Enzymatic processing of fumiquinazoline F: A tandem oxidative-acylation strategy for the generation of multicyclic scaffolds in fungal indole alkaloid biosynthesis

Article abstract of DOI:10.1021/bi1012029

Aspergillus fumigatus Af293 is a known producer of quinazoline natural products, including the antitumor fumiquinazolines, of which the simplest member is fumiquinazoline F (FQF) with a 6-6-6 tricyclic core derived from anthranilic acid, tryptophan, and alanine. FQF is the proposed biological precursor to fumiquinazoline A (FQA) in which the pendant indole side chain has been modified via oxidative coupling of an additional molecule of alanine, yielding a fused 6-5-5 imidazoindolone. We recently identified fungal anthranilate-activating nonribosomal peptide synthetase (NRPS) domains through bioinformatics approaches. One domain previously identified is part of the trimodular NRPS Af12080, which we predict is responsible for FQF formation. We now show that two adjacent A. fumigatus ORFs, a monomodular NRPS Af12050 and a flavoprotein Af12060, are necessary and sufficient to convert FQF to FQA. Af12060 oxidizes the 2′,3′-double bond of the indole side chain of FQF, and the three-domain NRPS Af12050 activates l-Ala as the adenylate, installs it as the pantetheinyl thioester on its carrier protein domain, and acylates the oxidized indole for subsequent intramolecular cyclization to create the 6-5-5 imidazolindolone of FQA. This work provides experimental validation of the fumiquinazoline biosynthetic cluster of A. fumigatus Af293 and describes an oxidative annulation biosynthetic strategy likely shared among several classes of polycyclic fungal alkaloids.

Full text of DOI:10.1021/bi1012029

8564 Biochemistry 2010, 49, 8564–8576
DOI: 10.1021/bi1012029
Enzymatic Processing of Fumiquinazoline F: A Tandem Oxidative-Acylation Strategy for
the Generation of Multicyclic Scaffolds in Fungal Indole Alkaloid Biosynthesis†
Brian D. Ames, Xinyu Liu,‡ and Christopher T. Walsh*
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston,
Massachusetts 02115. ‡Current address: Department of Chemistry, University of Pittsburgh, 219 Parkman Ave., Pittsburgh, PA 15232.
Received July 29, 2010; Revised Manuscript Received August 26, 2010
ABSTRACT: Aspergillus fumigatus Af293 is a known producer of quinazoline natural products, including the
antitumor fumiquinazolines, of which the simplest member is fumiquinazoline F (FQF) with a 6-6-6 tricyclic
core derived from anthranilic acid, tryptophan, and alanine. FQF is the proposed biological precursor to
fumiquinazoline A (FQA) in which the pendant indole side chain has been modified via oxidative coupling of
an additional molecule of alanine, yielding a fused 6-5-5 imidazoindolone. We recently identified fungal
anthranilate-activating nonribosomal peptide synthetase (NRPS) domains through bioinformatics ap-
proaches. One domain previously identified is part of the trimodular NRPS Af12080, which we predict is
responsible for FQF formation. We now show that two adjacent A. fumigatus ORFs, a monomodular NRPS
Af12050 and a flavoprotein Af12060, are necessary and sufficient to convert FQF to FQA. Af12060 oxidizes
the 20,30-double bond of the indole side chain of FQF, and the three-domain NRPS Af12050 activates
-Ala as
L
the adenylate, installs it as the pantetheinyl thioester on its carrier protein domain, and acylates the oxidized
indole for subsequent intramolecular cyclization to create the 6-5-5 imidazolindolone of FQA. This work
provides experimental validation of the fumiquinazoline biosynthetic cluster of A. fumigatus Af293 and
describes an oxidative annulation biosynthetic strategy likely shared among several classes of polycyclic
fungal alkaloids.
Filamentous fungi are prolific producers of bioactive second-
ary metabolites, including potent mycotoxins such as aflatoxin,
ochratoxin, gliotoxin, and cyclopiazonic acid (1), and molecules
of important therapeutic value such as the antibiotic cephalos-
porins/penicillins, immunosuppressant cyclosporins, and choles-
terol-lowering statins. One class of fungal natural products
consists of the quinazoline alkaloids (2, 3), a subclass of which
are the fumiquinazolines (FQs) which possess a pyrazino[2,1-
b]quinazoline-3,6-dione core scaffold derived from condensation
of anthranilic acid (Ant) with two additional amino acids (Figure
S1 of the Supporting Information) (4).
The isolation and structures of seven fumiquinazolines (A-G),
produced by a strain of Aspergillus fumigatus separated from the
gastrointestinal (GI) tract of the saltwater fish Pseudolabrus
japonicas, were originally described in an effort to discover
cytotoxic compounds from marine microorganisms (5). More
recently, analysis of secondary metabolites produced by 40
strains of A. fumigatus, including strain Af293 relevant to this
work, demonstrated that the fumiquinazolines make up one of
three families of secondary metabolites isolated from all tested
strains (6). In addition to their widespread production in A.
fumigatus, the isolation of FQs has also been reported in several
Penicillium species (7). The FQs are moderately cytotoxic and
have been reported to exhibit antitumor activity against several
cancer cell lines (8). Related pyrazinoquinazolinone metabolites
have been isolated from various fungi: fumiquinazoline H-I
(Acremonium sp.) (9), fiscalins A and B (Neosartorya fischeri)
(10), glyantrypine (Aspergillus clavatus) (11), cottoquinazoline A
(Aspergillus versicolor) (12), alantrypinone (Penicillium thymicola)
(13), spiroquinazoline (Aspergillus flavipes) (14), and verrucines
(Penicillium verrucosum) (15, 16) (Figure S1 of the Supporting
Information). Despite the widespread production of pyrazino-
quinazolinones by filamentous fungi and their isolation based on
bioactivity-guided approaches, their role in the chemical ecology
of the producing organism is yet to be determined.
The construction of the core scaffold of di- and tripeptidyl
alkaloids may be accomplished by nonribosomal peptide synthe-
tase (NRPS) assembly lines (17-19). NRPSs are often found as
multidomain megasynthases composed minimally of adenylation
(A), carrier protein/thiolation (T), and condensation (C) do-
mains arranged in repeating units termed modules. The general
process of building peptidyl frameworks begins with A domain-
catalyzed activation of amino acid building blocks as acyl-AMPs.
The acyl group of the acyl adenylate is then attached to the 40-
phosphopantetheine (ppt) prosthetic group of the T domain to
form an acyl-thioester intermediate. T domain-tethered inter-
mediates are then delivered to the active site of a C domain, which
catalyzes the coupling of building block units via amide bond
formation. Modifying domains sometimes present as part of the
NRPS include the C domain variant epimerization (E) and
heterocyclization domains and domains for N-methylation or
formylation. Following maturation and in-line modification of
the growing peptide chain, several mechanisms exist for chain
†This work was supported in part by National Institutes of Health
Grant GM20011 (to C.T.W.), F32GM090475 (postdoctoral fellowship
to B.D.A.), and the Ernst Schering Foundation (postdoctoral fellowship
to X.L.).
*To whom correspondence should be addressed: Department of
Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, 240 Longwood Ave., Boston, MA 02115. E-mail: christopher_
walsh@hms.harvard.edu. Phone: (617) 432-1715. Fax: (617) 432-0438.
r
pubs.acs.org/Biochemistry
Published on Web 08/30/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 39, 2010 8565
F
IGURE 1: (A) Fumiquinazoline gene cluster and (B) proposed biosynthetic route to fumiquinazoline A in A. fumigatus Af293.
release (20, 21). In bacterial NRPSs, and in the fungal ACV
tripeptide synthetase, a terminal thioesterase (TE) domain cata-
lyzes chain release via hydrolysis or macrocyclization. A distin-
guishing feature of many fungal NRPSs is the absence of a
terminal TE domain and instead the presence of a terminal
condensation, reductive, or thiolation domain (19). Particularly
relevant to the construction of the FQs and related fungal
alkaloids is the fact that the terminal C domains may use intra-
or intermolecular nucleophiles to attack the thioester bond of an
enzyme-tethered intermediate for chain release via cyclization.
Additionally, during chain elongation and after chain release, the
peptidyl scaffold is often modified by the action of discrete
tailoring enzymes (22, 23).
Because of the medicinal relevance of A. fumigatus as an
allergen and opportunistic pathogen in humans (24), this organism
was among the first of its genus to be sequenced. To date, two
clinical isolates of A. fumigatus have been sequenced: strain
Af293 (FGSC A110) (25) and strain CEA10 (FGSC A1163)
(26). The availability of sequenced fungal genomes has greatly
facilitated the identification of secondary metabolite gene clusters
through genome mining (27). On the basis of this approach, we
recently identified putative anthranilate-activating NRPS modules
in the genomes of Aspergilli (28).
Biochemical characterization of module 1 of AFUA_6g12080
from A. fumigatus Af293 (abbreviated as Af12080) validated its
anthranilate-dependent adenylation and thiolation activities and
prompted us to propose it as part of an eight-gene cluster
involved in fumiquinazoline biosynthesis (Figure 1A) (28). Mod-
ules 2 and 3 of Af12080 are predicted to activate and load Trp and
Ala, respectively, providing for the assembly of an Ant-Trp-Ala-
S-enzyme intermediate that would undergo double cyclization
for chain release and generation of the tricyclic 6-6-6 product
fumiquinazoline F (Figure 1B) (28). The presence of an E domain
predicted for module 2 of Af12080 is consistent with epimeriza-
on the holo form of a T domain and catalyze amide bond
formation by directed attack of the indole NH group of FQF on
the activated alanyl thioester carbonyl. Oxidation of the indole
C20-C30 bond could prime it for N-acylation and intramole-
cular cyclization en route to formation of FQA. Consonant with
this proposal, the putative fumiquinazoline gene cluster contains
a stand-alone, monomodular NRPS predicted to activate L-Ala
(Af12050, domain structure A-T-C) and also a predicted flavin
adenine dinucleotide-dependent monooxygenase (Af12060)
(Figure 1). While acylation of the indole NH group with L-Ala
could occur before indole oxidation (28), we demonstrate in this
work that oxidation of the indole 20,30-olefin by Af12060 is
required prior to alanine coupling by Af12050 in a manner
unprecedented for NRPS-based logic. This oxidative coupling
strategy diversifies and expands multicyclic scaffolds, and we
hypothesize that similar biosynthetic logic is likely involved in the
formation a number of fungal indole alkaloids (Figure 2).
EXPERIMENTAL PROCEDURES
Materials and General Methods. Triphenyl phosphite,
anhydrous pyridine, and anthranilic acid were purchased from
Sigma-Aldrich. N-Boc-
chased from EMD Chemicals.
was purchased from Toronto Research Chemicals.
L
-Ala-OH and N-Boc-Gly-OH were pur-
-Trp methyl ester hydrochloride
-[U-14C]Ala
D
L
(128 mCi/mmol) was from Sigma-Aldrich, [1-14C]acetyl-CoA1
(60 mCi/mmol) from Moravek Biochemicals, and [32P]PPi (100
mCi/mmol) from Perkin-Elmer. PCRs were conducted using
Phusion High-Fidelity PCR MasterMix (New England Biolabs).
Oligonucleotide primers were purchased from Integrated DNA
Technologies (Coralville, IA). Plasmid DNA was propagated in
Escherichia coli XL1 Blue (Stratagene), and plasmid DNA was
1Abbreviations: CoA, coenzyme A; cpm, counts per minute; EDTA,
ethylenediaminetetraacetic acid; ESI, electrospray ionization; FAD,
flavin adenine dinucleotide; HPLC, high-performance liquid chroma-
tion of L-Trp to D-Trp during assembly to generate the R-
stereocenter at C14 of FQF. Maturation of FQF to FQA would
involve further processing of the Trp side chain indole group
through oxidative coupling of L-Ala to give a 6-5-5 framework
known as an imidazoindolone (Figure 1B).
One strategy for accomplishing the net oxidation, acylation,
and cyclization for FQA assembly would be to utilize an NRPS
module to activate and install L-Ala as an S-pantetheinyl thioester
tography; IPTG, isopropyl β- -galactopyranoside; LC-MS, liquid
D
chromatography-mass spectrometry; LB, Luria-Bertani medium;
MeCN, acetonitrile; NAD(P)H, reduced nicotinamide adenine dinu-
cleotide (phosphate); NCBI, National Center for Biotechnology Infor-
mation; NMR, nuclear magnetic resonance; ORF, open reading frame;
PDB, Protein Data Bank; PCR, polymerase chain reaction; PPi,
inorganic pyrophosphate; SDS-PAGE, sodium dodecyl sulfate-
polyacrylamide gel electrophoresis; TCA, trichloroacetic acid; TCEP,
tris(2-carboxyethyl)phosphine; Tris, tris(hydroxymethyl)aminomethane.

8566 Biochemistry, Vol. 49, No. 39, 2010
Ames et al.
F
transformationofindolewithanadditionalamino acidina mannersimilar tothe generationofthe imidazoindolone moietyoffumiquinazoline A.
IGURE 2: Examples of fungal metabolites containing a multicyclic indolic scaffold likely derived in part or in whole from an oxidative coupling
The indolic C20 and C30 positions are labeled for the sake of clarity.
prepared using the QIAprep Spin Mini Kit (Qiagen). Automated
DNA sequencing was performed by Genewiz (South Plainfield,
NJ). Protein concentrations were determined spectrophotome-
trically using measured A280 values and theoretical extinction
coefficients obtained from the online ProtParam tool (29)
(Af12050/pET30 Ek-LIC, ε = 120780 M-1 cm-1; Af12060/
pET30 Ek-LIC, ε = 91250 M-1 cm-1). The concentrations of
FQF and FQA were determined using UV-vis spectroscopy and
published extinction coefficients of 13489 (277 nm) and 14791
20 min and a steady period of 95% B for 5 min with a flow rate of
1 mL/min. Under these conditions, FQA and FQF eluted at 18.1
and 19.3 min, respectively.
Cloning of Af12060 and Af12050. Growth of A. fumigatus
Af293, mRNA extraction, and cDNA preparation were per-
formed as previously reported (28). The identification of a new 50-
start site for Af12060 was determined as described in Results. The
start codon for this site is located at base 1700861 of the genomic
DNA for Af293 chromosome 6 (GenBank accession number
AAHF01000006.1), which is 210 bp upstream of the annotated
start in AFUA_6g12060. Sequence alignment of results from a
BlastP search indicated the presence of an intron in the newly
added 50-sequence and provided approximate intron boundaries.
Manual inspection of the nucleotide sequence led to the identi-
fication of a 69 bp intron starting at base 1700701 (donor site)
and ending at base 1700633 (acceptor site). PCR primers for
amplification of Af12060 from cDNA were designed on the basis
of this new start site and the annotated gene termination site:
forward, 50-GACGACGACAAGatgacaatcaacactgctctaccgactcc-30;
reverse, 50-GAGGAGAAGCCCGGtcactcgggactatatgtgtcctcc-30
(lowercase type signifies bases complementary to the gene, and
boldface type indicates the stop codon). The amplified DNA was
cloned using a pET-30 Ek-LIC vector kit (EMD Chemicals), for
expression as a 495-residue protein containing an N-terminal
His6-S tag. The boundaries of the new 50-intron were confirmed
by sequencing to be identical to that manually predicted. How-
ever, DNA sequencing revealed that the terminal 30-intron
(defined in the database as 21 bp, 1699627-1699605) was
incorrectly predicted, with the correct 60 bp intron instead at
bases 1699689-1699550.
M
-1 cm-1 (256 nm), respectively (5). FQF, FQA, and GAT stock
solutions (5 mM) were made with 50% DMSO in water. An
Agilent Technologies 6520 Accurate-Mass QTOF instrument
was used for high-resolution LC-MS analysis, an Agilent Tech-
nologies G1956 LC-MSD instrument for low-resolution analysis,
and a Beckman Coulter System Gold instrument equipped with
diode array detection for reverse-phase HPLC. Liquid scintilla-
tion counting was performed with a Beckman Coulter LS 6500
instrument. NMR data were collected on a Varian 600 MHz
spectrometer using the residual solvent peak from incomplete
deuteration as an internal standard (CDCl3, δ 7.26).
Isolation and Characterization of FQF and FQA from an
A. fumigatus Af293 Culture. An A. fumigatus Af293 spore
stock (50 μL) was used to inoculate 50 mL of potato dextrose
broth and the culture incubated at 37 °C for 24 h. Following
growth, cells were removed by filtration (Whatman #54), the
filtrate was applied to 5 mL of Amberlite XAD-16 resin (Sigma),
and 2 ꢀ 5 mL volumes of MeOH were applied to elute absorbed
compounds. The solvent was removed in vacuo and the resulting
residue dissolved in 1.5 mL of MeOH (termed “XAD extract”).
Solvent systems A (water with 0.1% formic acid) and B (MeCN
with 0.1% formic acid) were used for LC-MS and HPLC
analysis. For high-resolution LC-MS, 10 μL samples of XAD
extract were injected onto a Gemini-NX C18 column (50 mm ꢀ
2.0 mm) and resolved with a gradient from 2 to 98% B over 12
min and a steady period of 98% B for 2 min with a flow rate of
0.4 mL/min. Under these conditions, FQA and FQF eluted with
retention times of 8.3 and 8.6 min, respectively. For HPLC, 25 μL
samples were injected onto an Alltima C18 column (150 mm ꢀ
4.6 mm) and resolved with a gradient from 5 to 95% B over
Cloning of Af12050 also utilized a 50-start site different from
the annotated entry for ORF AFUA_6g12050 (as described in
Results). This new start site begins at base 1695552 of the
genomic DNA for Af293 chromosome 6, which is 582 nucleotides
downstream of the start site of NCBI database entry NC_007199.1.
PCR amplification of Af12050 from cDNA was accomplished
using the following primer pair: forward, 50-GACGACGACAA-
Gatgttggagacgacggagccaattg-30; reverse, 50-GAGGAGAAGC-
CCGGtcacaccgtaatctcagataacagagc-30. The amplified DNA was

Article
Biochemistry, Vol. 49, No. 39, 2010 8567
cloned into a pET-30 Ek-LIC vector to encode a 1153-residue
protein with an N-terminal His6-S tag, and the final construct
was confirmed by DNA sequencing.
identity of the synthetic FQF include high-resolution ESI/MS
([M þ H]þ expected, 359.1503; observed, 359.1492), UV-vis,
and 1H NMR data (5, 30) (see Figures S5 and S6 of the Support-
ing Information).
Expression and Purification of Af12050 and Af12060.
Both protein constructs were overproduced in E. coli BL21-
Gold(DE3) cells (Stratagene) in a similar manner. Cells (2 L)
were grown at 37 °C in LB with 50 μg/mL kanamycin to an
OD600 between 0.4 and 0.8, and the temperature was lowered to
16 °C prior to induction with 0.2 mM IPTG. Cells were harvested
18-24 h postinduction by centrifugation, suspended in lysis
buffer [25 mM Tris-HCl (pH 7.5), 300 mM NaCl, 20% glycerol,
0.1% Tween 20, and 1ꢀ protease inhibitor cocktail (SigmaFast,
EDTA-free)], and lysed using a EmulsiFlex-C5 homogenizer
(Avestin). Insoluble material was removed by centrifugation
(35000g) and the soluble protein applied to 1-2 mL of Ni-
NTA agarose (Qiagen) equilibrated in lysis buffer. We performed
Ni-affinity purification was performed by batch binding protein
for 30 min at 4 °C, washing Ni resin with 2 ꢀ 20 mL of buffer A
[50 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA] containing
20 mM imidazole, and then eluting protein using a step gradient
from 250 to 500 mM imidazole in buffer A (5 mL for each step).
The elution fractions containing target protein were pooled and
concentrated using a centrifugal filtration device (30K molecular
weight cutoff, Amicon), and concentrated protein was flash-frozen
in liquid N2 and stored at -80 °C.
GAT was synthesized using N-Boc-Gly (190 mg, 200 μmol) in
place of N-Boc-L-Ala, and other components and methods as
described for FQF. The crude reaction mixture was purified by
silica-gel flash chromatography (solvent system as described for
FQF) to yield GAT and its enantiomer. Data supporting the
identity of the synthetic GAT include high-resolution ESI/MS
([M þ H]þ expected, 345.1346; observed, 345.1364), UV-vis,
and 1H NMR data (30) (see Figures S12C and S14 of the
Supporting Information).
HPLC-Based Assays for Af12060. For determination of
cofactor requirements, 50 μL reaction mixtures contained 5 μM
Af12060 and 200 μM enantiopure FQF with or without 1 mM
NADPH or NADH in NaPi reaction buffer [50 mM sodium
phosphate (pH 7.4), 100 mM NaCl, and 5% glycerol]. Reactions
were initiated with enzyme, mixtures incubated at room tem-
perature for 30-60 min, and then reactions quenched by addition
of an equal volume of MeCN. Following incubation for an
additional 5 min at 25 °C, the precipitant was removed by
centrifugation, and 20 μL samples of the resulting supernatant
were injected for HPLC analysis using an Alltima C18 column
(150 mm ꢀ 4.6 mm), with detection at 274 nm. Solvent systems A
(water with 0.1% formic acid) and B (MeCN with 0.1% formic
acid) were used to apply a linear gradient from 0 to 60% B over
20 min, followed by a gradient from 60 to 95% B over 1 min, and
a steady period of 95% B for 5 min with a flow rate of 1 mL/min.
Under these conditions, a retention time of 21.9 min was
observed for FQF, and retention times of 18.2 and 24.9 min
were observed for products 3 and 4, respectively.
For time course analysis of FQF utilization, a 350 μL reaction
mixture was set up with 2 μM Af12060, 200 μM enantiopure
FQF, and 1 mM NADPH in NaPi reaction buffer. Reactions
were initiated with enzyme and time points taken between 2.5 and
20 min by quenching 50 μL portions with an equal volume of
MeCN. Quenched reactions were prepared for and analyzed by
HPLC as described in the previous paragraph. FQF peak
integration at 274 nm was used to generate a plot of FQF peak
area versus time to approximate the enzymatic rate. Initial rate
data, obtained as integration area per minute, were converted to
micromolar per minute using a standard curve generated from
20 μL injections of FQF samples at a known concentration.
Turnover was calculated on the basis of the enzyme concentra-
tion taking into account the fact that only ≈34% of the protein is
in the holo and hence catalytically active form [turnover = rate/
([enzyme] ꢀ 0.34)]. Additionally, both rate and turnover data
were calculated by taking into account the concentrations of
substrate and enzyme before the 2-fold dilution of the initial
reaction volume from the MeCN quench.
Synthesis and Characterization of Fumiquinazoline F
(FQF) and Glyantrypine (GAT). The synthesis of FQF and
GAT followed a previously described three-component one-pot
microwave-assisted protocol (30). For the synthesis of FQF,
anthranilic acid (140 mg, 200 μmol), N-Boc-L-Ala (190 mg,
200 μmol), and triphenyl phosphite (315 μL, 220 μmol) were
combined with anhydrous pyridine (5 mL) in a round-bottom
flask and heated in an oil bath for 16 h at 55 °C. After the sample
had cooled to room temperature,
D-Trp methyl ester hydro-
chloride (255 mg, 200 μmol) was added and the solution was
divided among five septum-sealed 10 mL reaction vials and
irradiated using a CEM Discover microwave reactor for 1.5 min
at 220 °C. The resulting crude reaction mixture was concentrated
in vacuo, and the residue was dissolved in CH2Cl2 and purified by
silica gel flash chromatography (eluting with a 7:3:0.1 CH2Cl2/
EtOAc/MeOH mixture). The desired fractions were concen-
trated, and the resulting residue was dissolved in 1 mL of DMSO
for further purification using preparative reverse-phase HPLC,
injecting 2 ꢀ 0.5 mL samples onto a Luna C18(2) column
(250 mm ꢀ 21.2 mm) with a solvent system of water and
acetonitrile, and running a linear gradient from 5 to 95% MeCN
over 40 min at a flow rate of 8 mL/min. The peak corresponding
to FQF and its enantiomer (tR = 29 min) was collected, the
MeCN removed in vacuo, and the remaining sample lyophilized.
Acidic modifiers such as TFA and formic acid were avoided
because of their contribution to epimerization at C3 of FQF. The
correct enantiomer of FQF was determined using analytical
chiral LC-MS (low-resoluton, Chiralcel OD-RH, 50 mm ꢀ
4.6 mm, 0 to 60% MeCN in water over 20 min, flow rate of
0.5 mL/min) with ESIþ mass detection by comparing the
retention time of synthetic material with that of biologically
derived FQF from the A. fumigatus XAD extract. The enantio-
meric excess (ee) of FQF was determined to be 14% by analytical
chiral HPLC (same column and gradient as described previously,
flow rate of 1 mL/min); we prepared enantiopure FQF by inject-
ing multiple samples of the scalemic mixture on the Chiralcel
column and collecting the appropriate peak. Data supporting the
ATP-[32P]PPi Exchange Assay for Af12050. This assay
was used to monitor the substrate-dependent exchange of the 32P
label of [32P]PPi into ATP from the adenylation reaction cata-
lyzed by the A domain of Af12050. Typical reaction mixtures
(100 μL) contained 2 mM ATP, 2 mM MgCl2, 3 mM Na4[32P]PPi
(0.19 μCi), 5 μM Af12050, and 2 mM amino acid substrate in Tris
reaction buffer [50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM
TCEP, and 5% glycerol]. Reactions were initiated by addition of
enzyme, mixtures incubated at 25 °C for 1.5 h, and then reactions
quenched by addition of a charcoal solution [1.6% (w/v)
activated charcoal, 100 mM sodium pyrophosphate, and 3.5%

8568 Biochemistry, Vol. 49, No. 39, 2010
Ames et al.
perchloric acid in water]. The charcoal was pelleted by centrifu-
gation and washed with a solution containing 100 mM sodium
pyrophosphate and 3.5% perchloric acid, and the charcoal-
bound radioactivity was detected by liquid scintillation counting.
[14C]Acetyl-CoA Labeling and [14C]-L-Ala Loading As-
says for Af12050. In vitro phosphopantetheinylation of
Af12050 to generate holoprotein was monitored using radiola-
beled acetyl-CoA and the phosphopantetheinyl transferase Sfp
from Bacillus subtilis (31). A typical 400 μL reaction mixture
contained 3 μM Af12050, 83 μM [1-14C]acetyl-CoA (0.25 μCi),
and 0.1 μM Sfp in Tris reaction buffer. Reactions were initiated
by addition of Sfp and mixtures incubated at 25 °C for 1, 2.5, 5,
10, 20, or 45 min; at each time point, a 50 μL volume was
quenched via addition to 0.5 mL of 10% TCA (with 50 μg of BSA
for the visualization of the precipitated protein). Protein precipi-
tate was pelleted by centrifugation, washed twice with 10% TCA,
and dissolved in 80% formic acid for liquid scintillation counting.
A ratio of nanomoles of radioactivity counted to nanomoles of
protein was used to calculate the percent conversion based on
1 equiv of [1-14C]acetyl-CoA labeling 1 equiv of Af12050.
at 18.2, 20.6, 21.9, and 24.8 min, respectively. Data supporting
the identity of the enzymatically prepared FQA include high-
resolution ESI/MS ([M þ H]þ expected, 446.1818; observed,
446.1823), UV-vis, and 1H NMR data (5, 30) (see Figures S10
and S11 of the Supporting Information).
A time course study was performed to obtain apparent rate
data of FQF utilization and FQA formation by combining
Af12050 and Af12060. A 250 μL reaction mixture was set up
to contain 1 μM Af12060, 5 μM holo-Af12050, 1 mM NADPH, 1
mM ATP, 2 mM MgCl2, 1 mM -Ala, and 200 μM FQF in NaPi
L
reaction buffer. Aliquots (50 μL) were taken at 5, 10, 25, and
60 min after the addition of Af12060 (to initiate reaction) and
reactions quenched with an equal volume of MeCN. Samples
(20 μL) were prepared for and analyzed by HPLC as described in
the previous paragraph. FQF and FQA peak integration and
initial velocity estimates were determined as described in HPLC-
Based Assays for Af12060 using FQF and FQA standards
(concentrations calculated using published extinction coefficients
as described in Materials and General Methods).
Spectrophotometric Assay for Substrate-Dependent
NADPH Consumption by Af12060 with or without Af12050.
For the assay of Af12060 alone, 200 μL reaction mixtures
contained 1 μM Af12060, 200 μM NADPH, and varying concen-
trations of FQF (5-200 μM) in NaPi reaction buffer. For the
assay of Af12060 in the presence of Af12050, 200 μL reaction
mixtures contained 1 μM Af12060, 5 μM holo-Af12050, 200 μM
The loading of radiolabeled L-Ala onto apo- or holo-Af12050
was performed to monitor the in cis thiolation activity of the
A-T pair of Af12050. Apo-Af12050 was obtained by protein
production in E. coli BL21-Gold(DE3) cells followed by Ni-
affinity purification. Holoprotein was generated in vitro with the
Ni-affinity-purified protein using CoA and Sfp (2 mM CoA,
1 mM MgCl2, and 2 μM Sfp added directly to purified Af12050,
incubation for 30 min at 25 °C). Then, a 400 μL reaction mixture
was set up to contain 3 μM holo-Af12050, 5 mM ATP, and
NADPH, 1 mM ATP, 1 mM L-Ala, 2 mM MgCl2, and varying
concentrations of FQF (5-200 μM) in NaPi reaction buffer.
Holo-Af12050 was generated in vitro using Sfp and CoA prior to
addition. Reactions were initiated by addition of Af12060 and
continuous spectrophotometric data collected at 340 nm (on a
Varian Cary 50 Bio instrument) in triplicate for each concentration
of FQF. Initial rate data (in absorbance units per minute)
representing NADPH consumption were converted to micromolar
per minute using the extinction coefficient for NADPH of 6200
M-1 cm-1. In the absence of substrate, and consistent with
observation from HPLC-based assays, oxidation of NADPH by
holo-Af12060 did not occur to any appreciable degree unless excess
FAD was included in the reaction mixture. Utilization of NADH
in place of NADPH provided similar rate data, and including the
(3R,14S) enantiomer of FQF did not decrease the measured rate.
Biosynthesis of FQA Analogues. For biosynthesis of FQA
analogue 1 [7 (Figure S12 of the Supporting Information)], a
100 μL reaction mixture consisted of 2 μM Af12060, 4 μM holo-
Af12050, 1 mM NADPH, 1 mM ATP, 2 mM MgCl2, 1 mM
50 μM
-[U-14C]Ala (0.32 μCi) in Tris reaction buffer. For time
L
point analysis, 50 μL volumes of the reaction mixture were
quenched 0.5, 1, 2.5, 5, 10, 20, and 30 min after addition of
[14C]Ala substrate with 10% TCA, and the precipitated protein
was collected and prepared for liquid scintillation counting as
described in the previous paragraph. The percentage loading of
[14C]Ala was calculated as the mole fraction of 14C-labeled
substrate covalently transferred to holo-Af12050 protein assum-
ing 1:1 stoichiometry.
Reconstitution of FQA Biosynthesis. To address the
requirement for tethering of L-alanine as a pantetheinyl inter-
mediate to the T domain of Af12050 for FQA production, 50 μL
reaction mixtures were set up to contain 2.5 μM Af12060, 2 mM
MgCl2, 2 mM NADPH, 1 mM -Ala, and 200 μM enantiopure
L
FQF in NaPi reacton buffer with varying combinations of 1 mM
ATP and 5 μM apo- or holo-Af12050. (1) To address the ability
of Af12050 to catalyze acylation of FQF with free
Af12050 was used without addition of ATP. (2) To address
coupling of -Ala-AMP to FQF by Af12050, the reaction mixture
included ATP and apo-Af12050. (3) To reconstitute -Ala-S-Enz
L
-Ala, apo-
L
-Ala, and 100 μM GAT in NaPi reaction buffer. After 2 h at
25 °C, the reaction was quenched by addition of 100 μL of MeCN
and a 10 μL sample was analyzed by high-resolution LC-MS
[Gemini-NX C18 column, 150 mm ꢀ 4.6 mm, 2 to 98% MeCN
in water (0.1% formic acid) over 13 min at a flow rate of 0.4 mL/
min]. Under these conditions, the retention times of 7 and GAT
were 9.7 and 10.2 min, respectively. A similar experimental design
was used to monitor biosynthesis of FQA analogue 2 [8 (Figure S13
of the Supporting Information)], but the reaction mixture
included 100 μM FQF (instead of GAT) and 1 mM glycine
L
L
formation (as tethered to the T domain of Af12050), the reaction
mixture included ATP and holo-Af12050 (generated in vitro by
preincubation of apo-Af12050 with CoA and Sfp). Reactions
were initiated by addition of Af12060 and mixtures incubated for
40 min at 25 °C prior to quenching with 50 μL of MeCN. The
precipitate was removed by centrifugation, and 20 μL of the
clarified supernatant was injected onto an Alltima C18 column
(150 mm ꢀ 4.6 mm) for HPLC analysis (274 nm detection). The
injected sample was separated using a gradient from 0 to 60%
MeCN over 20 min, a gradient from 60 to 95% MeCN over
1 min, and a steady period of 95% B for 5 min at a flow rate of
1 mL/min (solvent system of water and acetonitrile with 0.1%
formic acid). Under these conditions, 3, FQA, FQF, and 4 eluted
(instead of -Ala). Under the LC-MS conditions described for 7,
analogue 8 eluted at a retention time of 9.8 min.
L
RESULTS
Identification of Fumiquinazolines F and A from an A.
fumigatus Af293 Culture. Although the production of FQF

Article
Biochemistry, Vol. 49, No. 39, 2010 8569
and FQA from A. fumigatus Af293 has been previously repor-
ted (6), our own identification of these compounds from Af293
culture broth extracts proved to be useful in the characterization
of synthetic FQF and the enzymatic reconstitution product FQA.
A culture broth extract, prepared following growth of A.
fumigatus Af293 for 24 h at 37 °C in potato dextrose broth,
was analyzed by high-resolution LC-MS and analytical HPLC
(Figure S2 of the Supporting Information). Mass filtering of the
total ion current chromatogram identified FQA ([M þ H]þ
expected, 446.1823; observed, 446.1812) and FQF ([M þ H]þ
expected, 359.1503; observed, 359.1495) as two of the seven most
prominent peaks detected. Support for the positive identification
of these two peaks as FQA and FQF was provided by matching
the UV-vis spectra from diode array detection with previously
published data (5).
Cloning, Expression, and Purification of Af12050 and
Af12060. Genes encoding Af12050 and Af12060 were cloned
from A. fumigatus Af293 cDNA using start sites that varied from
the ORF annotations found in the NCBI database. The alternate
start sites were determined on the basis of multiple-sequence
alignment generated via BlastP and by analysis of homology
models generated using HHpred (32). For Af12060, sequence
alignment suggested that the ORF as annotated (AFUA_6g12060,
GenBank accession number XM_745992.1) would encode a
truncated version of the protein lacking ≈50 residues at the
N-terminus. Modeling based on PDB entry 2RGJ suggested that
these additional residues are necessary to form two β-strands and
an R-helix that are critical for interaction with the adenosine
pyrophosphate of FAD. Inspection of the 50-sequence upstream
of the annotated start site led to the identification of an additional
exon-intron pair that was used as the basis for designing the
forward primer for PCR amplification. For Af12050, an alter-
nate start site was identified following failed attempts to clone
the gene from cDNA using primers designed for the start site of
NCBI database entry AFUA_6g12050 (GenBank accession
number XM_745991.1). The new start site identified and utilized
for cloning is 582 nucleotides (mature transcript coding for 49
amino acids) downstream of the start codon of AFUA_6g12050,
eliminating the first exon-intron pair of the database entry. The
alternate start site for Af12050 is in agreement with the majority
of the top 100 hits returned from BlastP and is suggested to
encode a structurally complete A domain (as part of the full-
length A-T-C protein) based on homology modeling. Both
Af12060 and Af12050 were cloned into pET30 Ek-LIC vectors
to encode N-terminal His6-S tag proteins (Af12050, 128 kDa;
Af12060, 55 kDa), expressed using E. coli BL21-Gold(DE3), and
purified by Ni-affinity chromatography (Figure S3 of the Sup-
porting Information). Af12060 was purified as a bright yellow
protein to >90% purity with a yield of 32 mg/L, while Af12050
eluted from the Ni resin in moderate yield (12 mg/L) with a purity
of ≈60%, likely due to contamination by degraded and/or
truncated versions of the target protein.
the flavin cofactor as FAD was further confirmed by HPLC
analysis of the supernatant from heat-denatured Af12060 (Figure
S4C of the Supporting Information). The percent holoprotein
was determined to be 23% for the native enzyme using absor-
bance values specific for protein (A280) and flavin (A446) or 34%
when using the A446 of the released FAD following protein
denaturation (Figure S4A,B of the Supporting Information). The
discrepancy in percent holo-Af12060 may arise from quenching
of the FAD A446 signal when bound to enzyme.
Fumiquinazoline F, a candidate substrate for Af12060, was
chemically synthesized from anthranilic acid, N-Boc-
L-alanine,
and -tryptophan methyl ester hydrochloride using a modified
D
literature procedure (30). Enantiopure FQF was purified from
the crude reaction mixture by sequential silica gel flash chroma-
tography, semipreparative reverse-phase HPLC, and chiral-
phase HPLC (Figure S5A of the Supporting Information). The
identity of FQF was further supported by MS, UV-vis, and
NMR spectroscopy (Figures S5B and S6 of the Supporting
Information).
Incubation of enantiopure FQF with purified Af12060 and
reduced nicotinamide cofactor (NADPH or NADH) led to
the disappearance of FQF and the formation of two new
products (3 and 4) in an enzyme- and time-dependent manner
(Figure 3A,B). By monitoring the disappearance of FQF over
time, we obtained an apparent reaction velocity of 11 μM min-1
and turnover number of 16 min-1 for the experimental conditions
assayed (2 μM Af12060, 200 μM FQF, and 1 mM NADPH).
Further kinetic analysis of Af12060 was hampered because of
substrate inhibition (Figure S7 of the Supporting Information).
The UV-vis spectra of 3 and 4 are similar to those obtained for
FQF, but there are notable differences in the range of 275-
290 nm, suggesting possible modification of the indole moiety of
FQF (Figures S8 and S9 of the Supporting Information).
Although the instability of compounds 3 and 4 hindered struc-
tural identification by NMR spectroscopy, mass spectral analysis
suggested that 3 is a dihydroxylated version of FQF (FQF-
indoline-20,30-diol, [M þ Na]þ expected, 415.1386; observed,
415.1386) and that 4 is an oxidized dimer ([M þ H]þ expected,
749.2831; observed, 749.2845) (Figures S8 and S9 of the Support-
ing Information).
The biosynthetic intermediate leading to the formation of
either 3 or 4 is postulated to be an indole C30-hydroxyiminium
species (1) or a 20,30-epoxyindole (2) generated by the enzymatic
oxidation of FQF by Af12060 (Figure 3C). As an FAD- and
NAD(P)H-dependent monooxygenase, we postulate that Af12060
catalyzes the reduction of FAD to FADH-, which reacts with
molecular oxygen to form a C4a-peroxyflavin species that is then
protonated to yield C4a-hydroperoxyflavin. The 20,30-double
bond of the pendant indole could serve as the nucleophile for
capturing the electrophilic hydroxyl group of the hydroperoxy-
flavin to generate 1. Epoxide formation to form 2 may then occur
via intramolecular capture by the 30-OH group. In the absence of
downstream enzymatic machinery, and on the basis of the
electrophilic reactivity at indole-derived C20 of the oxy-FQF
intermediate, release of 1 or 2 from the enzyme active site could
lead to capture by water from bulk solvent to yield 3, or dimeriza-
tion to form some variety of 4. Additional details regarding
characterization of 3, including isotope labeling studies using
[18O]H2O, are provided as Supporting Information.
Characterization of Af12060. Gene annotation and se-
quence analysis suggested that Af12060 is a FAD-dependent
oxidoreductase. Homology modeling provided additional in-
sight, suggesting that Af12060 is a class A monooxygenase that
contains noncovalent but tightly bound FAD, requires NAD-
(P)H as a coenzyme, and should possess an overall structure (for
residues 1-420) similar to that of the aromatic hyroxylase PhzS
from Pseudomonas aeruginosa (33, 34). The UV-vis spectrum of
purified Af12060 supported the presence of bound flavin cofactor
(Figure S4A,B of the Supporting Information). The identity of
Characterization of Af12050 and Reconstitution of FQA
Biosynthesis. Af12050 is a monomodular three-domain (A-T-C)
NRPS that we predict to selectively activate and couple L-alanine

8570 Biochemistry, Vol. 49, No. 39, 2010
Ames et al.
F
IGURE 3: Biochemical characterization and proposed mechanism of the FAD- and NAD(P)H-dependent monooxygenase, Af12060. (A) HPLC
analysis (274 nm detection) of reaction mixtures containing 200 μM FQF and 5 μM Af12060 with or without 1 mM NADPH or NADH.
Reactions were quenched after incubation for 40min at 25°C via addition of 50% MeCN. (B) Time course for FQF utilization and accompanying
rate data obtained by integration of the FQF peak area. The reaction mixture contained 200 μM FQF, 2 μM Af12060, and 1 mM NADPH;
reactions were quenched at the indicated time point by addition of MeCN. (C) Proposed pathway for the conversion of FQF to 2 and 3 by
Af12060. Brackets denote putative intermediates not detected by HPLC or LC-MS analysis. Possible structures for the oxidized dimer species
(4) are provided in Figure S9A of the Supporting Information.
to the oxidized indole of FQF. The putative 10-residue substrate
specificity sequence for the A domain of Af12050 is 80% similar
to the L-Ala-specific A domain from module 11 of cyclosporin A
synthethase (35, 36). Experimental validation of Ala-specific
adenylation and thiolation activity was accomplished using
full-length protein, with the apo form obtained as purified from
E. coli and the holo form generated in vitro by incubation
with CoA and the phosphopantetheinyl transferase Sfp (31)
(Figure 4A).
couple an enzyme-tethered alanine to the indole-derived side
chain of oxy-FQF to reconstitute FQA biosynthesis. Evidence
that FQF is not a direct substrate for Af12050 is provided in
Figure 5A (bottom two traces); incubation of holo-Af12050,
L
-Ala, Mg2þ, ATP, and FQF does not lead to a decrease in the level
of FQF or to the formation of any new peaks that could represent
alanyl-FQF intermediates. The requirement for an alanyl-S-Enz
intermediate for productive coupling of Ala to oxy-FQF was
investigated using apo- or holo-Af12050, Af12060, NADPH,
The amino acid preference for adenylation by Af12050 was
investigated by monitoring the substrate-dependent exchange of
Mg2þ, and
L
-Ala with or without ATP (Figure 5A, top three
traces). The different combinations tested illustrate that (1) free
-Ala is not a substrate for coupling (middle trace, products 3 and
4), (2) -Ala-AMP is not a substrate for coupling (second trace
radioactive [32P]PPi into ATP. As illustrated in Figure 4B,
L
-Ala
is the preferred substrate for adenylation by the A domain of apo-
Af12050, while Gly, -Ser, -Ala, and -Val also promote exchange
above the background level (20, 10, 7, and 4% of the level
observed for -Ala, respectively). Conversion of the Af12050 T
L
L
L
D
L
from the top, products 3 and 4), and (3) including Af12060 and
holo-Af12050 with the necessary components for FQF oxidation
and alanyl-S-Enz formation leads to the production of a single
new product identified as FQA [top trace; [M þ H]þ expected,
446.1823; observed, 446.1818 (see Figures S10 and S11 of the
Supporting Information)]. A time course study monitoring the
conversion of FQF to FQA by analytical HPLC did not detect
intermediate compounds or the presumed shunt metabolites 3
and 4 (Figure 5B). The putative intermediates for this reaction
are likely short-lived and therefore not detected, but the absence
of detectable amounts of 3 and 4 suggests that (1) if formed, 3 and
4 are in equilibrium with the predicted on-pathway intermediates
[e.g., 1 and 2 (Figure 3C)] and/or (2) Af12060 and Af12050 are a
highly efficient pair for the net oxidative acylation of FQF to
form FQA. Intermediate channeling between an enzyme complex
L
domain from apo to holo was assayed by incubation with Sfp and
[14C]acetyl-CoA to achieve ≈22% conversion to the acetyl-S-
pantetheinylated holo form. Transfer of the alanyl group of the
activated
thioester was assayed using [14C]-
loading of 24% [14C]-
-Ala was reached between 5 and 10 min
after initiation of the reaction. When Sfp was omitted from the
reaction mixture, no labeling of Af12050 with [14C]-
-Ala was
L-Ala-AMP to the holo T domain as an aminoacyl
L-Ala (Figure 4C). A maximal
L
L
observed, demonstrating that Af12050 purified from E. coli is
in apo form.
The ability of Af12050 to selectively activate and load
L-Ala set
the stage for evaluation of the ability of this same enzyme to

Article
Biochemistry, Vol. 49, No. 39, 2010 8571
terminal C domain of Af12050 binds both the free oxy-FQF
intermediate (1 or 2) and the T domain-tethered, L-Ala-S-Enz
intermediate, to bring these two species into proximity for
reaction. For pathway A, the indoline NH group of intermediate
2 could serve as the nucleophile for attack at the thioester
carbonyl of the enzyme-tethered
be N-acylation of the epoxyindoline via amide bond forma-
tion and release of the -alanyl intermediate from Af12050.
L-Ala. The net result would
L
Subsequently, activation of the alanyl-R-amino group of inter-
mediate 5 by the C domain and attack at indoline C20 would
result in the favorable 5-exo-tet intramolecular cyclization that
is concomitant with epoxide ring opening. For pathway A, the
stereochemistry of the epoxide (as installed by Af12060) would
dictate the observed trans stereochemistry at positions C30 and
C20 of the resulting imidazoindolone by necessitating an SN2-
mediated attack of the alanyl NH2 group at C20. For pathway B,
position C20 of the indole-derived 30-hydroxyiminium (1) serves
as the electrophilic center for attack by the C domain-activated R-
amino group of the alanyl-S-Enz intermediate. At this point, the
alanyl-oxy-FQF intermediate (6) would remain tethered to the T
domain of Af12050, with chain release and the favorable
intramolecular 5-exo-trig ring closure occurring by attack of
the indoline NH group on the thioester carbonyl. This reaction
may occur in the active site of the C domain but may also be
spontaneous. For path B, the final stereochemistry at positions
C20 and C30 of the indoline could be influenced in part by the
stereochemistry of the hydroxylation step and/or the direction of
attack by the alanyl-R-amino group as dictated by the C domain
active site architecture.
Substrate Tolerance of Af12060 and Af12050 and Bio-
synthesis of FQA Analogues. The monooxygenase Af12060 is
sensitive to changes in the stereochemistry of FQF at C14
F
IGURE 4: Characterization of substrate-dependent adenylation ac-
tivity and L-Ala loading of Af12050. (A) Schematic for the two-step
process of adenylation (to generate an activated L-alanyl-AMP
intermediate) and subsequent loading of the L-alanyl group onto
the 40-phosphopantatheine group of the holoprotein. (B) ATP-
[32P]PPi exchange activity promoted by apo-Af12050 with various
amino acids tested as substrates (100% activity corresponds to
112000 cpm). Reactions were quenched with activated charcoal
following a 1.5 h incubation at 25 °C, and radioactivity was detected
by liquid scintillation counting. (C) Time course for the loading of
radiolabeled [14C]-L-Ala onto Af12050 protein that was purified
directly from E. coli BL21(DE3) cells (b, apoprotein) or following
in vitro incubation with B. subtilus Sfp and CoA for T domain
phosphopantetheinylation (9, holoprotein).
(functionality derived from
D-Trp) but not at position C3
(from -Ala); the enantiomer of FQF, with a (3R,14S) config-
L
uration, is not a substrate for Af12060, while FQG, a diaste-
reomer with a (3R,14R) configuration, is a substrate (data not
shown). Additionally, assays using a mixture of synthetic FQF
with its enantiomer (in a 57:43 ratio) with Af12060 (with or
without Af12050) did not result in altered production of 3, 4, or
FQA or hinder the rate of product formation, suggesting that the
enantiomer of FQF is not an inhibitor.
To further explore substrate tolerance as well as the potential
utility of applying the two enzymes Af12060 and Af12050 in
tandem to generate molecules with a 6-5-5 muticyclic imidazoin-
dolone scaffold, we reconstituted the production of two FQA
analogues: 7 (Figure S12 of the Supporting Information) and 8
(Figure S13 of the Supporting Information). Compound 7 arises
from the enzymatic processing of glyantrypine (GAT) (11). The
only difference between FQF and GAT is the change in
functionality at position C3 of the pyrazinoquinazolinone tri-
of Af12060 and Af12050 is one possible means of achieving
efficient product formation without off-pathway capture of the
reactive intermediate by water or through dimerization; however,
gel filtration chromatography did not detect formation of a
protein complex. Apparent rates associated with the conversion
of FQF to FQA were estimated using the data from the HPLC
time course experiment (right panel of Figure 5B); as anticipated
for efficient product formation, the apparent initial velocity of
FQF disappearance (4.0 μM min-1) matches the rate of FQA
appearance (3.8 μM min-1).
cycle; FQF contains a methyl substituent derived from L-Ala,
while GAT lacks functionality at this position because of
incorporation of Gly. GAT was synthesized in a manner similar
to that of FQF and obtained as a mixture of enantiomers. Upon
combining the holo versions of both Af12060 and Af12050 in a
We propose two alternative pathways for the enzymatic
conversion of FQF to FQA by Af12060 and Af12050
reaction mixture containing NADPH, ATP, and L-Ala with
(Figure 5C). Activation and loading of
L
-Ala by Af12050 primes
synthetic GAT, the formation of 7 was detected by LC-MS
(Figure S12 of the Supporting Information). The production of 7
indicates that a methyl substituent at C3 is not essential for
substrate recognition or productive binding by either Af12060 or
Af12050. On the basis of the exclusive preference of Af12060 for
(3S,14R) FQF over the (3R,14S) FQF enantiomer, we presume
this enzyme for coupling of alanine to oxy-FQF, where oxidation
of the indole 20,30-double bond of FQF is catalyzed by Af12060
via 20,30-epoxidation (2) or 30-hydroxylation (1) (Figure 3C). Sub-
sequently, and consistent with the role of C domains in the
chemistry of N-C bond formation (37), we anticipate that the

8572 Biochemistry, Vol. 49, No. 39, 2010
Ames et al.
F
IGURE 5: Reconstitution of fumiquinazoline A (FQA) biosynthesis by combining Af12060 (monooxygenase) and Af12050 (nonribosomal
peptide synthetase) with FQF as the substrate. (A) HPLC analysis of reaction mixtures containing 200 μM FQF, 1 mM NADPH, 1 mM L-Ala, 2
mM Mg2þ, and various combinationsof 2.5 μM Af12060 (abbreviated as 60), 5 μM Af12050 (abbreviated as 50), and 1 mM ATP. The (-) enzyme
control contained all reaction components except Af12050 and Af12060. Holo-50 denotes phosphopantetheinylated protein. (B) Time course of
FQA production and accompanying rate data for FQF utilization and FQA formation. The concentrations of components used were the same as
those for panel A, except for 1 μM Af12060. (C) Two proposed pathways for the conversion of FQF to FQA by Af12060 and Af12050. Brackets
denote putative intermediates not detected by HPLC or LC-MS analysis. The C domainof Af12050 is postulated to catalyze the two-stepprocess
of L-Ala coupling and subsequent intramolecular attack to generate the 6-5-5 imidazoindolone moiety of FQA.
that the stereochemistry of 7 is (14R). Additionally, though
is the preferred substrate for adenylation by Af12050, Gly is also
L
-Ala
(e.g., 4-OH-benzoate) that can generate an adjacent carbanion
equivalent, hydroxylation of the pendant indole of FQF may
involve participation of the pyrrole NH group. Conversion of 1
to the epoxy intermediate 2 may not necessarily occur; however,
the formation of 2 prior to alanine coupling and intramolecular
cyclization could control the observed stereochemistry of the
imidazoindolone scaffold of FQA. One possible precedent is
epoxidation of the terminal olefin of squalene by the two-
component FAD-dependent squalene epoxidase, proposed to
catalyze donation of electrophilic oxygen from hydroperoxy-
flavin for hydroxylation followed by intramolecular capture to
form an epoxide product (39). For Af12060, we note that
interconversion of 1 and 2 may occur in the enzyme active site.
Oxidation of the pyrrole ring of indoles by non-heme iron or
cytochrome-type oxgenases has been reported previously (40, 41),
activated and promotes a level of exchange that is ≈20% that
of L-Ala (Figure 4B). To investigate the ability of Af12050 to
activate, load, and couple Gly to oxy-FQF, holoenzymes Af12060
and Af12050 were combined with NADPH, ATP, FQF, and Gly,
and product analysis was performed by LC-MS (Figure S13 of
the Supporting Information). A small amount of the glycine-
derived product 8 was observed, while the majority of the product
formed was the indoline-20,30-diol (3). These results suggest that
the coupling of glycine to the N10 and C20 positions of the
oxidized indole of FQF occurs; however, this process is much less
efficient than when L-Ala is used as a substrate for Af12050 and
leads to the formation of the uncoupled product 3.
including the degradation of L-Trp to N-formylkynurenine by
DISCUSSION
two heme/O2-dependent dioxygenases (42). Additionally, the
20,30-epoxidation of indole by the P450 monooxygenase KtzM
is postulated to occur in the biosynthesis of the kutzneride
building block dichlorohydroxyhexahydropyrrole indole (43).
However, reports describing the flavin-dependent oxidation of
indole are much less common. Hydroxylation at indole C2 or C3
leading to indigo and indirubin has been reported for an engi-
neered variant of the class A flavoprotein 2-hydroxybiphenyl
3-monooxygenase, HbpA (44) (15% identical and 48% similar to
Af12060 for residues 1-452). The hydroxyindole reaction products
produced by HbpA are reported to be unstable and spontaneously
Oxidation of FQF by Af12060. This study indicates that
Af12060 is a single-component FAD-dependent monooxygenase
that acts on the pendant indole side chain of FQF. We propose a
mechanism that involves hydroxylation at C30 of the indole
moiety (1) and possible conversion to a 20,30-epoxyindole (2)
(Figure 3C). The initial hydroxylation of FQF by Af12060 may
be similar to flavoprotein-catalyzed hydroxylation of phenolic
compounds, where a C4a-hydroperoxy-FAD is the electrophilic
oxygenating species for a nucleophilic substrate (38). In analogy
to the hydroxyl activating substituent of phenolic compounds

Article
Biochemistry, Vol. 49, No. 39, 2010 8573
oxidize and dimerize to form pigments (44). Analogously, the
presumed oxy-FQF intermediates 1 and 2 were not isolated
during the course of experimentation; rather, the compounds
isolated were from spontaneous reaction with water (3) or
dimerization (4). With regard to modification of a pendant indole
group as part of a more complex natural product, the oxidation
at indole C2 of a bisindolepyrrolidone by VioC has been reported
in the biosynthesis of violacein (45). VioC and Af12060 are class
A flavoprotein monooxygenases that share moderate amino acid
sequence homology (16% identical and 50% similar). Addition-
ally, the flavoprotein oxidation of a Trp-derived indole via 20,30-
epoxidation has been proposed for the biosynthetic route to the
natural products notoamides C and D (46) [the recently reported
gene cluster contains two Af12060 homologous, NotB and
NotI (47)]. The characterization of Af12060 provides an intri-
guing example of flavoprotein-catalyzed oxidation of indole and
expands upon the small number of known or proposed flavin-
dependent monooxygenases that modify the C2 or C3 position of
indole or indole-derived metabolites.
Enzymatic Conversion of FQF to FQA. The FAD- and
NAD(P)H-dependent monooxygenase Af12060 and the alanine-
activating tridomain NRPS Af12050 are necessary and sufficient
to convert FQF to FQA, an enzymatic process requiring tailoring
of a Trp-derived indole via oxidation and dual N-C bond
formation to form a 6-5-5 imidazoindolone ring system. Initially,
the order of oxidation and alanine coupling was unknown. We
had previously proposed N-acylation of FQF via nucleophilic
attack of the indole NH group of FQF on an activated alanyl-S-
Enz thioester, and then oxidation of the indole 20,30-olefin and
intramolecular attack of the alanine R-amino group on C20 of the
N-acylindole (28). However, the ability of Af12060 to accept
FQF as a substrate and the observation that incubation of
that of prenylation in abolishing indole aromaticity, but in the
case of epoxidation, there is an oxygen neighboring the indole
NH group resulting in hemiaminal functionality. Because FQF is
not a substrate for Af12050, we hypothesize that if path A is
operant, the epoxidation likely enhances the nucleophilicity of
the indole NH group due to rehybridization of C20 and C30
from sp2 to sp3 and/or the formation of the epoxide is a critical
recognition determinant for L-Ala coupling by Af12050.
Acylation of oxy-FQF by Af12050 represents the first char-
acterized example of nonribosomal peptide synthetase-based
coupling of an amino acid to the pyrrole ring of indole. We
propose that the coupling step may be catalyzed by the terminal C
domain of Af12050 that would bind in cis a T domain-tethered
alanyl-S-pantetheinyl intermediate and in trans a free-standing
oxy-FQF intermediate [either 1 or 2 (see Figure 5C)]. This
functionality is in contrast to that traditionally observed for C
domains embedded within a multimodular NRPS (e.g., Trp-
specific module 2 of Af12080) that couple two T domain-tethered
amino acyl intermediates (one upstream and one downstream of
the C domain) via amide bond formation for chain elongation.
For embedded C domains, the thioester carbonyl of the upstream
intermediate acts as the electrophilic acceptor while the R-amino
group of the downstream intermediate is activated to be nucleo-
philic. Path A of Figure 5C fits this paradigm for the upstream
tethered intermediate as the electrophilic acceptor, but indole N1
of oxy-FQF (rather than an R-amino group) is the nucleophile
for amide bond formation. The reaction between an upstream
enzyme-bound thioester intermediate and free low-molecular
weight nucleophile (N-hydroxyhistamine) has also been charac-
terized in pseudomonine biosynthesis (49). Path B of Figure 5C
reverses the roles of the two proposed reaction intermediates and
also represents an N-C bond forming event unprecedented for
C domain chemistry; the upstream tethered alanyl-S-Enz inter-
mediate would be activated for nucleophilic attack on the
electrophilic C2 atom of the bound oxy-FQF intermediate. In
either case, the bicyclic indole moiety is thereby converted to the
tricyclic imidazoindolone ring system.
Other Fungal Alkaloids That Are Biosynthesized in Part
by Oxidative Acylation of Indole. The two-step process of
Trp-derived indole oxidation and amino acid coupling described
in this work for the conversion of FQF to FQA is likely shared
among several families of fungal indolic natural products
(Figure 2). The bioactivities of the compounds provided in
Figure 2 include the mycotoxin tryptoquivaline, a tremor-causing
metabolite originally isolated from A. clavatus (50, 51); and
potential therapeutics asperlicin, a cholecystokinin receptor
antagonist from Aspergillus alliaceus (52, 53), chaetominine, an
antitumor agent more potent than 5-fluorouracil (54), fiscalin A,
a substance P inhibitor from N. fischeri (10), and fumiquinazoline
I, a weak antifungal agent from an Acremonium sp. (9). Com-
pounds listed that do not have an attributed biological activity
include the pyrazinoquinazolinone cottoquinazoline A from
A. versicolor (12) and the diketopiperazine lumpidin from
Penicillium nordicum (55). Of interest is the fact that in cases
where bioactivities have been described for the natural product
before and after amino acid coupling to the indolic scaffold (FQF
vs FQA, fiscalin B vs fiscalin A, and asperlicin C vs asperlicin)
the coupled product is reported to be 2-15-fold more potent
(5, 10, 53).
L-alanyl-S-Af12050 with FQF did not result in the formation
of any new products suggested that the order of enzymatic events
actually starts with indole oxidation by Af12060 followed by
acylation with L-Ala and cyclization by Af12050. While this order
may be dictated to some degree by enzyme substrate specificity,
perhaps a more important consideration is the reactivity of the
substrate, with oxidation of the indole 20,30-olefin activating the
indole-derived functionality for N-acylation.
With regard to the timing of indole N-acylation, parallels may
be drawn between the N-alanylation of oxy-FQF (Figure 5C)
and the N-acetylation of aszonalenin (48) (Figure S15 of the
Supporting Information), based on the transformation process
and associated reactivity of the Trp-derived indole. In both cases,
the aromaticity of the pyrrole portion of indole is lost prior to
N-acylation due to modification at indole C3. For azonalenin,
there is prenylation by AnaPT at C3 coupled to intramolecular
cyclization (N12 of the benzodiazepine attacks the indole C2). The
indole-derived nitrogen of the resulting compound (aszonalenin)
is an aniline-like secondary amine, which is now presumed to be
sufficiently nucleophilic for attack on the thioester carbonyl of
acetyl-CoA to yield the N-acetylated product acetylaszonalenin.
For the conversion of FQF to FQA, the oxidation at indole C30
by Af12060 (either as the 3-hydroxyiminium or the 20,30-epoxide)
occurs prior to the coupling of L-alanine by Af12050 via two
consecutive N-C bond formations. For path B of Figure 5C, the
oxidation at indole C30 of FQF (similar to prenylation at indole
C3 of the benzodiazepinedione) abolishes aromaticity and results
in generation of a positively charged iminium species that renders
the indole C2 electrophilic and susceptible to nucleophilic attack.
For path A of Figure 5C, epoxidation serves a purpose similar to
Inspection of the compounds listed in Figure 2 provides
possible insight into the mechanism of indole oxidation and dual
N-C bond formation at N10 and C20 of the indole ring. Like that

8574 Biochemistry, Vol. 49, No. 39, 2010
Ames et al.
observed for FQA (Figure 1B), the side of attack of the R-amino
group of the coupled amino acid at C20 is opposite the side
observed for the hydroxyl group at C30 for chaetominine,
fumiquinazoline I, and asperlicin. In all these cases, the resulting
stereochemistry across the C20-C30 bond could result from
nucleophilic attack of an epoxyindole intermediate (e.g., path
A, Figure 5C) or directional, “opposite-side” attack of a 30-
hydroxyiminium intermediate (e.g., path B, Figure 5C). In
contrast, the stereochemistry across the C20-C30 bond for lumpi-
din, tryptoquivaline, fiscalin A, and cottoquinazoline A could be
obtained only by attack of the R-amino group of the coupled
amino acid at C20 on the same side as the hydroxyl functionality
at C30 and not by an attack on an epoxyindole intermediate. This
scenario suggests that a 30-hydroxyiminium species is the likely
intermediate prior to amino acid coupling to the indole scaffold
of this group of fungal alkaloids.
Fumiquinazoline Biosynthetic Gene Cluster and Similar
Clusters in Other Fungi. Our results characterizing the activ-
ities of Af12060 and Af12050 validate the identification of the
fumiquinazoline biosynthetic gene cluster of A. fumigatus Af293.
We anticipate that the fumiquinazoline cluster includes the trimodu-
lar NRPS Af12080, two putative FAD-dependent oxidoreduc-
tases (ORFs 12060 and 12070), a stand-alone monomodular
NRPS (12050), an anthranilate synthase homologue (12110), a
HET domain protein (12090), a nitrilase homologue (12100), and
a putative MFS transporter (12040) (Figure 1A). Of note is the
the Supporting Information). The N. fischeri cluster contains a
unique ORF between the monooxygenase and monomodular
NRPS, a 2-oxoglutarate-Fe(II)-dependent oxidoreductase. In
A. clavatus NRRL1, the homologous ORFs include ACLA_017890
(trimodular NRPS), ACLA_017900 (monomodular NRPS), and
ACLA_017910 and ACLA_017920 (both FAD monooxygenases).
In addition, this A. clavatus cluster contains an Af12070 homo-
logue that resides immediately upstream of the trimodular
NRPS. Characterized products from A. clavatus include the
tryptoquivalines (50, 51, 59), which could arise from enzymatic
processing of a fiscalin A-like precursor.
ORFs AFUA_6g12040-AFUA_6g12080 of the fumiquinazo-
line gene cluster are regulated by LaeA (60), a global regulator of
several secondary metabolite pathways (61); loss of LaeA results
in an A. fumigatus mutant with reduced virulence (62). Although
there is no reported connection between the production of
fumiquinazolines and A. fumigatus virulence, it is known that
many secondary metabolites produced by filamentous fungi are
important for their pathogenicity as toxins and immunosuppre-
sants (63). However, there is no single factor (small molecule or
gene product) that alone has been described as being essential and
sufficient for A. fumigatus pathogenicity (64). Future studies
aimed at understanding the biological function of FQs and their
possible connection to the pathogenicity of A. fumigatus can now
be undertaken.
In conclusion, the maturation of FQF to FQA in A. fumigatus
Af293 is an oxidative, dual N-C bond-forming process pre-
viously unprecedented for NRPS-based logic. Oxidation of the
pyrrole ring of FQF by the flavoprotein monooxygenase Af12060
is a prerequisite for alanine coupling to N10 and C20 of the
oxidized indole by the monomodular tridomain NRPS Af12050.
Overall, the four NRPS modules of the fumiquinazoline cluster
(three modules of Af12080 and one module of Af12050) use two
fact that the process for generating FQA from Ant,
L-Trp, and
L-Ala requires just three ORFs of this eight-gene cluster: Af12080,
Af12050, and Af12060. This leaves an additional oxidoreductase
(Af12070), a HET domain protein (Af12100), and a nitrilase/
amidohydrolase homologue (Af12110) for other possible trans-
formations. At least one additional enzyme, likely Af12070 or
Af12100, is required to transform FQA into the naturally
occurring fumiquinazolines C and D (Figure S1 of the Support-
ing Information), which appear to be formed via intramolecular
cyclization between the imidazoindolone OH or NH group and
C3 of the pyrazinoquinazolinone (5). The possible role of the
putative HET domain protein [or homologue of HET-6OR from
Neurospora crassa (56)] is a much greater mystery. HET domain
proteins are associated with mediating cell death in fungal
vegetative (heterokaryon) incompatibility (57, 58). A PSI-BLAST
search with residues 1-250 of Af121090 as the query sequence
indicates that there are seven HET domain genes in A. fumigatus
Af293. It is tempting to speculate that the clustering of this ORF
with the FQ biosynthetic machinery provides some link to FQ
biological function, but it may be that the FQ biosynthetic gene
cluster spans only ORFs 12040-12080.
proteinogenic amino acids, L-Trp and L-Ala, and one nonproteino-
genic amino acid, the aryl R-amino acid anthranilate, to build the
hexacyclic FQA molecule in a short efficient pathway. This work
validates the fumiquinazoline biosynthetic gene cluster of
A. fumigatus Af293 and represents a biosynthetic oxidative-acyla-
tion transformation that may be shared among several families of
bioactive fungal indolic alkaloids.
ACKNOWLEDGMENT
We thank the ICCB-Longwood Screening Facility at Harvard
Medical School for access to their microwave reactor. We thank
Thomas Gerken for providing A. fumigatus Af293 cDNA and
Elizabeth Sattely for purified Sfp. We are grateful to Stuart
Haynes for his careful reading of the manuscript.
As expected, an orthologous gene cluster is present in
A. fumigatus CEA10 spanning AFUB_078010-AFUB_078100.
The gene product for the amidohydrolase homologue has not
been annotated but is present and 100% identical to AFUA_
6g12100. Of greater potential interest is the presence of genes
homologous to Af12080, Af12050, and Af12060 clustered in the
genomes of N. fischeri NRRL181 and A. clavatus NRRL1. The
N. fischeri homologues include NFIA_057960 (trimodular
NRPS), NFIA_057990 (monomodular NRPS), and NFIA_057970
(FAD monooxygenase). N. fischeri is a known producer of
mambers of the fiscalin family of quinazoline alkaloids (10);
fiscalin A and FQA are highly similar, exhibiting two subtle
differences: the stereochemistry at C20 of the imidazoindolone
and the C3 substituent of the pyrazinoquinazolinone core
(methyl for FQA vs isopropyl for fiscalin A) (Figure S1 of
SUPPORTING INFORMATION AVAILABLE
Discussion of the mass spectral characterization and [18O]H2O
incorporation studies of the indoline-20,30-diol (3) and figures
providing examples of fungal alkaloids containing a pyrazino-
quinazolinone core scaffold (Figure S1), identification of FQF
and FQA from A. fumigatus Af293 culture broth (Figure S2),
SDS-PAGE gels of Ni-affinity-purified Af12060 and Af12050
(Figure S3), FAD binding by Af12060 (Figure S4), data char-
acterizing synthetic FQF (Figure S5), 1H NMR data for synthetic
FQF (Figure S6), a spectrophotometric assay of NADPH
consumption by Af12060 with or without Af12050 (Figure S7),
characterization of 3 (Figure S8), characterization of the oxidized
dimer of FQF (4) (Figure S9), data characterizing the enzymatic
product FQA (Figure S10), 1H NMR data for FQA (Figure S11),

Article
Biochemistry, Vol. 49, No. 39, 2010 8575
data describing the enzymatic production of FQA analogue 1
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