MCI3434 genome and found an additional gene, bapA, coding for a protein showing sequence similarity to DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 43% identity.. One of them w
Trang 1dipeptidase specific for b-alanyl dipeptides
Hidenobu Komeda and Yasuhisa Asano
Biotechnology Research Center, Toyama Prefectural University, Toyama, Japan
Many different kinds of microbial hydrolases acting
d-stereoselectively on amino acid amides or peptides
have been characterized, and some of them have been
applied to the production of optically active d-amino
acids from the corresponding racemic amino acid
amides [1] The d-stereoselective amidases and peptidases
known to date can be classified into four groups based
on their primary structures d-Aminopeptidase from Ochrobactrum anthropi C1-38 [2,3], d-amino-acid ami-dase from O anthropi SV3 [4,5], alkaline d-peptiami-dases from Bacillus cereus DF4-B [6,7] and AH559 [8], DmpB from O anthropi LMG7991 [9] and MlrB from Sphingomonassp [10] are active site serine hydrolases, which are classified into the penicillin-recognizing
Keywords
amidase; b-alanine; dipeptidase; DmpA;
Pseudomonas sp.
Correspondence
Y Asano, Biotechnology Research Center,
Toyama Prefectural University, 5180
Kurokawa, Kosugi, Toyama 939–0398,
Japan
Fax: +81 766 562498
Tel: +81 766 567500
E-mail: asano@pu-toyama.ac.jp
(Received 18 March 2005, revised 12 April
2005, accepted 18 April 2005)
doi:10.1111/j.1742-4658.2005.04721.x
We have determined the nucleotide sequence of a DNA fragment covering the flanking region of the R-stereoselective amidase gene, ramA, from the Pseudomonas sp MCI3434 genome and found an additional gene, bapA, coding for a protein showing sequence similarity to DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 (43% identity) The DmpA (called
l-aminopeptidase d-Ala-esterase⁄ amidase) hydrolyzes alanine-p-nitroani-lide, alaninamide, and alanine methylester with a preference for the d-con-figuration of the alanine, whereas the enzyme acts as an l-stereoselective aminopeptidase on a tripeptide Ala-(Gly)2, indicating a reverse stereoselec-tivity [Fanuel L, Goffin C, Cheggour A, Devreese B, Van Driessche G, Joris B, Van Beeumen J & Fre`re J-M (1999) Biochem J 341, 147–155]
A recombinant BapA exhibiting hydrolytic activity toward d-alanine-p-nitroanilide was purified from the cell-free extract of an Escherichia coli transformant overexpressing the bapA gene and characterized The purified enzyme contained two polypeptides corresponding to residues 1–238 (a-peptide) and 239–366 (b-peptide) of the precursor as observed for DmpA On gel-filtration chromatography, BapA in the native form appeared to be a tetramer It had maximal activity at 60C and pH 9.0– 10.0, and was inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, dithiothreitol, Zn2+, Ag+, Cd2+ or Hg2+ The enzyme hydrolyzed d-alanine-p-nitroanilide more efficiently than l-alanine-p-nitro-anilide the same as DmpA Furthermore, BapA was found to hydrolyze peptide bonds of b-alanyl dipeptides including b-Ala-l-Ala, b-Ala-Gly, b-Ala-l-His (carnosine), b-Ala-l-Leu, and (b-Ala)2 with high efficiency compared to d-alanine-p-nitroanilide b-Alaninamide was also efficiently hydrolyzed, but the enzyme did not act on the peptides containing pro-teinogenic amino acids or their d-counterparts for N-terminal residues Based on its unique substrate specificity, the enzyme should not be called
l-aminopeptidase d-Ala-esterase⁄ amidase but b-Ala-Xaa dipeptidase
Abbreviations
ORF, open reading frame; SD, Shine–Dalgarno.
Trang 2protein family together with dd-carboxypeptidases
involved in peptidoglycan biosynthesis and
b-lactamas-es for rb-lactamas-esistance against b-lactam antibiotics (group 1)
Zinc-containing d-alanyl-d-alanine-dipeptidases
inclu-ding VanX from vancomycin-resistant enterococci,
VanX from the glycopeptide antibiotic producer
Strep-tomyces toyocaensis and DdpX from Escherichia coli
are considered to be involved in vancomycin-resistance,
immunity to the self-produced antibiotic, and cell
sur-vival under condition of starvation, respectively [11]
(group 2) d-Aminopeptidase, DppA, from Bacillus
sub-tilis is a Zn2+-dependent self-compartmentalizing
pro-tease composed of 10 subunits [12,13] A thermostable
d-alanine amidase homologous to DppA has also been
found in Brevibacillus borstelensis BCS-1 [14] (group 3)
For the fourth group, DmpA acting on
d-alanine-p-nitroanilide (d-Ala-pNA) was found in O anthropi
LMG7991 [9] Unlike the above d-stereoselective
enzy-mes exhibiting strict d-stereoselectivity, DmpA shows
a peculiar substrate specificity acting on Ala-p-NA,
alaninamide and alanine methylester with a preference
for the d-configuration, but acting l-stereoselectively
on peptide substrates such as Ala-(Gly)2, and has
there-fore been called l-aminopeptidase d-Ala-esterase⁄
ami-dase [15] The activity of DmpA toward these d- and
l-substrates is, however, very weak, suggesting that
they are not the true substrates of the enzyme The
bio-logical role of DmpA also remains unclear
We recently found in Pseudomonas sp MCI3434 a
novel amidase, named R-amidase, acting
R-stereoselec-tively on piperazine-2-tert-butylcarboxamide and
isola-ted the gene coding for the enzyme, ramA [16] In this
study, we determined the nucleotide sequence of the
region flanking ramA and found six additional genes
One of them was named bapA and its deduced amino
acid sequence showed sequence similarity to the DmpA
from O anthropi LMG7991 The bapA gene was
expressed in an E coli host and the recombinant
pro-tein (BapA) acting on d-Ala-pNA was purified and
characterized BapA was found to show a unique
sub-strate specificity for b-alanyl dipeptides
Results
Characterization of the flanking region of ramA encoding an R-stereoselective amidase from Pseudomonas sp MCI3434
The R-stereoselective amidase-encoding gene, ramA, had been isolated from the Pseudomonas sp MCI3434 genome [16] Sequence analysis of the region down-stream from the termination codon of ramA suggested the presence of an ORF, which was preceded by a Shine–Dalgarno (SD) sequence located within a rea-sonable distance of the presumptive ATG start site This finding suggested that the ramA gene clustered together with some other genes The two plasmids pRTB1-Fba and pRTB1-Pst containing fragments cov-ering the flanking region of ramA had been construc-ted previously [16] Here, we determined the nucleotide sequence of the two inserted fragments to obtain a 6668-bp sequence and found four open reading frames (ORFs) designated ORF1, ORF2 (bapA), ORF3 and ORF4, in the upstream region of the ramA gene and two ORFs designated ORF6 and ORF7 in the down-stream region (Fig 1) ORF3, -4, and -7 would be transcribed in the opposite direction to the other ORFs It is interesting to note that the inverted repeat sequence, IR-1 (positions 1351–1497), found in the intergenic region of bapA-ORF3 shows 74% identity over 147-bp with the other sequence, IR-2 (positions 6154–6300), found between ORF6 and ORF7 (Fig 2) These two inverted repeats may rho-independent transcriptional terminators and not other genomic elements, for example, terminal repeats flanking trans-posable regions, because of absence of direct repeat sequences delineating the two inverted repeats
ORF2 designated bapA was found to be 1098 nucleo-tides long (positions 233–1330) and would encode a protein of 366 amino acids (molecular mass, 38123 Da)
A potential ribosome-binding site (AGAA) was located just seven nucleotides upstream from the start codon ATG In the upstream region of the bapA translational
Fig 1 Schematic view of the inserted fragments of pRTB1-Fba and pRTB1-Pst and structural organization of the 6668-bp DNA containing ORF1, ORF2 (bapA), ORF3, ORF4, ramA, ORF6 and ORF7 from Pseudomonas sp MCI3434 The location of the ORFs and the direction of transcription (arrows) are indicated Inverted repeat sequences are indicated by ‘IR-1’ and ‘IR-2’.
Trang 3start codon, sequences related to the )35 (TCGTCA)
and)10 (TACACT) consensus promoter regions were
identified A blast search indicated that the amino acid
sequence deduced from bapA was similar to those of
hypothetical protein PA1486 from Pseudomonas
aerugi-nosa PAO1 [17], putative d-aminopeptidase PP3844
from Pseudomonas putida KT2440 [18], hypothetical
protein PLU2258 similar to d-aminopeptidase from
Photorhabdus luminescens (ssp laumondii) TTO1 [19],
aminopeptidase Atu5242 from Agrobacterium
tumefac-iensC58 plasmid AT [20] and DmpA (l-aminopeptidase
d-Ala-esterase⁄ amidase) from O anthropi LMG7991
[15] (Table 1) Figure 3 shows the alignment of the
primary structures of BapA from Pseudomonas sp MCI3434 and its homologous proteins All the sequences except for DmpA in the figure are hypothet-ical proteins found in the genome sequence but yet to
be characterized functionally DmpA is organized as a homotetramer and each subunit contains two chains (a and b) which result from a probable autocatalytic cleavage of the Gly249-Ser250 peptide bond of the inac-tive precursor polypeptide [15] Not only this Gly-Ser dyad but also proposed active site residues, Tyr146 and Asn218, the possible two elements of an oxyanion hole [21], were well conserved in the BapA sequence (num-bering of the residues is based on DmpA)
Fig 2 Comparison of the nucleotide
sequences of IR-1 and IR-2 Identical bases
are marked by asterisks.
Table 1 Homology search analysis of seven ORFs in 6668-bp-long DNA region of Pseudomonas sp MCI3434 P putida, Pseudomonas put-ida; P aeruginosa, Pseudomonas aeruginosa; P luminescens, Photorhabdus luminescens; A tumefaciens, Agrobacterium tumefciens;
O anthropi, Ochrobactrum anthropi; P syringae, Pseudomonas syringae; S meliloti, Sinorhizobium meliloti; E coli, Escherichia coli; S flex-neri, Shigella flexneri.
Homology
ORF2
(bapA)
ORF4 Probable transcriptional regulator of luxR family 73 PP3847 P putida KT2440 Q88G78
ORF5
(ramA)
Trang 4As summarized in Table 1, the deduced amino acid
sequences of the other ORFs, ORF1, ORF3, ORF4,
ORF6 and ORF7, showed significant homology with
probable periplasmic polyamine-binding protein,
prob-able dipeptidase, probprob-able transcriptional regulator of
LuxR family, probable periplasmic polyamine-binding
protein and nickel ABC transporter, respectively
Comparison of the deduced amino acid sequences of
ORF1 and ORF7 with their homologous genes
indica-ted that both of the ORFs lack 5¢-terminus, probably
coding for about 320 amino acid residues for ORF1
and about 160 amino acid residues for ORF7 The
seven proteins encoded in the 6668-bp region of
Pseu-domonas sp MCI3434 showed 38–74% identity with
those encoded in P putida KT2440 genome Moreover,
ORF1-bapA and ORF4-ramA-ORF5 are also in equi-valent cluster arrangement in P putida KT2440 genome
Production of BapA in E coli and its purification
To express the bapA gene in E coli, we improved the sequence upstream from the ATG start codon by PCR, with the plasmid pRTB1-Fba as a template as described in Experimental procedures The resultant plasmid, p2DAPEX, in which the bapA gene was under the control of the lac promoter of the pUC19 vector, was introduced into E coli JM109 cells E coli JM109 harboring pUC19, which was cultured in LB medium supplemented with ampicillin and
isopropyl-Fig 3 Comparison of the amino acid sequences of BapA and homologous pro-teins Identical and conserved amino acids among the sequences are marked in black and in gray, respectively Dashed lines indi-cate the gaps introduced for better align-ment A cleavage site identified in BapA as well as in DmpA is marked by an arrow-head Proposed active site residues Tyr146 and Asn218 in DmpA are marked by aster-isks Pse-BapA, BapA from Pseudomonas
sp MCI3434; PA1486, hypothetical protein PA1486 from P aeruginosa PAO1; PP3844, putative D -aminopeptidase PP3844 from
P putida KT2440; Atu5242, aminopeptidase Atu5242 from Agrobacterium tumefaciens C58 plasmid AT; Oan-DmpA, DmpA ( L -amino-peptidase D -Ala-esterase ⁄ amidase) from
O anthropi LMG7991; PLU2258, hypothet-ical protein PLU2258 similar to D -aminopep-tidase from Photorhabdus luminescens (ssp laumondii) TTO1.
Trang 5b-d-thiogalactopyranoside for 15 h at 37C, exhibited
no hydrolytic activity toward d-Ala-pNA When
E coli JM109 harboring p2DAPEX was cultured
under the same conditions, the level of hydrolytic
activity toward the substrate was 0.776 units per
milli-liter of culture, suggesting that the BapA protein was
produced in an active form in E coli
Recombinant BapA was purified from the E coli
JM109 harboring p2DAPEX with a recovery of 10.3%
by ammonium sulfate fractionation and
DEAE-Toyo-pearl, Butyl-Toyopearl and MonoQ column
chromato-graphies (Table 2) The final preparation gave two
bands on SDS⁄ PAGE with molecular masses of 27
and 13 kDa (Fig 4) These polypeptides were
electro-blotted on to a poly(vinylidene difluoride) (PVDF)
membrane and submitted to N-terminal amino acid
sequencing, which yielded the MRIRE and SIVIT
sequences for the 27 and 13 kDa peptides, respectively
These sequences corresponded to residues 1–5 and
239–243 of the deduced amino acid sequence of BapA
This result clearly indicated that the mature enzyme
with two polypeptide chains (a and b) was formed
by the cleavage of Gly238-Ser239 peptide bond of the
366-residue precursor, as in the case of DmpA from
O anthropi LMG7991 The molecular mass of the
native enzyme was about 150 kDa according to
gel-filtration chromatography, indicating that the native
enzyme was active as a tetramer The purified enzyme
catalyzed the hydrolysis of d-Ala-pNA to d-alanine
and p-nitroaniline at 7.73 UÆmg)1 under standard
con-ditions
Effects of pH and temperature on stability and
activity of BapA
The stability of the enzyme was examined at various
pH values The enzyme was incubated at 30C for
10 min in the various buffers described in
Experimen-tal procedures Then a sample of the enzyme solution
was taken, and the remaining activity of BapA was
assayed with d-Ala-pNA as a substrate under standard
conditions The enzyme was most stable in the pH range 6.0–11.0 The stability of the enzyme was also examined at various temperatures After the enzyme had been preincubated for 10 min, the remaining activ-ity was assayed with d-Ala-pNA as a substrate under standard conditions It exhibited the following remain-ing activity: 60C, 0%; 55 C, 49%; 50 C, 87%;
45C, 100%; 40 C, 100%; 35 C, 100% The enzyme could be stored on ice without loss of activity for more than one month
The optimal pH for the activity of the enzyme was measured in the buffers used above The enzyme showed maximal activity at pH 9.0–10.0 The enzyme reaction was also carried out at various temperatures for 1 min in 0.1 m Tris⁄ HCl (pH 8.0), and enzyme activity was found to be maximal at 60C Above
75C, it decreased rapidly, possibly because of insta-bility of the enzyme at the higher temperatures
Effects of inhibitors and metal ions The BapA solution was dialyzed against 20 mm Tris⁄ HCl (pH 8.0) Various compounds were investi-gated for their effects on the enzyme activity We meas-ured the activity under standard conditions after incubation at 30C for 10 min with various compounds
Table 2 Purification of BapA protein from E coli JM109 harboring
p2DAPEX.
Total protein (mg)
Total activity (U)
Specific activity (U ⁄ mg)
Yield (%)
Fig 4 SDS ⁄ polyacrylamide slab gel electrophoresis of BapA Lane
1, molecular-mass standards [phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa)]; lane 2, purified BapA (10 lg) Polypeptides with a molecular mass of
27 kDa and 13 kDa were designated a- and b-peptide, respectively.
Trang 6at 1 mm The enzyme was inhibited by
p-chloro-mercuribenzoate (4.9%), N-ethylmaleimide (19.5%),
dithiothreitol (37.0%), HgCl2 (0.8%), ZnSO4 (1.5%),
ZnCl2(1.7%), AgNO3(5.2%) and CdCl2(9.5%): values
in parentheses indicate the relative remaining activity
Chelating reagents, e.g o-phenanthroline,
8-hydroxy-quinoline, ethylenediaminetetraacetic acid, and
a,a¢-dipyridyl had no significant effect on the enzyme
Carbonyl reagents such as hydroxylamine,
phenylhydr-azine, hydrphenylhydr-azine, d,l-penicillamine, and d-cycloserine
were not inhibitory toward the enzyme either A serine
protease inhibitor, phenylmethanesulfonyl fluoride, a
serine⁄ cysteine protease inhibitor, leupeptine, and an
aspartic protease inhibitor, pepstatin, did not influence
the activity Inorganic compounds such as LiCl,
H2BO3, NaCl, MgSO4, MgCl2, AlCl3, KCl, CaCl2,
CrCl3, MnSO4, MnCl2, FeSO4, FeCl3, CoCl2, NiCl2,
CuSO4, CuCl2, RbCl, Na2MoO4(NH4)6Mo7O24, SnCl2,
CsCl, BaCl2and PbCl2did not affect the activity
Substrate specificity
To study the substrate specificity, the purified BapA
was used to hydrolyze various amides and peptides and
the activity was assayed (Table 3) The enzyme
pre-ferred the d-configuration of Ala-pNA, hydrolyzing
d-Ala-pNA with 5.8 times higher efficiency than
l-Ala-pNA d-Alaninamide was, however, hydrolyzed by
BapA at a much lower rate than d-Ala-pNA, while the
hydrolysis of l-alaninamide was below the detection
limit The enzyme did not act on the peptides
contain-ing proteinogenic amino acids or their d-counterparts
for N-terminal residues Besides the three substrates
which could be hydrolyzed by BapA, dipeptides
con-taining b-alanine at the amino terminus, including
b-Ala-l-Ala, b-Ala-Gly, b-Ala-l-His (l-carnosine),
b-Ala-l-Leu and (b-Ala)2 were found to be efficiently
hydrolyzed by the enzyme b-Alaninamide was also
hydrolyzed by the enzyme The highest level of activity
was observed for b-Ala-l-Ala, being as much as
6.2 times that for d-Ala-pNA c-Aminobutyryl-l-His
(l-homocarnosine) was not hydrolyzed by BapA
These results indicated that the enzyme is specific for
N-terminal b-alanyl dipeptides (b-Ala-Xaa)
Discussion
In this paper, we determined the nucleotide sequence
of the flanking region of ramA coding for R-amidase
from Pseudomonas sp MCI3434, and found six ORFs
named ORF1, bapA, ORF3, ORF4, ORF6, and ORF7
upstream and downstream of ramA The amino acid
sequence deduced from bapA showed significant
similarity to that from dmpA of O anthropi LMG7991 DmpA has been reported to be a homo-tetrameric enzyme, each subunit of which is composed
of two polypeptides generated by a possible autocata-lytic cleavage of a precursor polypeptide Interestingly, DmpA changes its stereoselectivity depending on the substrate, catalyzing the l-stereoselective hydrolysis of peptides and d-stereoselective hydrolysis of amino acid amides and esters [15] The gel-filtration chromato-graphy and SDS⁄ PAGE analysis of the purified BapA revealed that the number of subunits in the native form and the polypeptide composition of each subunit were significantly similar to those for DmpA The pref-erence for the d-configuration of Ala-pNa and alanin-amide as substrates by BapA was also comparable to that exhibited by DmpA The substrate specificity of BapA was however, different from that of DmpA with respect to the activity toward peptide substrates BapA could not hydrolyze l-Ala-(Gly)2 which is a good sub-strate for DmpA Furthermore, a broader exploration
of the substrate range revealed that BapA acted with much higher efficiency on the b-alanyl dipeptides and b-alaninamide than d-Ala-pNA, and we therefore pro-pose that the enzyme be tentatively called b-Ala-Xaa dipeptidase (EC 3.4.13.-) It would be interesting to investigate whether DmpA can also hydrolyze b-alanyl dipeptides
Table 3 Comparison of the substrate specificity of BapA and DmpA The following compounds were not substrates for BapA: (Gly) 2 (Gly) 3 , D -Ala-Gly, D -Ala-(Gly) 2 ( D -Ala) 2 , D -Ala- L -Ala ( D -Ala) 3
( D -Ala)4, L -Ala-Gly, L -Ala-(Gly)2 ( L -Ala)2, L -Ala- D -Ala, L -Ala- D -Ala- L -Ala,
DL -Ala- DL -Asn, DL -Ala- DL -Ile, DL -Ala- DL - L eu, DL -Ala- DL -Met, DL
-Ala-DL -Phe, DL -Ala- DL -Ser, DL -Ala- DL -Val, L -Asp- D -Ala, L -Pro-Gly, L
-Pro-L -Phe, c-Aminobutyryl- L -His (homocarnosine), Gly-NH2, D -Phe-NH2,
D -Asp-NH2, D -Glu-NH2, D -Gln-NH2, D -Pro-NH2, L -Ala-NH2, L -Leu-NH2,
L -Phe-NH 2 , L -Tyr-NH 2 , L -Trp-NH 2 , L -Ser-NH 2 , L -Thr-NH 2 , L -Lys-NH 2
and L -Pro-NH 2 aData from reference [15]; N.D not detectable; N.I.
no information.
Substrate
Activity (UÆmg)1)
Trang 7Microbial peptidases acting on b-alanyl dipeptides
have been studied only in Pseudomonas aeruginosa and
Lactobacillus delbrueckiissp lactis DSM 7290 Van der
Drift and Ketelaars have isolated a bacterium
hydro-lyzing b-Ala-l-His (l-carnosine) and identified it as
P aeruginosa[22] They also investigated some
proper-ties of the dipeptidase, called carnosinase, using crude
cell-free extracts of the strain The carnosinase activity
in crude extracts was not affected by the addition of
EDTA and the enzyme could not hydrolyze
c-amino-butyryl-l-His (l-homocarnosine) Comparable
observa-tions were made for the purified BapA in this study,
suggesting that the carnosinase activity in P aeruginosa
is likely to correspond to the BapA activity in
Pseudo-monassp MCI3434 On the other hand, pepV has been
cloned from the genome of L delbrueckii ssp lactis
DSM 7290 and identified as a gene coding for
carnosin-ase activity [23] Crude extracts of an E coli
transform-ant producing PepV were subjected to native gel
electrophoresis and used for subsequent histochemical
staining with various peptides to study the substrate
specificity without purification of the enzyme Although
PepV and BapA have some overlap in substrate
speci-ficity especially for b-alanyl dipeptides, we also found
substrates that could be hydrolyzed by only PepV, such
as d-Ala-l-Leu and (l-Ala)2 There was no significant
homology between the amino acid sequences deduced
from pepV of L delbrueckii and from bapA BapA is
therefore the first example of a highly purified and
characterized enzyme specific for b-alanyl dipeptides
Experimental procedures
Bacterial strain, plasmids, and culture conditions
E coliJM109 (recA1, endA1, gyrA96, thi, hsdR17, supE44,
relA1,D (lac-proAB) ⁄ F¢ [traD36, proAB+, lacIq, lacZDM15])
was used as a host for the recombinant plasmids Plasmids
pRTB1-Fba and pRTB1-Pst [16] containing inserts of 5.3 kb
and 2.1 kb, respectively, were used for nucleotide
sequen-cing Plasmid pUC19 (Takara Bio Inc., Shiga, Japan) was
used as a cloning vector Recombinant E coli JM109 was
cultured in LB medium [24] containing 80 lgÆmL)1of
ampi-cillin To induce expression of the gene under the control of
the lac promoter, isopropyl thio-b-d-galactoside was added
to a final concentration of 0.5 mm
DNA sequence analysis
For routine work with recombinant DNA, established
pro-tocols were used [24] Nested unidirectional deletions were
generated from the plasmids, pRTB1-Fba and pRTB1-Pst,
with the Kilo-Sequence deletion kit (Takara Bio Inc.) An
automatic plasmid isolation system (Kurabo, Osaka, Japan) was used to prepare the double-stranded DNAs for sequen-cing Nucleotide sequencing was performed using the dide-oxynucleotide chain-termination method [25] with M13 forward and reverse oligonucleotides as primers Sequen-cing reactions were carried out with a Thermo SequenaseTM cycle sequencing kit and dNTP mixture with 7-deaza-dGTP from Amersham Biosciences K.K (Tokyo, Japan), and the reaction mixtures were run on a DNA sequencer 4000 L (Li-cor, Lincoln, NE, USA) Both strands of DNA were completely sequenced The nucleotide sequence data repor-ted in this paper will appear in the DDBJ⁄ EMBL ⁄ Gen-Bank nucleotide sequence databases with the accession number AB158573 Amino acid sequences were compared with the blast program [26]
Expression of the bapA gene in E coli
A modified bapA gene coding for the DmpA-homologous protein was obtained by PCR The reaction mixture for the PCR contained in 50 lL of 10 mm Tris⁄ HCl, pH 8.85,
25 mm KCl, 2 mm MgSO4, 5 mm (NH4)2SO4, each dNTP at
a concentration of 0.2 mm, a sense and an antisense primer each at 1 lm, 2.5 U of Pwo DNA polymerase from Roche Diagnostics GmbH (Mannheim, Germany) and 0.1 lg of plasmid pRTB1-Fba as a template DNA Thirty cycles were performed, each consisting of a denaturing step
at 94C for 30 s (first cycle 2 min 30 s), an annealing step at
55C for 30 s, and an elongation step at 72 C for 2 min The sense primer contained an HindIII-recognition site (underlined sequence), a ribosome-binding site (double underlined sequence), and a TAG stop codon (lowercase let-ters) in-frame with the lacZ gene in pUC19, and spanned positions 230–259 in the sequence from GenBank with acces-sion number AB158573 The antisense primer contained an XbaI site (underlined sequence) and corresponded to the sequence from 1320 to 1353 The two primers were as fol-lows: sense primer, 5¢-CACTTGAAGCTTTAAGGAGGA AtagACCATGCGTATCCGTGAGCTTGGCATCACC-3¢; antisense primer, 5¢-ACGCAATCTAGAGTCAGCCCTCA GGGGGCTTTCG-3¢ The amplified PCR product was digested with HindIII and XbaI (Takara Bio Inc.), separ-ated by agarose-gel electrophoresis and purified from the gel by use of a QIAquickTM gel extraction kit from QIAGEN (Tokyo, Japan) The amplified DNA was inserted downstream of the lac promoter in pUC19, yielding p2DAPEX, and which was then used to transform E coli JM109 cells
Purification of BapA from E coli transformant
E coli JM109 harboring p2DAPEX was subcultured at
37C for 12 h in a test tube containing 5 mL of LB medium supplemented with ampicillin The subculture
Trang 8(5 mL) was then inoculated into a 2-L Erlenmeyer flask
containing 500 mL of LB medium supplemented with
ampicillin and isopropyl thio-b-d-galactoside After a 16-h
incubation at 37C with rotary shaking, the cells were
harvested by centrifugation at 8000 g for 10 min at 4C
and washed with 0.9% (w⁄ v) NaCl All the purification
procedures were performed at a temperature lower than
5C The buffer used throughout this purification was
Tris⁄ HCl buffer (pH 8.0) containing 5 mm
2-mercaptoeth-anol and 0.1 mm ethylenediaminetetraacetic acid Washed
cells from the 2.5-L culture were suspended in 100 mm
buffer and disrupted by sonication for 10 min (19 kHz;
Insonator model 201M; Kubota, Tokyo, Japan) For the
removal of intact cells and cell debris, the sonicate was
centrifuged at 15 000 g for 20 min at 4C To the cell-free
extract was added 5% protamine sulfate, at a
concentra-tion of 0.05 g of protamine sulfate to 1 g of protein After
stirring for 30 min, the precipitate formed was removed
by centrifugation at 15 000 g for 20 min at 4C The
resulting supernatant was fractionated with solid
ammo-nium sulfate The precipitate obtained at 20–40%
satura-tion was collected by centrifugasatura-tion and dissolved in
20 mm buffer The resulting enzyme solution was dialyzed
against 10 L of the same buffer for 24 h The dialyzed
solution was applied to a column (/1.6· 14 cm) of
DEAE-Toyopearl 650M previously equilibrated with
20 mm buffer After the column had been washed
thor-oughly with 20 mm buffer, the enzyme was eluted with
80 mL of 20 mm buffer containing 75 mm NaCl To the
active fractions was added ammonium sulfate to 30%
sat-uration The enzyme solution was applied to a column
(/1.6· 6.5 cm) of butyl-Toyopearl 650M previously
equil-ibrated with 20 mm buffer containing ammonium sulfate
to 30% saturation The active fractions were eluted with a
linear gradient of ammonium sulfate (30–0% saturation)
in 20 mm buffer The active fractions were combined and
dialyzed against 10 L of 20 mm buffer for 12 h The
enzyme solution was applied to a MonoQ HR 10⁄ 10
col-umn (Amersham Biosciences K.K) equilibrated previously
with 20 mm buffer After the column had been washed
with 30 mL of 20 mm buffer, the enzyme was eluted with
a linear gradient of NaCl (0–0.5 m) in 20 mm buffer using
the A¨kta-FPLC system (Amersham Biosciences K.K) The
active fractions were combined and dialyzed against 10 L
of 20 mm buffer for 12 h and used for characterization
N-terminal amino acid sequencing of the purified enzyme
was performed at APRO Life Science Institute, Inc
(Tokushima, Japan) with a Procise 494 HT protein
sequencing system
Enzyme assay
Activity of BapA was assayed routinely at 30C by the
formation of p-nitroaniline from d-Ala-pNA as follows
A reaction mixture (1.0 mL) containing 5 mm
d-Ala-pNA, 100 mm Tris⁄ HCl (pH 8.0), and the enzyme was monitored by the change in absorbance at 405 nm with
an Hitachi U-3210 spectrophotometer One unit of enzyme activity was defined as the amount catalyzing the formation of 1 lmol p-nitroaniline per minute from
d-Ala-pNA under the above conditions Protein was determined by the method of Bradford [27] with BSA as standard, using a kit from Bio-Rad Laboratories Ltd (Tokyo, Japan)
To investigate the pH profile of the enzyme stability and activity, the following buffers (final concentration, 100 mm) were used: acetic acid⁄ sodium acetate (pH 4.0–6.0), Mes ⁄ NaOH (pH 5.5–6.5), potassium phosphate (pH 6.5–8.5), Tris⁄ HCl (pH 7.5–9.0), ethanolamine ⁄ HCl (pH 9.0–11.0), and glycine⁄ NaCl ⁄ NaOH (pH 10.0–13.0)
The substrate specificity was examined qualitatively by thin-layer chromatography with a solvent system (1-buta-nol⁄ acetic acid ⁄ water, 4 : 1 : 1, v ⁄ v ⁄ v) first, and then quan-titatively assayed using an HPLC system as follows The reaction mixture (1 mL) contained 100 lmol Tris⁄ HCl buf-fer (pH 8.0), 20 lmol substrate and an appropriate amount
of the enzyme The reaction was performed at 30C for 5–30 min and stopped by the addition of 1 mL of ethanol The amount of product formed in the reaction mixture was determined with an HPLC apparatus equipped with a Sumichiral OA-5000 or OA-5000 L column (/0.46· 15 cm; Sumika Chemical Analysis Service, Osaka, Japan) at a flow rate of 1.0 mLÆmin)1, using 2 mm CuSO4 as a solvent system The absorbance of the eluate was monitored at
254 nm
Analytical measurements
To estimate the molecular mass of the enzyme, the sample (3 lg) was subjected to HPLC on a Superdex 200 HR10⁄ 30 column (Amersham Biosciences K.K) at a flow rate of 0.4 mLÆmin)1 with 20 mm potassium phosphate (pH 7.0) containing 150 mm NaCl at room temperature The absorb-ance of the eluate was monitored at 280 nm The molecular mass of the enzyme was then calculated based on relative mobility (retention time) using the standard proteins glu-tamate dehydrogenase (290 kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), adenylate kinase (32 kDa), and cytochrome c (12.4 kDa) (products of Oriental Yeast Co., Tokyo, Japan) SDS⁄ PAGE analysis was performed
by the method of Laemmli [28] Proteins were stained with Brilliant blue R-250 and destained in ethanol⁄ acetic acid ⁄ water (3 : 1 : 6, v⁄ v ⁄ v)
Acknowledgements
This work was partly supported by a Grant-in-aid for Scientific Research (16780059 to H.K.) from JSPS (Japan Society for the Promotion of Science)
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