Identification and characterization of a new gene from
Variovorax
paradoxus
Iso1 encoding
N
-acyl-
D
-amino acid amidohydrolase
responsible for
D
-amino acid production
Pei-Hsun Lin
1
, Shiun-Cheng Su
1
, Ying-Chieh Tsai
2
and Chia-Yin Lee
1
1
Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan;
2
Graduate Institute of Biochemistry,
Yang-Ming University, Taipei, Taiwan
An N-acyl-
D
-amino acid amidohydrolase (N-D-AAase) was
identified in cell extracts of a strain, Iso1, isolated from an
environment containing N-acetyl-
D
-methionine. The bac-

D
-leucine and
D
-alanine and against N-chloroacetyl-
D
-phe-
nylalanine. Importantly, the enzyme does not act on the
N-acetyl-
L
-amino acid derivatives. The enzyme was inhibited
by chelating agents and certain metal ions, but was activated
by1m
M
of Co
2+
and Mg
2+
. Thus, the N-D-AAase from
V. paradoxus can be considered a chiral specific and metal-
dependent enzyme.
Keywords: N-acyl-
D
-amino acid amidohydrolase;
D
-amino
acid; LC/MS/MS; Variovorax paradoxus.
D
-Amino acids are important materials for chiral chemical
synthesis of such things as semi-synthetic antibiotics [1–3],
bioactive peptide [4–6], pyrethrods, pesticides and some

enzyme is inhibited by metallic ions such as Zn
2+
,Hg
2+
,
Cu
2+
and by EDTA. Notably, the enzymes purified from
Streptomyces olivaceus and Amycolatopsis orientalis
IFO12806 are activated by Co
2+
(1 m
M
). Some purified
enzymes have been found to contain between 2.06 g and
2.61 g Zn per mole and it is considered that zinc ions may
play a role in the catalytic activity and stability of the
enzyme structure [23–25].
Up to this point, it is not clear what the function of
N-D-AAase is in bacteria, and gene sequence information is
available only from Alicaligenes species [26–28].
D
-amino
acids are very important for the synthesis of intermediate
chiral compounds as mentioned earlier and some reports
have described enzymatic methods for the synthesis of
D
-amino acids [29–32], including the coupling in a process of
N-D-AAase and N-acylamino acid racemase. However,
some N-D-AAases isolated from bacteria have some

D
-methionine. It was
grown at 30 °C in TSB (Difco) medium and used as a
source of its chromosomal DNA. E. coli XL1-Blue [34] and
E. coli Top10 grown at 35 °C in Luria–Bertani broth
(Difco) were used as the host for gene cloning and
expression. Luria–Bertani medium supplemented with
100 mgÆmL
)1
ampicillin (Sigma) was used for plasmid
maintenance. Two plasmids pBluescript II KS(+) (Stra-
tagene) and pTrcHis2A (Invitrogen) were used as gene
cloning and expression vectors, respectively. For protein
expression, E. coli Top10 containing recombinant plasmids
was grown in 2YT medium supplemented with
200 mgÆmL
)1
ampicillin. Under the trc promoter and lac
q
repressor of pTrcHis2A, isopropyl thio-b-
D
-galactoside was
added to a final concentration of 1 m
M
.
Materials, enzymes and chemicals
Restriction enzymes and T4 DNA ligase were from New
BioLabs and Gibco BRL. Pfu DNA polymerase and
alkaline phosphatase were from Promega and Boehringer
Mannheim, respectively.

TREE VIEW
32wasusedtoviewthe
phylogenic trees [38]. The reliability of the each tree node
was confirmed by bootstrapping (1000 trees) and a consen-
sus tree was constructed using
SEQBOOT
and
CONSENSE
from
the
PHYLIP
package. The GenBank accession number for
the strain Iso1 is AY127900.
Cloning of
V. paradoxus N
-D-AAase gene
Recombinant DNA technology was carried out by the
standard methods of Sambrook et al.[39].Totalgenomic
DNA was prepared from V. paradoxus Iso1 by a modified
method, and partially digested with Sau3AI. The 3 kb to
9 kb DNA fragments were purified from 1.2% (w/v) low-
melting-point agarose gels (FMC SeaPlaque agarose), and
eluted by heating to 67 °C followed by phenol extraction
twice and ethanol precipitation. The DNA was ligated into
BamHI-digested and dephosphorated pBluescript II KS(+)
using T4 DNA ligase. Competent cells E. coli XL1-Blue
were transformed by electroporation according to the
protocol manual of the Gene Pulser II (Bio-Rad). White
colonies were selected into an ELISA microplate containing
50 lL Luria–Bertani medium supplemented with

replacing the substrate with buffer. The one positive E. coli
transformant contained a 12.3-kb plasmid, designated pBK-
damD4.
Southern analysis
Chromosomal DNA completely digested by EcoRI or
HindIII was separated on a 0.8% agarose gel. DNA
fragments were transferred onto Zeta-Probe membrane [39].
A SacI–PstI DNA fragment of pBK-damH1 was labelled
using a random-primer labelling kit (Roche) with
[a-
32
P]dCTP. After hybridization at 65 °Candwashing,
the membrane was exposed to X-ray film at )70 °C.
Nucleotide and amino acid sequence analysis
For sequencing, the N-D-AAase gene, pBK-damD4 was
digested with various restriction enzymes and subcloned
into pBluscript II KS(+) to obtain the clone pBK-damH1
that carried the smallest insert fragment that retained high
enzyme activity. The pBK-damD4 and pBK-damH1 were
used as sequencing templates to double confirm both
strands of the gene. The nucleotide sequencing was carried
out using an ABI Prism 3770 DNA sequencer (Perkin
Elmer). The nucleotide sequence was analysed by using the
DNASIS
(Hitachi, Japan) and
GNEYTEX
(Hitachi, Japan)
programs. The amino acid sequence was compared with
known protein sequences in the nucleotide/protein sequence
databases by the

plasmid was named pTrc2A-damA3 without His-tag.
Expression and purification of the
N
-D-AAase
from the
E. coli
transformant
The E. coli Top10 harbouring the pTrc2A-damA3 was
subcultured at 35 °C for 8–12 h in a test tube containing
3 mL 2YT medium supplemented with 200 lgÆmL
)1
ampi-
cillin. The subculture was diluted 1 : 50 into a 500-mL flask
containing 150 mL of the same medium and incubated at
35 °C, 150 r.p.m. At OD
600
¼ 0.6, isopropyl thio-b-
D
-galactoside was added to a final concentration of 1 m
M
and the culture was quickly shifted to a temperature of
20 °C and induced for 30 h with shaking. The cells from a
total of 3 L culture were harvested by centrifugation
(8000 r.p.m., 10–20 min) and washed twice with 50 m
M
Tris/HCl pH 7.5. All purification procedures were per-
formed at 4 °C except the FPLC-Mono Q chromatography,
which was carried out at room temperature. The pellets
were resuspended in lysis buffer (50 m
M

buffer A containing 15% saturated ammonium sulfate. The
active fractions were combined, concentrated by Centriprep
YM-10 (Amicon) and applied to a column of Sephacryl HR
S-200 equilibrated with buffer C (50 m
M
Tris/HCl, 0.15
M
NaCl, 0.01% 2-mercaptoethanol, pH 7.5). The eluted
fractions were made up to a final concentration of 10%
glycerol and the active fractions were combined for dialysis
against buffer C containing 10% glycerol then concentrated
using a Centriprep YM-10. Finally, the sample was added
to a FPLC Mono Q (Pharmacia) at a flow rate of
0.5 mLÆmin
)1
. All fractions were assayed for enzyme
activity and the active fractions were further analysed by
Western blotting.
Enzyme activity assay
The standard reaction mixture (0.5 mL) for the determin-
ation of N-D-AAase activity contained 50 m
M
Tris/HCl
pH 7.5 and 25 m
M
N-acetyl-
D
-methionine to which an
appropriate amount of the enzyme was added. The
reactions were incubated at 40 °C for 10–30 min and

amide) as described by Laemmli [41]. For Western blotting,
the proteins were transferred to poly(vinylidene fluoride)
membrane using 10 m
M
Caps containing 10% methanol by
a semidry transfer device (Pharmacia) for 1–2 h at 50 mA
and 5 V. After transfer, the membrane was immersed in 6
M
urea-PBST (phosphate buffer/saline/Tween-20) solutions
with overnight shaking. The membrane was washed three
timeswithPBSTfor10minthenblockedwithGelatin-
NET (NaCl/EDTA/Tween-20) for 1–2 h. The primary
antibody (1 : 20000 anti N-D-AAase from Alicaligenes
faecalis DA1) was incubated with the membrane at room
temperature for 1 h, and then washed three times. The
diluted second antibody (1 : 5000 anti-rabbit horseradish
peroxidase) was then added and the membrane was
incubated for 1 h followed by three washes. Following the
protocol supplied with the peroxidase substrate kit (Vector
Lab, Inc.), signal bands appeared after the membrane was
incubated at room temperature for 5–20 min.
Peptide sequencing by LC/MS/MS analysis
and isoelectric focusing
After separation by SDS/PAGE, the proteins were detected
by staining the gel with Coomassie blue R250 and then
destained. Proteins to be identified were excised from the gel
and processed for mass spectrometric analysis by the ion
trap mass spectrometry processes including in-gel reduction,
S-carboxyamidomethylation, and trypsin digestion. The
reaction mixture was then introduced directly into the

activity was measured by the colorimetric assay.
Influences of chelating reagents and metal ion
on enzyme activity
Chelating reagents and metal ions were added to the enzyme
reaction which was then preincubated at 35 °Cfor30min
followed by the addition of 25 m
M
N-acetyl-
D
-methionine
and the residual enzyme activity was measured by the
Chirobiotic T HPLC method using
D
-methionine as stand-
ard [44]. The test concentration of chelating reagents and
metal ions used for assay were 1 m
M
and 10 m
M
, respectively.
Substrate specificity analysis
Various substrates (25 m
M
) were added to the enzyme in the
standard reaction described previously and incubated at
40 °C for 20 min. The amount of
D
-amino acids produced
was determined by the Chirobiotic T HPLC method and the
appropriate

-D-AAase from
V. paradoxus
Iso1
A V. paradoxus Iso1 total genomic library was constructed
in E. coli XL1-Blue. One positive clone (pBK-damD4) was
found among 1840 clones tested and it developed a faint red
Fig. 1. Phylogenetic relationships of the 16S rDNA sequence of the
strain Iso1 with other bacteria. The GenBank accession numbers for the
organisms used in this analysis were as follows: V. paradoxus
MBIC3839, AB008000; V. paradoxus IAM12373, D88006; V. para-
doxus E4C, AF209469; V. paradoxus VAI-C, AF250030; Aquaspiril-
lum delicatum, AF078756; Xylophilus ampelinus, AF078758;
Acidovorax facilis, AF078765; Rhodoferax fermentans,RHYFR2D;
Hydrogenophaga taeniospiralis, AF078768; Aquaspirillum sinuosum,
AF078754; Comamonas acidovorans, AF149849; Ralstonia campinen-
sis, AF312020; Leptothrix mobilis, X97071; Brachymonas denitrificans,
D14320; Pandoraea pnomenusa, AF139174; Burkholderia brasilensis,
AJ238360; E. coli, A14565. The phylogenetic tree was based on the
alignment of the 16S rDNA sequences. The 16S rDNA sequence of
E. coli was used as an outgroup.
Ó FEBS 2002 V. paradoxus N-D-AAase gene (Eur. J. Biochem. 269) 4871
colour in the ELISA microplate N-D-AAase enzyme
activity assay system after blue-white selection. The pBK-
damD4 plasmid contained an insert of  9kbandthiswas
used for Southern hybridization and subcloning to generate
deletion plasmids for nucleotide sequencing. The Southern
hybridization analysis indicated that the insert fragment was
derived from V. paradoxus chromosomal DNA (data not
shown). At the same time, degenerate primers for the N-D-
AAase gene were developed using alignment analysis of the

and Swiss-Port databases
using the
CLUSTAL W
program showed the primary structure
of N-D-AAase to be similar to N-acyl-
D
-amino acid
amidohydrolase (56.7% identity and 63.6% similarity),
N-acyl-
D
-glutamate amidohydrolase (44.8% identity and
51.2% similarity) and N-acyl-
D
-asparate amidohydrolase
(48.5% identity and 56.5% similarity) from Alicaligenes
xylosoxydans ssp. xylosoxydans A-6 and the
D
-aminoacylase
from Alicaligenes faecalis DA1 (54.6% identity and 62.5%
similarity). These results are summarized in Table 1. The
N-D-AAase protein was also similar to the genes from the
complete genome sequences of Pyrococcus abyssi (42.8%
identity and 53.3% similarity), Streptomyces coelicolor
(35.8% identity and 42.3% similarity) and Mycobacterium
tuberculosis (33.5% identity and 41.9% similarity) [46,47].
Fig. 3 shows the N-D-AAase protein of V. paradoxus
compared to the other protein sequences in the database
and using a motif search program [48] at least seven specific
motifs were identified. Among these motifs, all except
M. tuberculosis had motif 1, 2 and 6 while Streptomyces

higher than the E. coli
XL1-Blue containing pBK-damH1 (6.0 mU mg
)1
). From
3 L bacterial culture, 0.18 mg protein was obtained. The
specific activity and the recovery of the N-D-AAase were
516.7 UÆmg
)1
and 8%, respectively (Table 2). The purified
protein appeared as a single band with a few minor
contaminants on SDS/PAGE with a molecular mass of
54.2 kDa (Fig. 4A). The value was consistent with the
predicted molecular mass. Western blotting analysis
gave similar results (Fig. 4B). The native molecular mass
of N-D-AAase protein was determined by Sephacryl HR
S-200 gel filtration to be 52.8 kDa and this indicated
that the enzyme was monomeric. IEF of the purified
N-D-AAase gave a band at a pI 5.12, which was closed
to predicted pI of 5.8. To confirm the protein sequence,
high resolution LC/MS/MS (Finnigan LCQ) analysis
was used. The results gave a similarity of 100% when
compared to the predicted amino acid sequence of the
N-D-AAase protein.
Influence of temperature and pH on enzyme activity
The optimal temperature for N-D-AAase was 50 °C
(Fig. 5A). The enzyme still had 80% activity after
Table 1. Comparison of the amino acid sequence similarity of putative N-D-AAases from V. paradoxus and other species. Thegeneaccessionnumber
and the strains are the same as in Fig. 4.
Accession number or strain
Amino acid

Iso1 N-acyl-
D
-amino acid amidohydrolase;
P. abyssi: Pyrococcus abyssi N-acyl-
D
-amino
acid amidohydrolase; S. coelicolor: Strepto-
myces coelicolor N-acyl-
D
-amino acid amido-
hydrolase; M. tuberculosis: Mycobacterium
tuberculosis hypothetical protein Rv2913c.
Sequence alignment by
CLUSTAL W
[37]. The
identical, conserved and semi-conserved amino
acid residues are marked by asterisks, dots and
colons, respectively. The numbers represent
amino acid positions. Gaps were introduced to
optimize the alignment. The amino acid resi-
dues in the box were the motifs identified using
the
MOTIFSEARCH
program [48].
Ó FEBS 2002 V. paradoxus N-D-AAase gene (Eur. J. Biochem. 269) 4873
30 min preincubation. When the temperature was 55 °C,
the treatment resulted in a 60% loss of activity (Fig. 5B).
Above 55 °C, activity decreased rapidly reflecting the
instability of the enzyme at higher temperatures. The
optimal pH for enzyme activity was pH 7.5 (Fig. 6A).

purification; lane 5, protein after Sephacryl HR S-200 purification; lane
6, protein after FPLC-MonoQ purification step. (B) Western blotting.
Lanes 1–6 are as described in (A).
Fig. 5. Optimal temperature and thermostability of N-D-AAase. (A)
The optimal temperature of purified enzyme. Enzyme activity mea-
surements were performed at various temperatures for 20 min. The
highest activity was taken as 100%. (B) Thermostability of purified
enzyme. The purified enzyme was preincubation for 30 min at various
temperatures. Then the substrate (N-acetyl-
D
-methionine, 25 m
M
,
pH 7.5) was added to the reaction and the activity was measured at
40 °C for 20 min. The highest activity was taken as 100%. The results
were the means of duplicate determinations.
4874 P H. Lin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Influences of chelating reagents and metal ions
on enzyme activity
It has been reported that the enzyme activity of N-D-AAase
is affected by the presence of metal ions. Thus, the enzyme
was treated with EDTA, EGTA, 1,10-phenanthroline and
metal ions at concentrations of 1 and 10 m
M
. The presence
of the metal ions, Fe
2+
,Cu
2+
,Zn

To study the substrate specificity of the N-D-AAase protein,
the activity of the enzyme against N-acyl-
D
-or
L
-amino acids
and other
D
-amino acid derivates was determined (Table 3).
The substrates analysed were a range of hydrophilic,
hydrophobic and aromatic N-acyl or derivative
D
-amino
acids that could easily be purchased from commercial source
such as Sigma and Bachem. The enzyme activity was 50%
higher towards N-acetyl-
D
-methionine, N-acetyl-
D
-alanine,
N-acetyl-
D
-leucine and N-chloroacetyl-
D
-phenylalanine
than towards N-acetyl-
D
-valine, N-acetyl-
D
-phenylalanine,

compound and this suggests that the chloride atom of the
N-chloroacetyl-
D
-phenylalanine substrate may promote
substrate binding to the enzyme.
DISCUSSION
This study has identified a strain, Ios1, of V. paradoxus,
formally Alicaligenes paradoxus, belonging to the subclass
b-Proteobacteria and the family Comamonadaceae.At
present, the Variovoras group consists of only V. paradoxus
[49], divided into biovar I and biovar II strains. The
difference between Alicaligenes and Variovorax is that
the Variovorax group releases a yellow pigment into the
medium, whereas Alicaligenes does not. Strain Iso1 also
shows nitrate reduction activity and as such is considered to
be a biovar II strain [45]. In addition, strain Iso1 was shown
to be resistant to ampicillin (100 lgÆmL
)1
) and to contain a
polyhydroxyalkanoates synthase gene (phaC)byPCR
amplification [50].
The N-D-AAase gene expressing N-acyl-
D
-amino acid
amidohydrolase activity was cloned from V. paradoxus and
Fig. 6. Optimal pH and pH stability of N-D-AAase protein. (A) Opti-
mal pH. The enzyme reactions were determined at 35 °Cinthefol-
lowing buffers (50 m
M
): acetate buffer (d, pH 4.0–5.6); phosphate

D
-leucine 84 ± 2.2
N-acetyl-
D
-phenylalanine 24 ± 2.2
N-acetyl-
D
-tryptophan 5 ± 0.2
N-acetyl-
D
-tyrosine 4 ± 0.3
N-chloracetyl-
D
-phenylalanine 201 ± 2.2
N-acetyl-
D
-asparagine 19 ± 2.2
N-acetyl-
L
-methionine 0
N-acetyl-
L
-leucine 0
Ó FEBS 2002 V. paradoxus N-D-AAase gene (Eur. J. Biochem. 269) 4875
its nucleotide sequence determined. Upstream of the ORF,
three possible promoter regions were identified (Fig. 2).
These were the )35 regions TTGGCA )192 to )187 bp,
TGGTCA )152 to )147 bp, CTGAGC )99 to )104 bp
and the )10 regions TATGGT )165 to )160 bp, GACACT
)131 to )126 bp and TACATC )73 to )68 bp. The

Additionally, the first histidine residue of motif 6 is con-
sidered to be involved in metallic ion binding and enzyme
catalytic function [23]. The function of the N-D-AAase
protein in the bacterium is not very clear, but recently
one study of V. paradoxus has suggested that the amino-
acylase may be used to hydrolyse N-butanoyl-homoserine
lactones (C4-HSL) to produce HSL and fatty acids, which
are then used as the sole energy and nitrogen sources [51].
The acyl-HSL signalling molecules may be biologically
inactivated by specific soil bacteria. Here, the N-D-AAase
from V. paradoxus may possibly play a role in the
degradation of acyl-HSL molecules and this needs to be
tested in the future.
According to the results of the peptide sequencing
determined by LC/MS/MS analysis, some methionine
residues seemed to be modified because there was a
molecular weight increase of 16. These methionine residues
were Met39, Met171, Met254, Met273 and Met352. It is
known that the common sites of oxidation in proteins are
histidine, lysine, proline, cysteine, arginine and methionine
residues [52]. Methionine oxidation can be caused by
protein damage or aging by endogenous or oxidizing agents
[53,54] and maybe the reason why the N-D-AAase enzyme
of V. paradoxus purified from E. coli is unstable when the
enzyme is stored at 4 °C. Under these conditions, enzyme
activity decreased very rapidly over a few days. Although
some reports have shown that methionine oxidation has no
influence on protein function [55,56], others have shown
that inhibition of biological function or loss of enzyme
activity can occur [57–59]. In future studies, it might be

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