Identification and characterization of a new gene from VariovoraxPei-Hsun Lin1, Shiun-Cheng Su1, Ying-Chieh Tsai2and Chia-Yin Lee1 1 Graduate Institute of Agricultural Chemistry, Nationa
Trang 1Identification and characterization of a new gene from Variovorax
Pei-Hsun Lin1, Shiun-Cheng Su1, Ying-Chieh Tsai2and Chia-Yin Lee1
1
Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan;2Graduate 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-terium was classified as Variovorax paradoxus by
phylo-genetic analysis The gene was cloned and sequenced The
gene consisted of a 1467-bp ORF encoding a polypeptide of
488 amino acids The V paradoxus N-D-AAase showed
significant amino acid similarity to the N-acyl-D-amino acid
amidohydrolases of the two eubacteria Alcaligenes
xylo-soxydans A-6 (44–56% identity), Alcaligenes facelis DA1
(54% identity) and the hyperthermophilic archaeon
Pyro-coccus abyssi(42% identity) After over-expression of the
N-D-AAase protein in Escherichia coli, the enzyme was
purified by multistep chromatography The native molecular
mass was 52.8 kDa, which agreed with the predicted
molecular mass of 52 798 Da and the enzyme appeared to be
a monomer protein by gel-filtration chromatography A
homogenous protein with a specific activity of 516 UÆmg)1
was finally obtained After peptide sequencing by LC/MS/
MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and
50C, respectively After 30 min heat treatment at 45 C, between pH 6 and pH 8, 80% activity remained The N-D-AAase had higher hydrolysing activity against N-ace-tyl-D-amino acid derivates containing D-methionine,
D-leucine andD-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
b y 1 mMof Co2+and Mg2+ Thus, the N-D-AAase from
V paradoxuscan 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
food additives such as altimate [7,8] They can also be used
to synthesizeD-configuration specificD-amino acid
deriva-tives [9,10].D-amino acids also are important constituents of
eubacterial cell walls [11] They are found in
microorga-nisms, plants and animals and their function and
physio-logical roles have been investigated and identified [12,13]
N-acyl-D-amidohydrolase (EC.3.5.1.81, N-D-AAase) is an
enzyme capable of catalysing the hydrolysis of
N-acyl-D-amino acids to yield the correspondingD-amino acid and
the organic acid They have been found in a number of
bac-terial species, including members of the Alcaligenes,
Strep-tomyces, Pseudomonas, Stenotrophomonas, Amycolatopsis and Sebekia [14–22] So far, all N-D-AAases characterized consist of monomeric proteins of 45–55 kDa except for the Pseudomonas sp 1158 enzyme which has a molecular mass of 100 kDa and the Amycolatopsis enzyme which has
a molecular mass of 36 kDa They have similar optimal temperatures (45–50C) and pHs [7,8] but show a variety of different specific activities towards different substrates The enzyme is inhibited by metallic ions such as Zn2+, Hg2+,
Cu2+and by EDTA Notably, the enzymes purified from Streptomyces olivaceus and Amycolatopsis orientalis IFO12806 are activated by Co2+ (1 mM) 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
L-aminoacylase activity [18,33] Therefore, it is necessary
to avoid -aminoacylase interference if the enzyme is to be
Correspondence to C.-Y Lee, Graduate Institute of Agricultural
Chemistry, National Taiwan University, 1, Sec 4, Roosevelt Road.,
Taipei 106, Taiwan.
Fax: +886 2 2366 0581, Tel.: +886 2 2363 0231, extn 2816.
E-mail: m477@ccms.ntu.edu.tw
Abbreviations: N-D-AAase, N-acyl- D -amidohydrolase; HSL,
homo-serine lactone; C4-HSL, N-butanoyl-homohomo-serine lactone.
Enzyme: N-acyl- D -amidohydrolase (N-D-AAase, EC.3.5.1.81).
(Received 30 June 2002, revised 14 August 2002,
accepted 20 August 2002)
Trang 2used in industrial applications In this study, a strain, Iso1,
with N-D-AAase enzyme activity, was isolated from the
environment and the gene for the enzyme was cloned and
then sequenced The recombinant protein, N-D-AAase, was
also produced in the Escherichia coli, purified and
charac-terized
M A T E R I A L S A N D M E T H O D S
Bacterial strains, plasmids and conditions
Variovorax paradoxusIso1 was isolated from an
environ-mental situation containing N-acetyl-D-methionine It was
grown at 30C in TSB (Difco) medium and used as a
source of its chromosomal DNA E coli XL1-Blue [34] and
E coli Top10 grown at 35C 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)1ampicillin Under the trc promoter and lacq
repressor of pTrcHis2A, isopropyl thio-b-D-galactoside was
added to a final concentration of 1 mM
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.D-Amino acid oxidase (EC 1.4.3.3)
from porcine kidney and horseradish peroxidase were
purchased from Sigma Chemical Co DEAE-Toyopearl
650 M and Butyl-Toyopearl 650 M were from Tosoh
(Tokyo, Japan) FPLC-Mono Q was from Pharmacia
Substrates and standards were from commercial sources
such as Sigma or Bachem All other reagents were the
highest grade available
16S rDNA gene sequence analysis
The nucleotide sequence of the 16S rDNA from strain Iso1
was amplified by PCR using proof reading Pfu DNA
polymerase The universal primers 5F (5¢-TGAAGAGTTT
GATCATGGCT-3¢) and 1540R (5¢-AAGGAGGTGAT
CCAACCGCA-3¢) numbered according to the E coli 16S
rRNA sequence were used The PCR product was purified
and ligated into the p-GEM-T Easy Vector system
(Promega) [35] DNA sequencing was carried out using
an ABI Prism 3770 DNA sequencer (Perkin Elmer)
Comparison with other 16S rDNA sequences was
per-formed by theBLASTprogram [36] against GenBank The
sequence alignment analysis was carried out usingCLUSTAL
W[37] ThePHYLIPsoftware package was used for
phylo-genetic analysis and TREE VIEW32 was used to view the
phylogenic trees [38] The reliability of the each tree node
was confirmed by bootstrapping (1000 trees) and a
consen-sus tree was constructed usingSEQBOOTandCONSENSEfrom
thePHYLIP 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] Total genomic DNA was prepared from V paradoxus Iso1 by a modified method, and partially digested with Sau3AI The 3 kbto
9 kbDNA fragments were purified from 1.2% (w/v) low-melting-point agarose gels (FMC SeaPlaque agarose), and eluted by heating to 67C 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
100 lgÆmL)1ampicillin in each well using a sterile toothpick and incubated at 37C overnight The transformants were screened for enzyme activity by adding to each well
10 lL 2 mgÆmL)1 lysozyme and incubating at 37C for
30 min This was followed by the addition 110 lL 25 mM
N-acetyl-D-methionine and incubation at 40C overnight Then, 40 lL of the colour reagent (50 mM Tris/HCl
pH 7.5, 3 UÆmL)1 D-amino acid oxidase, 10 UÆmL)1 horseradish peroxidase, 4 lL phenol, 0.2 mgÆmL)1 4-amino-antipyrine) was added and the plate was incubated at room temperature for 10–20 min Wells positive for the enzyme should develop a red colour and V paradoxus Iso1 was used as the positive control, whereas E coli containing pBluescript II KS(+) plasmid was used as the negative control Any positive clones were then confirmed by replacing the substrate with buffer The one positive E coli transformant contained a 12.3-kbplasmid, 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-32P]dCTP After hybridization at 65C and washing, 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 BLAST program from the Swiss-Prot database Sequence alignment was carried out using the programCLUSTAL W[37] The accession number of the gene reported in this paper is AY126714
Trang 3Construction of a plasmid to produce the recombinant
proteinN-D-AAase in the E coli
A DNA fragment coding for the N-D-AAase was obtained
by PCR using the Pfu DNA polymerase (Promega) PCR
amplification was carried out as follows: 94C for 5 min
followed by 30 cycles of 30 s at 94C, 30 s at 62 C and
2 min 30 s at 72C and then a further 5 min extension at
72C The PCR mix before amplification contained a final
concentration of 5% acetylamine The PCR product was
purified, digested with EcoRI and HindIII, ligated into
pTrcHis2A and finally transformed into E coli Top10 The
plasmid pTrcHis2A carrying the whole of the N-D-AAase
coding sequence was digested with EcoRI and NcoI, treated
with mung bean nuclease and self-ligated This step was
to optimize the distance between the vector-borne
Shine-Dalgarno sequence and the N-D-AAase start codon The
sequence upstream of the N-D-AAase gene is:
5¢…AGGACAGACGAATG…3¢ (The Shine-Dalgarno
sequence and start codon are in bold) The recombinant
plasmid was named pTrc2A-damA3 without His-tag
Expression and purification of theN-D-AAase
from theE coli transformant
The E coli Top10 harbouring the pTrc2A-damA3 was
subcultured at 35C 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
35C, 150 r.p.m At OD600¼ 0.6, isopropyl
thio-b-D-galactoside was added to a final concentration of 1 mM
and the culture was quickly shifted to a temperature of
20C 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 mM
Tris/HCl pH 7.5 All purification procedures were
per-formed at 4C except the FPLC-Mono Q chromatography,
which was carried out at room temperature The pellets
were resuspended in lysis buffer (50 mM Tris/HCl, 10%
glycerol, 0.01% 2-mercaptoethanol, 1 mM
phenyl-methanesulfonyl fluoride pH 7.5) and disrupted by a
French press cell (12 000–20 000 psi), followed by the
immediate addition of 1 mM phenylmethanesulfonyl
fluo-ride and protease inhibitor (Merck, 10 gÆmL)1E coli) Cell
debris were removed by centrifugation (12000 r.p.m.,
1–2 h), heated to 40C for 15 min, and then centrifuged
to remove any unstable protein After dialysis with buffer A
(50 mM Tris/HCl, 10% glycerol and 0.01%
2-mercapto-ethanol, pH 7.5), the crude protein was loaded onto a
DEAE-Toyopearl 650 M column (2.6· 15 cm)
pre-equili-brated with buffer A After washing with 2.5 bed vols buffer
A, the adsorbed protein was eluted stepwise with buffer A
over a linear gradient containing 0–0.25M NaCl The
pooled active fractions were brought to 20% ammonium
sulfate saturation and applied to a Butyl-Toyopearl 650 M
column (1.6· 7.5 cm) pre-equilibrated with buffer B
(buf-fer A containing 20% ammonium sulfate) After washing
with 3 bed vols buffer B, the enzyme was eluted with the
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 Tris/HCl, 0.15
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 mM Tris/HCl
pH 7.5 and 25 mM N-acetyl-D-methionine to which an appropriate amount of the enzyme was added The reactions were incubated at 40C for 10–30 min and then stopped by heat treatment at 100C for 10 min
D-methionine was determined using the colorimetric assay carried out as follows: 100 lL of the enzyme assay solution was mixed with 60 lL of 50 mMTris/HCl pH 7.5, 20 lL
D-amino acid oxidase (3 UÆmL)1) and 20 lL colorimetric solution containing peroxidase (10 UÆmL)1), 4-aminoanti-pyrine (0.04 lgÆmL)1) and 0.8 lL phenol This was then incubated at room temperature for 10 min and measured
at 520 nm using D-methionine as the standard Protein concentration was determined by the Bradford method with BSA as the standard [40] One unit of N-D-AAase enzyme activity was defined as the formation of 1 lmol
D-methionineÆmin)1
SDS/PAGE and Western analysis The proteins were separated by SDS/PAGE (10% acryl-amide) as described by Laemmli [41] For Western blotting, the proteins were transferred to poly(vinylidene fluoride) membrane using 10 mMCaps 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 6M
urea-PBST (phosphate buffer/saline/Tween-20) solutions with overnight shaking The membrane was washed three times with PBST for 10 min then b locked with Gelatin-NET (NaCl/EDTA/Tween-20) for 1–2 h The primary antibody (1 : 20000 anti N-D-AAase from Alicaligenes faecalisDA1) 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
Trang 4electrospray ionization (ESI) source of a quadrupole ion
trap mass spectrometer (Finnigan LCQ) by a reverse phase
microcapillary column [42] Peptides were eluted at a flow
rate of 500 nLÆmin)1 and the MS/MS spectra of each
peptide was identified by comparison with known peptide
sequences [43] IEF determination was performed using a
Pharmacia Ampholine PAGplate (pH 3–9 gradient gel)
using a broad pI calibration kit
Influences of temperature and pH on enzyme activity
For the determination of the optimal temperature of the
enzyme, the reaction was carried out at 25, 30, 35, 40, 45, 50,
55, 60, 65 and 70C and the enzyme activity measured as
described above Pre-incubation at the indicated
tempera-ture for 30 min was followed by the determination of the
residual enzyme activity was used as a measure the
thermostability of the enzyme The substrate 25 mM
N-acetyl-D-methionine in various buffers was used to
determine the optimal pH The buffers used were: 50 mM
acetate buffer (pH 4.0–5.6), phosphate buffer (pH 6.0–7.2),
Tris/HCl buffer (pH 7.0–8.6) and glycine/NaOH buffer
(pH 8.8–10.2) To measure enzyme stability at the various
pH values, the enzyme was preincubated at 35C for
30 min in the different buffers and the residual enzyme
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 35C for 30 min
followed by the addition of 25 mM N-acetyl-D-methionine
and the residual enzyme activity was measured by the
Chirobiotic T HPLC method usingD-methionine as
stand-ard [44] The test concentration of chelating reagents and
metal ions used for assay were 1 mMand 10 mM, respectively
Substrate specificity analysis
Various substrates (25 mM) were added to the enzyme in the
standard reaction described previously and incubated at
40C for 20 min The amount ofD-amino acids produced
was determined by the Chirobiotic T HPLC method and the
appropriateD-amino acids were used as the standards
R E S U L T S
Identification and phylogenetic analysis
of the strain Iso1
The nucleotide sequence of the 16S rRNA of strain Iso1 was
determined and compared with other bacterial 16S rRNA
sequences corresponding to the E coli 16S rRNA from
positions 28–1489 ABLASTsearch of GenBank showed that
the strain Iso1 had the highest similarity to various
V paradoxusspecies The 16S rRNA of strain Iso1 was
99% similar to those of other V paradoxus strains By using
the neighbour-joining, parsimony and
maximum-likehood (Fig 1) methods from PHYLIP and testing the
resulting trees using bootstrap analysis, the strain Iso1
specifically associated with V paradoxus strains 100% of
the time (1000 bootstraps) Other biochemical activity
analyses [45] and Biolog system kit (Biolog Inc.) identifica-tion also showed that the strain Iso1 was V paradoxus For example, the stain Isol was positive for catalase, oxidase and nitrate reduction but negative of hydrolysis for gelatin and starch Therefore, according to all above analysis results, the strain Iso1 was clearly a strain of V paradoxus
Cloning and nucleotide sequencing analysis
of theN-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.
Trang 5colour in the ELISA microplate N-D-AAase enzyme
activity assay system after blue-white selection The
pBK-damD4 plasmid contained an insert of 9 kb and this was
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
other N-D-AAase protein gene sequences in the GenBank
database A single band was obtained after PCR
amplifi-cation with these degenerate primers using the plasmid
pBK-damD4 as DNA template (data not shown) and this
was used to completely re-sequence the N-D-AAase gene
The nucleotide sequence of the open reading frame of the
N-D-AAase gene was 1467 bp and encoded 488 amino acid
residues with a predicted molecular weight of 52 798
(DNASIS software) (Fig 2) The GC content was about
64.21%, which is consistent with the genome of V
para-doxus (66.8–69.4%) A poorly conserved Shine-Dalgarno
sequence and three possible )10 and )35 regions were
predicted in the region upstream from the start codon
(GENTYEX software) Downstream of the stop codon, a
terminator was found and the pI was predicted to be 5.80 by
the use of the N-D-AAase amino acid composition in the
DNASISsoftware package
Sequence comparison of theV paradoxus
N-D-AAase protein
Alignment by theBLASTP,FASTAand Swiss-Port databases
using theCLUSTAL Wprogram 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
xylosoxydansssp xylosoxydans A-6 and theD-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 tuberculosishad motif 1, 2 and 6 while Streptomyces
coelicolordid not have motif 3 (Table 1 and Fig 3) All
other motifs were present in all the proteins The histidine
residues of motifs 1 and 3 have already been found to be
involved in the enzyme active site or structure of the
N-D-AAase protein [23–25] The function of the other motifs is
still unknown and it will be worthwhile to further investigate
N-D-AAase protein structure/function in the future
Expression and purification of theN-D-AAase protein
fromE coli Top10
E coli harbouring pTrc2A-damA3 was cultivated in the
presence of isopropyl thio-b- -galactoside (1 m ) at 20C
Fig 2 Nucleotide and deduced amino acid sequence of N-D-AAase from V paradoxus The putative termination codon is indicated by asterisk Three possible )35 and )10 regions of putative promoter sequences are shown as a box Double underlining showed the potential Shine-Dalgarno sequence The putative transcription termi-nator is underlined.
Trang 6to avoid forming inclusion body and enzyme activity could
be detected in the supernatant of the cell lysate The
enzyme activity was 1.4 UÆmg)1 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)1and 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 50C (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 The gene accession numb er and the strains are the same as in Fig 4.
Accession number or strain
Amino acid residues
Amino acid Identity (%) Similarity (%) Motif
Fig 3 Sequence alignment of amino acid
sequences of N-D-AAase from V paradoxus
and other homologous proteins A-6-D45918:
Alcaligenes xylosoxydans ssp xylosoxydans
A-6 N-acyl- D -amino acid amidohydrolase;
A-6-D45919: Alcaligenes xylosoxydans ssp.
xylosoxydans A-6 N-acyl- D -Asparate
amidohydrolase; A-6-D50061: Alcaligenes
xylosoxydans ssp xylosoxydans A-6
N-acyl-D -glutamate amidohydrolase; Alicaligenes
faecalis-DA1: Alcaligenes faecalis DA1
N-acyl– D -amino acid amidohydrolase;
V paradoxus Iso1: Variovorax paradoxus
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 program [48].
Trang 730 min preincubation When the temperature was 55C,
the treatment resulted in a 60% loss of activity (Fig 5B)
Above 55C, activity decreased rapidly reflecting the
instability of the enzyme at higher temperatures The
optimal pH for enzyme activity was pH 7.5 (Fig 6A)
In addition, when the N-D-AAase was preincubated at
35C for 30 min at various different pH values, the
greatest stability was from pH 6 to pH 8 (Fig 6B) Beyond these values, in both directions, the enzyme was highly unstable
Table 2 Purification of the N-D-AAase from E coli pTrc2A-damA3 Enzyme activity was assayed by colorimetric assay as described in Materials and methods.
Steps
Protein (mg)
Total activity (U)
Specific activity (UÆmg)1)
Recovery (%)
Purification fold
Fig 4 SDS/PAGE and Western blotting of N-D-AAase (A) SDS/
PAGE (10% acrylamide) M, Molecular mass standards; lane 1, crude
extract; lane 2, heat treatment of crude extract; lane 3, protein after
DEAE-Toyopearl purification; lane 4, protein after Butyl-Toyopearl
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.
Trang 8Influences 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 mM The presence
of the metal ions, Fe2+, Cu2+, Zn2+, Hg2+and Fe3+, at
1 mM gave rise to significant inhibition of between 90%
and 100% Additionally, 10 mM Ca2+, Mn2+and Ni2+
inhibited the enzyme by 50% In contrast, significant
activation or increased stability was observed with 1 mM
Co2+and with 1 mMMg2+ These results indicate that the
N-D-AAase protein of V paradoxus is possibly a
metal-dependent enzyme
Substrate specificity analysis
To study the substrate specificity of the N-D-AAase protein, the activity of the enzyme against N-acyl-D- orL-amino acids and otherD-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, N-acetyl-D-tryptophan, N-acetyl-D-tyrosine and
N-acetyl-D-asparagine However, the enzyme did not hydrolyse substrates such as N-acetyl-L-methionine and
N-acetyl-L-leucine The results indicated the N-D-AAase protein may prefer hydrophobic amino acids such asD-methionine and D-leucine N-acetyl derivates to aromatic amino acid such as D-phenylalanine and D-tryptophan N-acetyl deri-vates and that there is chiral specificity A comparison of N-acetyl-D-phenylalanine and N-chloroacetyl-D -phenylala-nine showed an increased activity against the latter compound and this suggests that the chloride atom of the N-chloroacetyl-D-phenylalanine substrate may promote substrate binding to the enzyme
D I S C U S S I O N
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) b y PCR 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 C in the
fol-lowing buffers (50 m M ): acetate buffer (d, pH 4.0–5.6); phosphate
buffer (n, pH 6.0–7.2); Tris/HCl b uffer (r, pH 7.0–8.6), and Glycine/
NaOH (j, pH 9.0–10.8) The highest activity was taken as 100%.
(B) pH stability The purified enzyme was preincubated for 30 min at
35 C in the various buffers Substrate was then added (N-acetyl- D
-methionine, 25 m M , pH 7.5) and the enzyme activity determined for 20
min at 40 C The highest activity was taken as 100% The results were
the means of duplicate determinations.
Table 3 Substrate specificity of purified N-D-AAaase Relative enzyme activity was assayed by the Chirobiotic T HPLC method [44] The activity for N-acetyl- D -methionine was taken as 100% Results are the means of duplicate determinations.
Substrate (25 m M ) Relative activity (%) N-acetyl- D -methioine 100 ± 4.1
N-acetyl- D -alanine 53 ± 5.2 N-acetyl- D -valine 18 ± 1.3 N-acetyl- 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
Trang 9its 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
plasmid pBK-damD4 showed enzyme activity indicating
that one or more of these promoter regions could be
recognized by an E coli RNA polymerase Interestingly,
another ORF was found on the complementary strand of
the N-D-AAase gene: its gene length was 1149 bp encoding
382 amino acid residues; however, this showed no
signifi-cant similarity to any gene in the GenBank database
When N-D-AAase was expressed in E coli originally,
active soluble protein production could not be obtained at
37C even at an isopropyl thio-b-D-galactoside
concentra-tion lower than 1 mM When the temperature was
down-shifted to 20C at 1 mM isopropyl thio-b-D-galactoside
induction, high soluble protein activity was detected This
indicated that the lower temperature might help the cells to
fold the active protein correctly
Some conserved motifs could be identified when
N-D-AAase protein sequence was compared with other
similar proteins with greater than 50% similarity (Table 1
and Fig 3) Among these motifs, the first histidine residue
of motif 1 (DXHXH) is considered to be involved in the
catalytic site of the enzyme and the second histidine residue
may play a role in maintaining the enzyme structure
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 4C 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
possible to use site-directed mutagenesis to replace the
methionine residues with other amino acids and thus
perhaps improve enzyme stability and enzyme activity for
industrial production
A C K N O W L E D G E M E N T S
We thank Dr M.C Pan, and Dr K.D Lee, (National Taiwan University) for the preparation of cell lysate This work was supported
by a grant NSC 89-2311-B-002-064 from the National Science Council
of Taipei, Taiwan, Republic of China.
R E F E R E N C E S
1 Kohno, K., Miura, H., Hirakawa, Y., Ueki, T & Morikuni, S (1988) Antibacterial lyophilized preparation of aspoxicillin U.S Patent No 4,966,899.
2 Lawen, A & Zocher, R (1990) Cyclosporin synthetase The most complex peptide synthesizing multienzyme polypeptide so far described J Biol Chem 265, 11355–11360.
3 Blackburn, R.K & Van Breemen, R.B (1993) Application of an immobilized digestive enzyme assay to measure chemical and enzymatic hydrolysis of cyclic peptide antibiotic lysobatin Drug Metab Dispos 21, 573–579.
4 Bodanszky, M & Perlman, D (1969) Peptide antibiotics Science.
163, 352–358.
5 Kreil, G (1997) D -amino acids in animal peptides Annu Rev Biochem 66, 337–345.
6 Finberg, R.W., Diamond, D.C., Mitchell, D.B., Rosenstein, Y., Soman, G., Norman, T.C., Schreiber, S.L & Burakoff, S.J (1990) Prevention of HIV-1 infection and preservation of CD4 function
by the binding of CPFs to gp120 Science 249, 287–291.
7 Collins, A.N., Sheldrake, G.N & Crosby, J (1994) Membrane bioreactors for the production of enantiomerically pure D -amino acids In Chirality Industry, pp.372–397 John Wiley and Sons Inc New York.
8 Van Regenmortel, M.H & Muller, S (1998) D -peptides as immunogens and diagnostic reagents Curr Opin Biotechnol 9, 377–382.
9 Taylor, P.P., Pantaleone, D.P., Senkpeil, R.F & Fotheringham, I.G (1998) Novel biosynthetic approaches to the production of unnatural amino acids using transaminases Trends Biotechnol 16, 412–418.
10 Yagasaki, M & Ozaki, A (1998) Industrials biotransformations for the production of D -amino acids J Mol Catal B 4, 1–11.
11 Cleifer, K.H & Kandler, O (1972) Peptidoglycan types of bac-terial cell walls and their taxonomic implications Bacbac-terial Rev.
36, 407–477.
12 Hashimoto, A., Oka, T & Nishikawa, T (1995) Extracellular concentration of endogenous free D -serine in the rat brain as revealed by in vivo microdialysis Neuroscience 66, 635–643.
13 Corrigan, J.J (1969) D -Amino acids in animals Science 164, 142– 149.
14 Moriguchi, M & Ideta, K (1988) Production of D -aminoacylase from Alcaligenes denitrificans subsp xylosoxydans MI-4 Appl Environ Microbiol 54, 2767–2770.
15 Sakai, K., Oshima, K & Moriguchi, M (1991) Production and characterization of N-acyl- D -glutamate amidohydrolase from Pseudomonas sp strain 5f )1 Appl Environ Microbiol 57, 2540– 2543.
16 Mun˜iz-Lozano, F.E., Domı´nguez-Sa´nchez, G., Dı´az-Viveros, Y.
& Barradas-Dermitz, D.M (1998) D -aminoacylase from a novel producer: Stenotrophomonas maltophilia ITV-0595 J Ind Microbiol Biotechnol 21, 296–299.
17 Moriguchi, M., Sakai, K., Katsuno, Y., Maki, T & Wakayama,
M (1993) Purification and characterization of novel
N-acyl-D -asparate amidohydrolase from Alcaligenes xylosoxydans subsp xylosoxydans A-6 Biosci Biotechnol Biochem 57, 1145– 1148.
18 Sugie, M & Suzuki, H (1978) Purification and properties of
D -aminoacylase of Streptomyces olivaceus Agric Bio Chem 42, 107–113.
Trang 1019 Tsai, Y.C., Tseng, C.P., Hsiao, K.M & Chen, L.Y (1988)
Pro-duction and purification of D -aminoacylase from Alcaligenes
denitrificans and taxonomic study of the strain Appl Environ.
Microbiol 54, 984–989.
20 Tokuyama, S (1999) D -Aminoacylase US Patent 5,916,774.
21 Tokuyama, S (2000) D -Aminoacylase European Patent
60,950,706,A2.
22 Kubo, K., Ishikara, T & Fukagawa, Y (1980) Deacetylation of
PS-5, a new beta-lactam compound II Separation and purification
of L -amino acid acylase and D -amino acid acylase from
Pseudo-monas sp 1158 J Antibiotic 33, 550–555.
23 Wakayama, M., Yada, H., Kanda, S., Hayashi, S., Yatsuda, Y.,
Sakai, K & Moriguchi, M (2000) Role of conserved histidine
residues in D -aminoacylase from Alcaligenes xylosoxydans subup.
xylosoxydans A-6 Biosci Biotechnol Biochem 64, 1–8.
24 Wakayama, M., Miura, Y., Oshima, K., Sakai, K & Moriguchi,
M (1995) Metal-characterization of N-acyl- D -glutamate
amido-hydrolase from Pseudomonas sp strain 5f )1 Biosci Biotechnol.
Biochem 59, 1489–1492.
25 Wakayama, M., Tsutsumi, T., Yada, H., Sakai, K & Moriguchi,
M (1996) Chemical modification of histidine residue of
N-acyl-D -glutamate amidohydrolase from Pseudomonas sp 5f )1 Biosci.
Biotechnol Biochem 60, 650–653.
26 Wakayama, M., Watanabe, E., Takenaka, Y., Miyamoto, Y.,
Tau, Y., Sakai, K & Moriguchi, M (1995) Cloning, expression
and nucleotide sequence of the gene of N-acyl- D -asparate
amido-hydrolase from Alcaligenes xylosoxydans subsp xylosoxydans
A-6 J Ferment Bioeng 80, 311–317.
27 Wakayama, M., Ashika, T., Miyamoto, Y., Yoshikawa, T.,
Sonoda, Y., Sakai, K & Moriguchi, M (1995) Primary structure
of N-acyl- D -glutamate amidohydrolase from Alcaligenes
xylosoxy-dans subsp xylosoxydans A-6 J Biochem (Tokyo) 118, 204–209.
28 Wakayama, M., Katsuno, Y., Hayashi, S., Miyamoto, Y., Sakai,
K & Moriguchi, M (1995) Cloning and sequencing of a gene
encoding D -aminoacylase from Alcaligenes xylosoxydans subsp.
xylosoxydans A-6 and expression of the gene in Escherichia coli.
Biosci Biotechnol Biochem 59, 2115–2119.
29 Chien, H.R., Jih, Y.L., Yang, W.Y & Hsu, W.H (1998)
Identi-fication of open reading frame for the Pseudomonas putida
D -hydantoinase gene and expression of the gene in Escherichia coli.
Biochim Biophy Acta 1395, 68–77.
30 Galkiw, A., Kulakova, L., Yoshimura, T., Soda, K & Esaki, N.
(1997) Synthesis of optically active amino acids from a-keto acids
with Escherichia coli cells expressing heterologous genes Appl.
Environ Microbiol 63, 4651–4656.
31 Ozaki, A., Kawasaki, H., Yagasaki, M & Hashimoto, Y (1992)
Enzymatic production of D -alanine from DL-alaninamide by
novel D -alaninamide specific amide hydrolase Biosci Biotechnol.
Biochem 56, 1980–1984.
32 Tokuyama, S & Hatano, K (1996) Overexpression of the gene for
N-acylamino acid racemase from Amycolatopsis sp TS-1-60 in
Escherichia coli and continuous production of optically active
methionine by a bioreactor Appl Microbiol Biotechnol 44, 774–
777.
33 Kubo, K., Ishikara, T & Fukagawa, Y (1980) Deacetylation of
PS-5, a new beta-lactam compound III Enzymological
char-acterization of L -amino acid acylase and D -amino acid acylase
from Pseudomonas sp 1158 J Antibiotic 33, 556–565.
34 Lee, C.Y., Su, S.C & Liaw, R.B (1995) Molecular analysis of an
extracellular protease gene from Vibrio parahaemolyticus
Micro-biology 141, 2569–2576.
35 Ausbel, F.N., Brent, R., Kingstone, R.E., Moore, D.D., Seidman,
J.G., Smith, J.A & Struhl, K (1993) Current protocols in
mole-cular biology John Wiley and Sons Inc., New York.
36 Altschul, S.F., Gish, W., Miller, W., Myers, E.W & Lipman, D.J.
(1990) Basic local alignment search tool J Mol Biol 215, 403–
410.
37 Thompson, J.D., Higgins, D.G & Gibson, T.J (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucl Acids Res 22, 4673– 4680.
38 Felsenstein, J (1989) Phylogeny inference package Cladistics 5, 164–166.
39 Smbrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
40 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254.
41 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacterialphage T4 Nature (Lond.) 227, 680–685.
42 Nash, H.M., Bruner, S.D., Scharer, O.D., Kawate, T., Addona, T.A., Spooner, E., Lane, W.S & Verdine, G.L (1996) Cloning of
a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily Curr Biol 6, 968– 980.
43 Chittum, H.S., Lane, W.S., Carlson, B.A., Roller, P.P., Lung, F.D., Lee, B.J & Hatfiled, D.L (1998) Rabbit b-globin
is extended beyond its UGA stop codon by multiple suppres-sions and translational reading gaps Biochemistry 37, 10866– 10870.
44 Su, S.C & Lee, C.Y (2002) Cloning of the N-acylamino acid racemase gene from Amycolatopsis azurea and biochemical char-acterization of the gene product Enzyme Microb Technol 30, 647–655.
45 Holding, A.J (1986) Bergeys’s Manual Systematic Bacteriology Williams & Wilkins Co, Baltimore, MD.
46 Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry, C.E., III Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, S., Osborne, J., Quail, M.A., Rajandream, M.A., Rogers, J., Rutter, S., Seeger, K., Skelton, S., Squares, S., Sqares, R., Sulston, J.E., Taylor, K., Whitehead, S & Barrell, B.G (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence Nature (Lond.) 393, 537–544.
47 Redenbach, M., Kieser, H.M., Denapaite, D., Eichner, A., Cul-lum, J., Kinashi, H & Hopwood, D.A (1996) A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3 (2) chromosome Mol Microbiol 21, 77–96.
48 Bailey, T.L & Gribskov, M (1998) Combining evidence using p-values: application to sequence homology searches Bioinfor-matics 14, 48–54.
49 Willems, A., Deley, J., Gillis, M & Kersters, K (1991) Comamonadaceae, a new family encompassing the Acidovorans rRNA complex, including Variovorax paradoxus General nov., comb nov., for Alcaligenes paradoxus Int J Syst Bacteriol 41, 445–450.
50 Sheu, D.S., Wang, Y.T & Lee, C.Y (2000) Rapid detection of polyhydroxyalkanoate accumulating bacteria isolated from the environment by colony PCR Microbiology 146, 2019–2025.
51 Leadbetter, J.R & Greenberg, E.P (2000) Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax para-doxus J Bacteriol 182, 6921–6926.
52 Stadtman, E.R (1993) Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions Ann Rev Biochem 62, 797–821.
53 Stadtman, E.R (1992) Protein oxidation and aging Science 257, 1220–1224.