δ1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline

Article abstract of DOI:10.1021/jf0700576

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Δ¹-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21-(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of L-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.

Full text of DOI:10.1021/jf0700576

J. Agric. Food Chem. 2007, 55, 5097−5102 5097
δ
1-Pyrroline-5-carboxylic Acid Formed by Proline
Dehydrogenase from the Bacillus subtilis ssp. natto Expressed
in Escherichia coli as a Precursor for 2-Acetyl-1-pyrroline
TZOU-CHI HUANG,† YI-WEN HUANG,† HUI-JU HUNG,‡ CHI-TANG HO,§ AND
MEI-LI WU*,‡
Institute of Biotechnology, National Pingtung University of Science & Technology, 912, Pingtung,
Taiwan, Department of Food Science, National Pingtung University of Science & Technology, 912,
Pingtung, Taiwan, and Department of Food Science, Rutgers University, 65 Dudley Road, New
Brunswick, New Jersey 08901-8520
Proline dehydrogenase (PRODH) catalyzes the biosynthesis of ∆1-pyrroline-5-carboxylic acid (P5C).
The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and
expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading
frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The
deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli.
The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21-
(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to
homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C.
Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-
1-pyrroline. Our data showed that P5C, derived from the conversion of L-proline by the purified
recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway
represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by
PRODH.
KEYWORDS: Proline dehydrogenase; B. subtilis subsp. natto; 2-acetyl-1-pyrroline; δ1-pyrroline-5-
carboxylic acid
INTRODUCTION
vicinal diimine as a key intermediate (5). Recently, another
synthetic procedure for the preparation of 2-acetyl-1-pyrroline
from 2-pyrrolidinone was published. The described process
provided access to 2-AP in gram quantity and 22% overall yield
in four steps (6).
2-Acetyl-1-pyrroline (2-AP) had been proposed as a character
impact odorant causing the roasty note of bread crust and
aromatic rice (1) at an extremely low odor threshold of 0.02
ng/L. It has been identified in a great variety of processed and
cooked food products. 2-AP is reported to be formed by the
nonenzymatic browning reaction between reducing sugars and
free amino acids, such as proline and ornithine (2).
Various syntheses of 2-AP have been described in the
literature (3). The first synthesis (4) of 2-AP was achieved by
an oxidation of 2-(1-hydroxyethyl)pyrrolidine with a large
excess of silver carbonate on Celite in benzene. Methods
entailing the cyclization of 6-amino- or 6-azidohexane-2,3-dione
to form 2-AP have been published. A short synthetic strategy
was developed depending on an amino-protected functionalized
However, 2-AP has also been found in biological systems.
Several strains of Bacillius cereus strains were reported to
produce 2-AP at about 0.030-0.075 mg/kg after 2 day incuba-
tion on a standard plate-count agar (PCA) (7). Recently, some
wine lactic acid bacteria were characterized as producing 2-AP
at about 0.001-0.005 mg/L. The biosynthesis of 2-AP was
studied by the wine lactic acid bacterium, Lactobacillus hilgardii
DSM 20176 (8). Recently, 2-AP was identified by gas chro-
matography-mass spectrophotometry (GC-MS) from the cul-
tures of the fungal strains Acremonium nigricans, Aspergillus
awamori, and Aspergillus oryzae and a yeast strain, KluVero-
myces maxianus var. marxianus (Hansen) (9). In plants, 2-AP
has been detected in leaves from pandan (Pandanus amarylli-
folius) (10), taro corms (11), uncooked Khao Dawk Mali 105
brown rice (12), and fresh bread flowers (Vallaris glabra Ktze)
(13).
* To whom correspondence should be addressed. Fax: 886-87740213.
E-mail: mlwu@mail.npust.edu.tw.
† Institute of Biotechnology, National Pingtung University of Science
& Technology.
‡ Department of Food Science, National Pingtung University of Science
& Technology.
§ Rutgers University.
10.1021/jf0700576 CCC: $37.00
© 2007 American Chemical Society
Published on Web 05/31/2007

5098 J. Agric. Food Chem., Vol. 55, No. 13, 2007
Huang et al.
was determined following the method of Mezl and Knox (16). To 1.35
mL of reaction mixture and 1.5 mL of 10% trichloroacetic acid was
added 0.15 mL of 0.1 M o-aminobenzaldehyde in 40% (v/v) ethanol.
After 25 min at room temperature, the sample was cleared by
centrifugation, and the absorbance in a 1 cm cuvette at 440 nm was
On the basis of the evidence above, we hypothesized that
methylglyoxal may react with ∆1-pyrroline-5-carboxylic acid
(P5C) to form 2-AP. P5C can be synthesized from the oxidation
of L-proline by proline dehydrogenase (BnPRODH) in Bacillus
subtilis subsp. natto. In this study, BnPRODH gene was
expressed in Escherichia coli BL21 and was purified to
homogeneity. A model system composed of methylglyoxal and
P5C produced by BnPRODH was set up to study the formation
of 2-AP. The present results provide the direct evidence that
BnPRODH is responsible for the biosynthesis of 2-AP in B.
subtilis subsp. natto.
determined. The extinction coefficient of P5C was 2.58 mM-1 cm-1
.
The characterization of P5C was determined by using a Hitachi model
6000A HPLC equipped with a photodiode array detector (Model
L-4500, Hitachi, Japan. 220-550 nm) following the method of
O’Donnell et al. (17) with a slight modification.
Liquid Chromatography-Mass Spectrometry (LC-MS). All
analyses (using 5 µL samples) were carried out in duplicate on a Thermo
Finnigan LCQ DECA XP MAX single quadrupole instrument (Walth-
am, MA) with an ESI interface. A C18 column (150 × 2.0 mm,
Phenomenex Co., Torrance, CA) with a Finnigan Surveyor HPLC pump
was used throughout the analyses. Separation was achieved with an
isocratic elution using 33:67 methanol/water solution for 30 min. The
mass spectrometer was configured for positive ion chemical ionization
with coronal discharge at ∼4.5 kV. Nitrogen at 35 psi was used as the
sheath gas. Mass parameters comprised a full scan mode m/z 100-
220 in 1.5 s and selected ion monitoring m/z ((0.15 amu) with a dwell
time of 0.25 s/window.
Synthesis and Characterization of 2-AP. The solution containing
P5C (5 mmole) was mixed with methylglyoxal (5 mmole) in 100 mL
phosphate buffer (0.1 M pH 7.2). After stirring for 30 min at room
temperature (25 °C), the mixture was extracted eight times with
dichloromethane (total volume ) 250 mL). After drying with anhydrous
sodium sulfate, the extract was further concentrated to a volume of 2
mL for GC-MS analysis. L-Proline, R-ketoglutarate, pyridoxal-5′-
phosphate, and methylglyoxal were purchased from Sigma-Aldrich (St.
Louis, MO).
Gas Chromatography-Mass Spectrophotometry (GC-MS). An
aliquot (0.5 µL) of the extracted sample was injected into a gas
chromatograph-mass spectrometer (Agilent 6890 and HP 5973 mass-
selective detector, Agilent Technologies, Palo Alto, CA) equipped with
a fused silica capillary column, HP-5MS, with (5%-phenyl)-methylpol-
ysiloxane as nonpolar stationary phase (30 m × 0.25 mm i.d. × 0.25
µm). The sample was injected with spilt ratio of 20:1 and the flow rate
of helium was 1 mL/min. The injection port temperature was 200 °C.
The temperature of the HP-5MS column was programmed starting at
50 °C after injection. After 1 min, the temperature was increased at a
rate of 5 °C/min from 50 to 250 °C and was held for 15 min. The
mass spectrometer was operated in electron impact (EI) mode with an
electron energy of 70 eV; ion source temperature, 250 °C; mass range
m/z 40-250; scan rate, 0.68 s/scan; and EM voltage, 1832 V. The mass
transfer line was set to 280 °C. Linear retention indices (RTs) were
calculated using n-alkanes as reference.
MATERIALS AND METHODS
Bacterial Strains, Plasmids, and Cultivation Conditions. E. coli
JM109 and XL1-Blue (Stratagene, La Jolla, CA) were used as hosts
for cloning, and E. coli BL21(DE3):pLysS (Novagen, Madison, WI)
was used as the host for protein production. All E. coli strains were
grown in Luria-Bertani (LB) medium at 37 °C. When necessary, the
medium was supplemented with ampicillin (50 µg mL-1). B. subtilis
subsp. natto was cultivated at 30 °C in a medium containing 0.3% beef
extract and 0.5% peptone. All E. coli strains were grown in LB medium
at 37 °C. Plasmids pGEM-7Zf (+) (Promega, Madison, WI), pGEM-T
easy vector (Promega, Madison, WI), and pET21b (Novagen, Madison,
WI) were used as a cloning and expression vector.
Cloning of BnPRODH Gene. On the basis of the similarity of the
nucleotide sequence of proline dehydrogenase (PRODH) gene among
bacteria, the forward primer (5′-AGG GAG TCA TAT GTT GAG ACA
TGT GTT TTT-3′) and reverse primer (5′-CTG CTC GAG CTT TTT
CAA AAT TCC TTT TAA-3′) were designed. PCR was performed
with a model PE2400 automatic thermocycler (Perkin-Elmer Cetus,
Norwalk, CT). The PCR products were ligated to pGEM7Zf (+) vector,
generating pGEM-BnPRODH. The nucleotide sequence was confirmed
by sequencing with an automated laser fluorescence sequencer (Model
377, ABI PRISM, Foster, CA).
Expression and Purification of BnPRODH in E. coli. The coding
sequence of BnPRODH was amplified using the forward primers (5′-A
GGG AGT CATATG TTG AGA CAT GTG TTT TT-3′; the NdeI is
underlined) and reverse primers (5′-CTG CTCGAG CTT TTT CAA
AAT TCC TTT TAA-3′; the XhoI site is underlined). The PCR products
were digested with NdeI and XhoI and were fused in frame to the His6
tag of pET21b. E. coli BL21 (DE3) containing pET21-BnPRODH was
grown at 37 °C. When the optical density at 595 nm reached 0.6-0.8,
the culture was induced with 1 mM IPTG (isopropyl-â-D-thiogalacto-
pyranoside) for 4 h. The cells were harvested and resuspended in 20
mM Tris (pH 8.0) and were disrupted by sonication. After centrifuga-
tion, the crude extracts were applied to a 5 mL HiTrap metal chelating
column (GE Healthcare, Milwaukee, WI) charged with Ni2+. The
protein was eluted with a linear gradient of 0-600 mM imidazole
containing 0.5 M NaCl and 20 mM Tris-HCl (pH 7.9). The eluted
protein fractions were dialyzed against 10 mM ammonium bicarbonate
buffer (pH 7.9) to remove salts, and the purified proteins were
lyophilized and stored at -70 °C. The purified proteins were separated
by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis
(SDS-PAGE). The protein concentration was measured according to
the Bradford method (14).
Nucleotide Sequence Accession Number. The nucleotide sequence
of BnPRODH has been deposited at the NCBI database under accession
no. AF497244.
RESULTS AND DISCUSSION
Cloning and Sequence Analysis of the BnPRODH Gene.
The gene encoding BnPRODH has been cloned from B. subtilis
subsp. natto by PCR. The complete 909 bp sequence was
determined, and it was found that the open-reading frame (ORF)
encodes a polypeptide of 302 amino acids with an estimated
molecular mass of 34.5 kDa. A search through the available
protein sequence databases revealed that BnPRODH shared
significant amino acid sequence similarity with bacterial
PRODH, such as Geobacillus kaustophilus HTA426 (YP_148863,
63% identity), Exiguobacterium sibiricum (EAM86525, 55%
identity), and Thermus thermophilus HB27 (YP_005183, 48%
identity) (Figure 1). BnPRODH shows less similarity to
PRODH domain of PutA (proline utilization A) from E. coli
(18), but the degree of identity (approximately 30% identical
at the amino acid sequence) still suggests that these two proteins
were structurally related. PutA is a 1320 amino acid modular
Assay of BnPRODH Activity. BnPRODH activity was determined
with the spectrophotometric method described by Scarpulla and Soffer
(15). The reaction was carried out at 25 °C in 50 mM Tris-HCl (pH
7.5) containing 100 mM L-proline, 10 mM KCN, 10 µM FAD, 0.2
mM phenazine methosulfate, 0.2 mM p-iodonitrotetrazolium (Sigma-
Aldrich, St. Louis, MO), 0.75% Triton X-100, and enzyme. One unit
of PRODH activity was defined as the amount of activity yielding
reduction of 1.0 µmol p-iodonitrotetrazolium per min under the assay
condition using the extinction coefficient 11.5 mM-1 cm-1
.
Synthesis and Characterization of P5C. For synthesis of P5C,
purified BnPRODH (289 units) was incubated with 1-5 mmole
L-proline, 5 mmole R-ketoglutarate, and 15 mmole pyridoxal-5′-
phosphate in 100 mL potassium phosphate buffer (pH 7.2). The mixture
was kept under constant agitation at 25 °C for 1 h. P5C concentration

∆
1-Pyrroline-5-carboxylic Acid Formed by Proline Dehydrogenase
J. Agric. Food Chem., Vol. 55, No. 13, 2007 5099
Figure 1. Optimal alignment of Bacillus subtilis subsp. natto proline dehydrogenase (bn-pdh) with those of other proteins. The sequences listed are
proline dehydrogenase (PRODH) from Geobacillus kaustophilus HTA426 (gk-pdh), Exiguobacterium sibiricum (es-pdh), Thermus thermophilus HB27
(tt-pdh), and PutA from Escherichia coli (ec-puta). The amino acid numbers are listed on the right, and they are numbered from Met-1 of the proteins.
The black background regions indicate highly conserved amino acid residues. Dashes represent gaps introduced during the alignment process.
enzyme consisting of a DNA binding domain (residues 1-47),
PRODH domain (residues 261-612), and ∆1-pyrroline-5-
carboxylate dehydrogenase (P5CDH) domain (residues 650-
1130) (19). According to the crystal structure of the PRODH
domain of E. coli PutA (20), and the studies of site-directed
mutagenesis (19), Glu289, Lys329, Asp370, Arg431, Leu432,
Tyr437, Leu514, Tyr540, Arg555, Arg556, and Arg559 were
likely to be involved in catalytic mechanism for PRODH
activity. Sequence analysis revealed that all residues corre-
sponding to these catalytic residues are clearly present in
BnPRODH.
Purification and Characterization of the Recombinant
BnPRODH. The coding region of the BnPRODH gene was
cloned into pET21b vector under the control of the T7 promoter.
The protein product contained a C-terminal His6 tag, derived
from pET 21b. After expression in E. coli BL21 (DE3), the
protein product was purified from cell extract by nickel ion
affinity column chromatography. The yield of the recombinant
protein was 22 mg/L E. coli culture. The purified protein product
showed a single band with a molecular mass of 35 KDa on
SDS-PAGE (Figure 2). It is in good agreement with the value
estimated from the deduced amino acid sequence of BnPRODH.
The enzyme activity of recombinant BnPRODH was determined
with p-iodonitrotetrazolium as the electron acceptor under
saturating substrate concentration to show the formation of P5C.
This indicated that BnPRODH underwent a dehydrogenation
to remove two hydrogen atoms, leading to the transformation
of L-proline to P5C. The final specific activity of the purified
enzyme was 28.9 units/mg. The optimal pH for the purified
BnPRODH was measured using a series of biological buffers
Figure 2. SDS-PAGE analysis of purified BnPRODH. Lane M, molecular
mass standard; lane 1, crude cell extract from E.coli BL21/pET21-
BnPRODH; lane 2, BnPRODH purified from E.coli BL21/pET21-BnPRODH
by nickel ion affinity column chromatography. The proteins were analyzed
by 10% SDS-PAGE, and the gel was stained with coomassie brilliant
blue R-250.
with various pH’s. The purified recombinant BnPRODH
exhibited the maximum activity within a pH range of 7.5-8.0
and a temperature of 25 °C (Figure 3A and 3B). The enzyme
is temperature sensitive, losing 70% of enzymatic activity upon
incubation at 45 °C for 15 min.
Synthesis of ∆1-Pyrroline-5-carboxylic Acid. The formation
of P5C was confirmed by reaction with ï-aminobenzaldehyde

5100 J. Agric. Food Chem., Vol. 55, No. 13, 2007
Huang et al.
is shown in Figure 4 with absorption maxima at 230, 294, and
436 nm, which are all in good agreement with the data reported
by O’Donnel et al. (17). The MS spectrum is shown in Figure
5 with M+1 ion 218. The formation of P5C in the model system
is dose-dependent on proline concentration as shown in Figure
5. A 100 mL reaction mixture containing 5 mmol of L-proline
and purified BnPRODH produced 2.2 mmol of P5C, which
presented 44% conversion to P5C. It has been suggested that
L-proline might be converted to P5C in large quantities by
proline oxidase (21), but successful application of this approach
has not been reported. Enzymatic method for synthesizing gram
quantities of P5C using purified ornithine aminotransferase has
been reported (22). L-Ornithine is converted to P5C with the
simultaneous conversion of R-ketoglutarate to L-glutamic acid.
It has previously been synthesized chemically by the hydrolysis
of γ,γ-dicarbethoxy-γ-acetamidobutyraldehyde with hydrochlo-
ric acid (16). This produces a relatively pure compound which
is a mixture of the D- and L-stereoisomers. Another chemical
method employing oxidation of hydroxylysine to P5C has been
reported, but it most probably produces a similar mixture of
the D- and L-stereoisomers. The periodate oxidation of hydroxy-
lysine offers a simple and efficient route to form ∆1-pyrroline-
5-carboxylate (23).
Characterization of 2-Acetyl-1-pyrroline in the Reaction
Mixtures. In the model system of P5C and methylglyoxal,
volatile compounds were extracted with methylene chloride and
were analyzed by GC-MS. The mass spectrum for 2-AP
(Kovats’ RT index, HP-5ms, 918) from the reaction mixture
shows some major ions at molecular ion at m/z 111 (12) and
other major ions at 43 (100), 41 (61), 83 (51), 42 (23), 68 (14),
69 (8), 55 (3.6), 52 (2.2), 67 (2.9), and 54 (1.5). The breakdown
of the methylene moiety CH2dCH2 andacetyl moiety CH3-
CO leads to the formation of the m/z 83 fragment and the m/z
54 pyrroline ring. It was essentially identical to 2-AP reported
from pandan leaf extract (10).
Figure 3. Effect of pH (A) and temperature (B) on the activity of
recombinant BnPRODH. Recominant BnPRODH was incubated with
L-proline in 1 mL buffers at various pH’s and temperatures. Vertical bars
indicate standard deviation (n 3).
)
using chemically synthesized P5C (16) as a standard. The Schiff
base product between P5C and ï-aminobenzaldehyde was
characterized as a dihydroquinazolium compound with molec-
ular weight of 217. Absorption spectrum of the product by the
reaction between P5C and ï-aminobenzaldehyde was recorded
by an HPLC coupled with a photodiode array detector, and the
molecular weight was characterized by LC-MS. The spectrum
By using the internal standard 2,4,6-trimethylpyridine, con-
centration of 2-AP in the model system of P5C/methylgyoxal
was estimated to be 0.27 ( 0.06 mmole which accounted for
the 6.3% conversion rate of P5C to 2-AP.
+
Figure 4. UV
−
vis spectrum and LC mass spectra (M 218.02) of the product formed by the reaction between
∆
1-pyrroline-5-carboxylic acid and
o-aminobenzaldehyde.

∆
1-Pyrroline-5-carboxylic Acid Formed by Proline Dehydrogenase
J. Agric. Food Chem., Vol. 55, No. 13, 2007 5101
cellular compounds and thereby develop toxicity. It was found
that even moderate concentrations of L-proline are toxic for
Arabidopsis in axenic culture. They hypothesized that toxicity
is not due to the existence of L-proline, but rather P5C is the
effector of proline-induced cell death. P5C might also act as a
single molecule activating processes related to apoptosis (28).
Results of stable isotope tracer experiments using 15N-proline
indicated that the nitrogen source of 2-AP was L-proline in B.
cereus (7) and rice callus (25). We cloned the gene encoding
B. subtilis subsp. natto BnPRODH, which can convert L-proline
into P5C. 2-AP was found in the mixture of P5C and
methylglyoxal kept at room temperature (25 °C) for 30 min.
The production of 2-AP by Bacillus PRODH at ambient
temperature supported the possibility of a nonthermal formation
pathway for 2-AP by other microorganisms, reported previously,
such as B. cereus (7), Saccharomyces cereVisiae (1), and several
lactic acid bacteria, such as Lactobacillus hilgardii and Lacto-
bacillus breVis (8).
Figure 5. Synthesis of
-proline concentration.
∆
1-pyrroline-5-carboxylic acid as a function of
L
Methylglyoxal (MG) has been used as one of the major
intermediates for 2-AP synthesis in model system. Ahmed et
al. (29) pointed out that acetone, threonine, and glucose may
generate methylglyoxal. Among them, formation of methylg-
lyoxal from glucose through triosephosphate pathway deserves
special interest. Methylglyoxal is predominantly generated by
the nonenzymatic degradation of triose phosphate intermediate
through fragmentation and removal of phosphate from 1,3-
dihydroxyacetone phosphate. Phillips and Thornalley (30)
ascribed the formation of methylglyoxal to the excess glucose
metabolized through Embden-Meyerhof pathway.
In B. cereus, increase of glucose in PCA medium from 0.2
to 1% leads to a 3-fold increase of 2-AP (145 to 458 µg/kg).
We postulated that a high level of glucose resulted in the
accumulation of MG following a similar pathway described by
Phillips and Thornalley (30). By adding 1% of 13C6-glucose,
Romanczyk et al. (7) demonstrated that two carbons from
glucose were incorporated within 2-AP. The R-dicarbonyls, such
as methylglyoxal, are extremely reactive as glycating agents
for collagen, enzymes, and other important cellular components,
and they are toxic to cultured cells. They are elevated in the
plasma of diabetic individuals and are found in increased levels
in patients showing evidence of early diabetic nephropathy and
retinopathy (31). We hypothesized that the added glucose was
metabolized by B. cereus via glycolysis pathway and reacted
with the P5C synthesized from L-proline to form 2-AP.
Figure 6. Proposed mechanism for 2-acetyl-1-pyrroline production by
proline dehydrogenase (BnPRODH) from the Bacillus natto gene expressed
in Escherichia coli.
Proposed Biosynthesis Pathway for 2-Acetyl-1-pyrroline.
Although a significant amount of 2-AP (3.3% and conversion
from 1-pyrroline) in 1-pyrroline/methylglyoxal model system
under boiling condition has been detected (1), we demonstrated
here for the first time that P5C which is the metabolite from
L-proline is the key precursor for 2-AP synthesis. Romanczyk
et al. (7) reported that supplement with L-proline in plate count
agar (PCA) medium inoculated with B. cereus induced a dose-
dependent production of 2-AP. Suprasanna et al. (24) found
that L-proline is an effective precursor for 2-AP synthesis in
cell and callus cultures of rice. Among the three amino acids,
L-glutamic acid, L-ornithine, and L-proline, L-proline can best
produce 2-AP in both rice callus and seeding in a dose-
dependent manner (25). Recently, L-proline was found to
enhance the in vitro synthesis of 2-AP in semidifferentiated
callus culture of Pandanus amaryllifolius (26). Iyer and Caplan
(27) reported the induction of stress-related genes in rice by
P5C but did not clarify whether this induction was due to P5C
being toxic. Cyclic P5C is in equilibrium with glutamate-γ-
semialdehyde and is unstable in aqueous solution (16). Since
aldehydes are highly reactive, P5C might react with various
Costello and Henschke (8) proposed that acetylation at the
C-2 position of the 1-pyrroline intermediate by the accumulated
acetyl CoA may lead to the formation of the 2-AP. Hypothetical
pathway leading from 1-pyrroline and methylglyoxal was
proposed by Hofmann and Schieberle (32). Our data showed
that P5C, derived from L-proline by the purified recombinant
BnPRODH, might react directly with methylglyoxal and lead
to the formation of 2-AP. On the other hand, P5C may also
degrade to 1-pyrroline and condense with methylglyoxal to form
2-AP (Figure 6). P5C/methylglyoxal pathway represents the
first report of a biological mechanism by which 2-AP may be
synthesized in vitro by BnPRODH.
ACKNOWLEDGMENT
We thank Dr. Gan-Nan Cheng, Department of Biotechnology,
Tajen University, and Chung-Ho Yeh, Department of Food
Science, National Pingtung University of Science and Technol-
ogy, for their assistance with BnPRODH gene cloning.

5102 J. Agric. Food Chem., Vol. 55, No. 13, 2007
Huang et al.
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Received for review January 8, 2007. Revised manuscript received April
18, 2007. Accepted April 20, 2007. This work was funded by a grant
from the National Science Council, Taiwan, Republic of China (NSC
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