALAS and ALA-
CoA Cyclase. It has been reported that the keto-form of ALA
was in large excess compared to the enolic tautomers at neutral
1
pH. Using 13C and H NMR, no direct observation of the enol
forms of ALA had been achieved,28 arguing against a ready
supply of the C5 carbon nucleophile required for spontaneous
intramolecular capture of ALA-CoA to C5N. Therefore, to favor
C5N formation, it seemed likely that activation of C5 adjacent
to the amine and sequestration of the reactive terminal amine
of ALA-CoA might be required for cyclization. As no putative
cyclase was found in the gene cluster, we hypothesized that
the PLP-dependent ORF34 ALAS was the best candidate for
“cyclase” considering its catalytic mechanism. The ALAS
reaction involves binding of glycine to PLP to yield a PLP-
glycine complex, and a resonance-stabilized nucleophilic quinon-
oid intermediate is formed following pro-R proton subtraction.29
Some ALASs can also catalyze labilization of the pro-R proton
at C5 of the ALA product and generate detectable PLP-ALA
quinonoid upon exposure of the enzyme to ALA.30 These
chemical roles of coenzyme PLP in both the forward and reverse
reactions meet the prerequisites for C5N cyclization as mentioned
above, particularly the ability to labilize the C5 hydrogen.
To test our hypothesis, purified ORF34 was added to the
reaction mixture of ORF35, and the reaction was quenched with
compound with a UV absorbance maximum at 321 nm (λmax
)
300 nm for OTEA) (Supporting Information Figures S9 and
S10), along with the formation of AMP and attenuation of both
amine and acid substrate peaks. HRMS (m/z ) 234.1108 [M +
H]+, ∆ ) 1.7 mmu) and mass fragmentation (two major mass
fragments: 114.0553, 121.0643) suggested the new product to
be 2,4,6-octatrienyl-C5N (OTEA-C5N). The identity of the
product was further confirmed by comparison to the synthetic
standard, produced from synthetic C5N and OTEA as described
in the Materials and Methods (Supporting Information Figure
S11). Notably, ORF33 was not able to ligate ALA to OTEA in
our assays. These results confirmed the role of ORF33 as a C5N
amide synthetase.
ATP-[32P]PPi exchange assays demonstrated that ORF33
adenylated carboxylate substrates as predicted on the basis of
its homology to AMP-forming enzymes (Figure 6C). The kinetic
data further revealed that the enzyme preferred acids of longer
(28) Jaffe, E. K.; Rajagopalan, J. S. Bioorg. Chem. 1990, 18, 381–394.
(29) Nandi, D. L. J. Biol. Chem. 1978, 253, 8872–8877.
(30) Hunter, G. A.; Ferreira, G. C. Biochemistry 1999, 38, 3711–3718.
9
J. AM. CHEM. SOC. VOL. 132, NO. 18, 2010 6407

A R T I C L E S
Zhang et al.
Figure 5. Characterization of PLP-dependent ORF34 as an ALA-CoA cyclase. (A) UV traces (250 nm) during LC-MS analysis of ORF34/ORF35 activity
showing reaction with ORF34 only (trace i), ORF35 only (trace ii), ORF34 + ORF35 with [4-12C]ALA (trace iii), ORF34 + ORF35 with [4-13C]ALA (trace
iv), and C5N synthetic standard (trace v). (B) Schematic of cyclization of ALA-CoA to C5N catalyzed by ORF34 via the PLP-quinonoid intermediate. (C)
HPLC trace (250 nm) of ORF34 reaction producing C5N and CoA using synthetic ALA-CoA as the sole substrate (trace ii). No enzyme control is shown
in trace i. (D) Kinetic parameters for the ALA-CoA substrate determined by DTNB assays. Error bars represent standard deviations from at least three
independently performed experiments.
chain length and higher degree of unsaturation (OTEA > 2,4-
hexadienoic acid .2-butenoic acid > octanoic acid) consistent
with resemblance to the presumed C56 native substrate. It is
notable that the turnover rates were slow (kcat ) 0.3 min-1 for
OTEA) in the assays, perhaps limited by slow PPi off rates.
The kinetic parameters of the enzyme toward C5N and OTEA
were then further examined by monitoring the rate of OTEA-
C5N formation at 355 nm (Figure 6D). This wavelength was
selected to minimize the inference of the substrate OTEA on
UV absorption (Supporting Information Figures S9 and S10).
With a C5N concentration of 1 mM, the enzyme showed a Km
the reaction mixture was monitored by LC-MS. The OTEA-
C5N was successfully produced (Figure 6B, trace ii), and was
also detected using ALA as a starting material instead of glycine
and succinyl-CoA (data not shown). Omission of any of the
three enzymes completely abolished the production of OTEA-
C5N (Figure 6B, trace iv), confirming the necessity of all three
enzymes in the biosynthesis and incorporation of the C5N
moiety. In addition, no protein complexes were observed
between any of the three enzymes by gel filtration chromato-
graphy (data not shown), suggesting that all of the intermediates
produced during each transformation are freely diffusible. This
is in agreement with the above results that every step can be
reconstituted individually in Vitro.
of ∼7.8 mM for OTEA with an apparent kcat of ∼10.2 min-1
,
indicating the poor affinity of this substrate analog. In contrast,
ORF33 had a low Km of ∼95 µM for C5N and an apparent kcat
of ∼1.8 min-1 at an OTEA concentration of 2 mM (poor OTEA
solubility precluded reproducible assays at higher concentra-
tions). These results demonstrated the capability of ORF33 in
recognizing the presumably weak amine nucleophile of C5N.
The overall turnover rate was an order of magnitude greater in
the product assays than in the ATP-PPi exchange assays
performed without the amine donor C5N, indicating that acid
activation and amine binding likely accelerate release of the
PPi coproduct.
Discussion
In this work, we have studied the biosynthesis of 2-amino-
3-hydroxycyclopent-2-enone (C5N) and its incorporation via an
amide linkage to a partial polyenoate scaffold of the antifungal
natural product ECO-02301. The condensation between glycine
and succinyl-CoA catalyzed by ORF34 (ALAS) leads to
formation of ALA, which is then ligated to CoASH yielding
ALA-CoA promoted by the ATP-dependent ORF35 (ACL).
ALA-CoA is not stable and can undergo spontaneous intramo-
lecular cyclization with the amine group acting as an internal
nucleophile to form the shunt product 2,5-piperidinedione.
Reconstitution of the Entire C5N Biosynthetic Pathway in
Vitro. All three enzymes, ORF33, ORF34, and ORF35, were
incubated together with the essential cofactors (Mg2+, ATP,
CoA) and substrates (glycine, succinyl-CoA and OTEA), and
9
6408 J. AM. CHEM. SOC. VOL. 132, NO. 18, 2010

Three Enzyme Pathway for 2-Amino-3-hydroxycyclopent-2-enone
A R T I C L E S
Figure 6. Characterization of ORF33 as an amide synthetase (AMS). (A) Schematic of ligation of C5N to 2,4,6-octatrienoic acid (OTEA) catalyzed by
ORF33. (B) UV traces (321 nm) during LC-MS analysis showing OTEA-C5N formation using ORF33 and synthetic C5N (trace i), the entire reconstituted
pathway using ORF33-35, glycine, succinyl-CoA and CoA (trace ii). OTEA-C5N synthetic standard is shown in trace iii and no enzyme control is shown
in trace iv. It is notable that all of the no enzyme controls, including the control without ORF33 for trace i and the controls without either of the three
enzymes for trace ii, were the same as the one shown in trace iv. (C) ATP-[32P]PPi exchange assays monitoring the reversible formation of acyl-AMP as the
first half reaction of ORF33. Calculated rates: k(OTEA) ) 0.30 min-1, k(2,4-hexadienoic acid [HDEA]) ) 0.21 min-1, k(2-butenoic acid [BEA]) ) 0.009
min-1, k(octanoic acid [C8]) ) 0.005 min-1. (D) Kinetic parameters measured for ORF33 by monitoring the formation of OTEA-C5N at 355 nm. ORF33
concentration was maintained at 5 µM and concentrations of OTEA and C5N were fixed at 2 mM and 1 mM, respectively. Error bars represent standard
deviations from at least three independently performed experiments.
Figure 7. Characterized biosynthetic pathway for C5N involving ORF33-35 from ECO-02301 gene cluster.
Alternatively, ALA-CoA can be directed by the PLP-dependent
ORF34 to form C5N through promotion of a carbon nucleophile
at C5. Finally, ORF33 (AMS) catalyzes ligation of C5N to a
polyenoic acid in an ATP-dependent manner (Figure 7). We
suspect that this three enzyme pathway constitutes a general
biosynthetic route for C5N formation and incorporation into
other natural products. This supposition is supported by the
presence of the same trigene cassette encoding ALAS, ACL,
and AMS in reported gene clusters of C5N-containing natural
products, including ECO-0501 and moenomycin.15,31
The discovery of ALAS-catalyzed cyclization of ALA-CoA
to C5N solves the mechanistic problem regarding the formation
of C5N from ALA. Interestingly, the ALAS enzyme catalyzes
(31) Banskota, A. H.; McAlpine, J. B.; Sorensen, D.; Ibrahim, A.; Aouidate,
M.; Piraee, M.; Alarco, A. M.; Farnet, C. M.; Zazopoulos, E. J.
Antibiot. 2006, 59, 533–542.
9
J. AM. CHEM. SOC. VOL. 132, NO. 18, 2010 6409

A R T I C L E S
Zhang et al.
two distinct reactions in this pathway: (1) ALA synthesis from
glycine and succinyl-CoA, and (2) cyclization of ALA-CoA to
afford C5N. ALAS belongs to the family of a´-oxoamine
synthases and, because of its long known role in providing
building blocks for heme biosynthesis, its PLP-dependent
catalytic mechanism has been studied extensively. Catalysis
involves two bond cleavage steps (CR-H and CR-COO) and one
bond forming step (between CR and the thioester carbonyl of
succinyl-CoA).32 To initiate catalysis, binding of glycine induces
transaldimination, leading to the formation of Gly-PLP aldi-
mine complex after deprotonation of the CR-HR of glycine by
the ε-NH2 of a lysine residue in the active site. This generates
a CR-PLP carbanion stabilized as the para-quinonoid species.
The electrophilic thioester carbonyl is then attacked by the CR
carbanion of the activated Gly-PLP, which yields a proposed
R-NH2-ꢀ-ketoadipyl-PLP intermediate upon C-C bond forma-
tion from a thio-Claisen type condensation with concomitant
release of CoASH. The subsequent decarboxylation of the ꢀ-keto
acid via CR-COO- cleavage and CR reprotonation lead to the
formation of PLP bound ALA that is finally released by
transaldimination with the active site lysine (Figure 8A).
We propose a related mechanism for the novel cyclization
of ALA-CoA (Figure 8B). The CoA moiety of ALA-CoA is
assumed to be a key recognition element, and after its binding
in the well-defined CoA binding site,33 the terminal -NH2 of
ALA-CoA can reach the PLP-Lys internal aldimine and undergo
transaldimination (see Supporting Information Figure S12). The
C5 proton of the ALA moiety can be abstracted (presumably
by the same lysine residue) as proposed for the Gly-PLP
intermediate in the first step of ALA formation.30 Now the C5-
ALA-PLP stabilized carbanion can intramolecularly attack the
thioester carbonyl, releasing CoASH as a leaving group. The
immediate result is the cyclization of the ALA chain to the C5N
group in aldimine linkage to the PLP in the active site.
Transaldimination by lysine would release free C5N and
regenerate the resting form of the enzyme. Compared to ALA
synthesis by this same PLP enzyme, cyclization of ALA-CoA
does not have the decarboxylation step, and the nucleophilic
attack is intramolecular instead of intermolecular, which may
account for the observed higher catalytic efficiency of the
enzyme for cyclization. This is an exquisite example of a PLP-
dependent mechanism being employed by Nature to sequester
and deactivate the reactive terminal amine of ALA and to
simultaneously activate the adjacent carbon to generate a
competent carbon nucleophile.
Figure 8. Proposed PLP-dependent catalytic mechanism of ORF34. (A)
Mechanism for ALA synthesis. In the substrate-free state, PLP is bound
by enzyme (internal aldimine). The incoming glycine induces transaldimi-
nation, leading to the PLP-Gly adduct (external aldimine). The electrophilic
substrate succinyl-CoA is attacked by the PLP-activated glycine, leading
to C-C bond formation with the release of CoASH. Decarboxylation and
reprotonation result in formation of the PLP-ALA adduct that is finally
released to regenerate the internal aldimine. (B) Mechanism for C5N
synthesis. ALA-CoA induces transaldimination, yielding the ALA-
CoA-PLP complex. The thioester carbonyl in ALA-CoA is then attacked
by the PLP-activated ALA moiety intramolecularly, leading to C-C bond
formation with the release of CoASH. The PLP-C5N complex is finally
released to regenerate internal aldimine. (C) Three-dimensional structural
model of the ORF34 active site. The structure was modeled on the
Rhodobacter capsulatus ALAS using the online program HHpred, and the
substrates were docked using the software GOLD.
X-ray structures of the R. capsulatus ALAS have been
recently reported, showing the positions of both Gly-PLP and
of succinyl-CoA.33 The electron density for the 3′,5′-ADP
portion of the CoA and the carboxylate of the succinyl moiety
was well resolved, allowing the placement of the CR of the
Gly-PLP adduct within 3 Å of the thioester carbonyl when
modeled into the active site. With the use of the solved ALAS
structure as the template (PDB: 2BWN), the structure of ORF34
was modeled. It showed high similarity to and shared almost
identical active site with ALAS from R. capsulatus (Supporting
Information Figure S12). ALA-CoA was docked into the active
site of ORF34 with the CoA adenosine binding site constrained
at the same location as that of succinyl-CoA in the R. capsulatus
ALAS structure. In the modeled ALA-CoA-PLP complex, C5
of the ALA moiety was positioned approximately 3 Å from
Lys248 and the thioester carbonyl, demonstrating that the ALA
moiety can readily fit into the active site pocket for cyclization
while retaining the same CoA adenosine binding site at the
protein surface (Figure 8C). On the basis of the high similarity
of protein structures, we further propose that the “cyclase”
activity of the ALAS may be general for all of the ALASs,
including those from primary metabolism involved in heme
(32) Zhang, J. S.; Ferreira, C. J. Biol. Chem. 2002, 277, 44660–44669.
(33) Astner, I.; Schulze, J. O.; van den Heuvel, J.; Jahn, D.; Schubert, W. D.;
Heinz, D. W. EMBO J. 2005, 24, 3166–3177.
9
6410 J. AM. CHEM. SOC. VOL. 132, NO. 18, 2010

Three Enzyme Pathway for 2-Amino-3-hydroxycyclopent-2-enone
A R T I C L E S
biosynthesis. Such an activity would, however, be revealed only
in a cell where ALA has been processed to the ALA-CoA
thioester.
suggests a critical role in efficacy. This is especially apparent
in the cases such as limocrocin, Sch 725424 and reductiomycin
(Figure 1), where C5N is the major functional group in these
structures. It is possible that C5N serves as a Michael acceptor
for covalent inactivation of enzymes, which is a common
inhibition mechanism of many other natural and synthetic
molecules, including resorcylic acid lactones and acrylamides.38,39
Our work presented here successfully closes the knowledge gap
around the biosynthesis of C5N, reveals a novel cyclization
activity of ALA synthase, and defines a minimal, presumably
portable cassette for the formation and amide-mediated incor-
poration of this unique pharmacophore.
The two other enzymes in the pathway (ORF35 and ORF33)
are both confirmed to be AMP-forming enzymes. ORF35
catalyzes the ligation between acid and CoA substrates to form
the C-S thioester bond, while ORF33 generates the connectivity
between acid and amine substrates to afford the C-N amide
bond found in the natural product. ORF35 showed substrate
promiscuity toward various acids and demonstrated higher
specificity toward fatty acids than ALA, indicating that the
enzyme might have evolved from a fatty acid acyl-CoA ligase.
In contrast, ORF33 had low affinity for all the acid substrate
analogs tested, suggesting the strong preference of the enzyme
toward longer chain polyketide acids. In addition, unlike many
AMP-forming enzymes including acyl-CoA ligases and ade-
nylation domains of NRPSs which activate acids to yield acyl-
AMP intermediates independently in the first half reaction,
ORF33 activates acids much more efficiently upon the binding
of the amine, suggesting that C5N binding likely induces
conformation change to render the enzyme in a more favorable
state for acid activation.
In addition to the manumycin family compounds, many
bioactive metabolites, including reductiomycin (antitumor),34
senacarcin (antitumor),35 limocrocin (reverse transcriptase in-
hibitor),10 virustomycin (antivirus),36 enopeptin (antibiotic)37 and
Sch 725424 (antibiotic)11 also contain the C5N ring system.
Although there has been no direct study of the structure-activity
relationship of C5N to date, the prevalence of this functionality
in natural products with a broad range of biological activities
Acknowledgment. This work was supported in part by an NIH
Grant GM20011 (C.T.W.) and NIH Grant GM066174 (D.K.). We
thank Dr. Albert Bowers for help with HRMS measurements. We
also thank Prof. Sheryl Tsai and Dr. Brian Ames for assistance
with protein structure modeling and substrates docking.
Supporting Information Available: SDS-PAGE of ORF33-
35 purified from E. coli; UV profile of purified ORF34;
titration of ORF34 with PLP; detection of ALA formation
by OPTA derivatization; HRMS of ALA-CoA; determination
of ORF35 kinetic parameters for various acid substrates;
HPLC traces of ORF35 reactions with selected acid sub-
strates; spectroscopic characterization of C5N; UV profile of
2,4,6-octatrienoic acid (OTEA); spectroscopic characteriza-
1
tion of OTEA-C5N; H and 13C NMR characterization of
synthesized OTEA-C5N standard; modeled three-dimensional
structure of ORF34. This material is available free of charge
via the Internet at http://pubs.acs.org.
(34) Cho, H.; Beale, J. M.; Graff, C.; Mocek, U.; Nakagawa, A.; Omura,
S.; Floss, H. G. J. Am. Chem. Soc. 1993, 115, 12296–12304.
(35) Laursen, J. B.; Nielsen, J. Chem. ReV. 2004, 104, 1663–1685.
(36) Omura, S.; Imamura, N.; Hinotozawa, K.; Otoguro, K.; Lukacs, G.;
Faghih, R.; Tolmann, R.; Arison, B. H.; Smith, J. L. J. Antibiot. 1983,
36, 1783–1786.
JA1002845
(38) Schirmer, A.; Kennedy, J.; Murli, S.; Reid, R.; Santi, D. V. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 4234–9.
(37) Koshino, H.; Osada, H.; Yano, T.; Uzawa, J.; Isono, K. Tetrahedron
Lett. 1991, 32, 7707–7710.
(39) Deng, W.; Guo, Z.; Guo, Y.; Feng, Z.; Jiang, Y.; Chu, F. Bioorg.
Med. Chem. Lett. 2006, 16, 469–72.
9
J. AM. CHEM. SOC. VOL. 132, NO. 18, 2010 6411