Vol.
176,
No.
April
15,
1991
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
321-327
Pages
Nucleotide Hiroki
sequence
Ohnishi,
Tamotsu
Department Tohoku
Received
of pnl
of
March
from
Nishida, and Kazuo
Agricultural University,
1,
gene
Erwinia
Aruto Izaki
Chemistry, Amamiya-machi,
Er
mmtovora
Yoshida,
Yoshiyuki
Kamio
Faculty Sendai,
of Agriculture 981 Japan
1991
SUMMARY:
The nucleotide sequence of pnl gene encoding pectin lyase (PNL; EC4.Z.Z.lO)from Erwinia carotovora Er was determined. The structural gene of pnl consisted of 942 base pairs. An open reading frame that could encode a 33,700 dalton polypeptide consisting 314 amino acids was assigned. The molecular size of the polypeptide predicted from the amino acid composition was close to the value of PNL determined in E.carotovora Er. The nucleotide sequence of the 5’-flanking region showed the presence of the consensus sequence of ribosome binding site, Pribnow box and the RNA polymerase recognition site in E.carotovora and Escberichia coli. Between the presumed Pribnow box and the ribosome binding site, two pairs of inverted repeats were found. By comparing the predicted amino acid sequences of pnl, several reported bacterial pectate lyases and Aspergillus niger pectin lyase, short regions of homology were found despite the different 0 1991 Academic B-e**, Inc. substrate specificities of these enzymes.
Many
vora (PL; ing
Er EC the
produce ing
secrete
the
4.2.2.2)
which
soft-rot.
In
pectin agents
most
such
are
as
of
Exarotovora
a
We
have
and
expressed
(4).
In
the
the
response
mitomycin
study,
pnl
gene
by
In
PNL
the
addition,
using we and
pnl
gene
tic
promoter
determined its
321
flanking
enzymes
caus-
Exarotovora DNA-damag-
UV cell
in
In
lysis
recA
and in gene
E.carotovora
Er
Escherichia
coli
complete region
light(l).
production
functional from
the
lyase
to
C or
a
pectate
of
accompanied
requires
caroto-
the
in
carotovora cloned
of
4.2.2.10)
is
by
one
strains
(2).
gene
be
Erwinia as
some
acid,
bacteriocin
present of
EC
such
PL,
production
recently the
to
to
nalidixic
subsp.
(3).
thought
(PNL;
including
enzymes
addition
PNL
production
species
pectolytic
lyase
strains,
sequence
Erwinia
phytopathogenic
nucleotide of
Copyright f3 I991 All rights of reproduction
E.cartovora 0006-291X/91 $1.50 by Academic Press* Inc. in any form reserved.
Vol.
176,
No.
BIOCHEMICAL
1, 1991
Er to study chemical
the
mechanism
structure
AND
of the
strains
and
A(lac-proAL?), x, [F’,traD36, proAB,lacP
expression
plasm!&
endAl,
gyrA96,
Escherichia thi, hsdRl7,
and
gene
and
the
Ml3KO7 were for preparation
coli
JMlO9
irecAl,
relA1, supE44, was used as a host strain for pUCll8 and pUCll9 were used as {ara,A (lac-pro), ), [F’,traD36,
A(.srl-recA)306::Tn10(tetr
respectively
of pnl
COMMUNICATIONS
AND METHODS
Bm5]} recombinant plasmids. Plasmid cloning vectors. E.coli MVll84 lac,ZWfl5), Akfls]},
RESEARCH
of PNL. MATERIALS
Hacterial
BIOPHYSICAL
strA, proA&
thi, (@80 la& Z-
used as a host and a helper of single stranded DNA.
phage,
DNA sequencing. The 2.1 kb of StuI-EcoRI fragment of pTN2159 (4) was subcloned into Smal site of pUCll9 and pUCl18 after EcoRI site of the fragment was treated with Sl nuclease. A series of deletion derivatives of each subclones were obtained by exonuclease III and mung bean nuclease digestion, according to the procedures described by Henikoff (5). DNA sequencing was performed by the dideoxy chain termination method of Sanger et al.
(61. RESULTS Nucleotide-ence
AND DISCUSSION
of the*@
gene
fragment
in pTN2159
contains
We have
determined
the
nucleotide
pd
gene
which
contained
the
following
the
sequencing
sequence
of
the
the
was
its
this
sequence,
frame
which
begins
with
an
ATG
codon
at
position
and
sequence
w EcoRI
with
Physical fragment.
TAA
map
we
of
can
1,286 identify
codon
strategy
the
bp
in
regions
1. The
nucleotide
as shown open
in
reading 290
amino
1286
(4).
fragment
position
The
of
the
an at
1232.
Er
3’-flanking
in Fig.
comprising
EcoRI-StuI
E.cartovora
S’- and
shown
2. Within
The
of
sequence
Fig.
terminates
gene
pnl
and
strategy
fragment
from~~~NZl59.
acid
bp
and se-
StuI-
Lines and arrows indicate the direction and extent of a portion of DNA fragment of which the nucleotide sequence have been cIetermined and are aligned in the 5’ to 3’ direction. Restriction sites: D, DraI; E, EcoRV; P, PvaII. 322
Vol.
176,
No.
1,
1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
-70 80 90 100 110 GGTAAAAGGCTGGTGATGATAATCGTAGCQCTGCCATTTTACTAAAAQATGG~TAT
190 200 210 230 220 ATGAAAT~TTTCTATACAATTTTTAATTGTC~A~GTATTATTT~OTCTCAATTAAA
COMMUNICATIONS
120
240 -
260 260 270 280 290 300 TAATA~CT~AAGACATTATTATTCACTCATTGTAAA~AAACTTAT~CTTAT~ MetAlaTyrF’ro 310 320 330 340 350 AACAACAAATCTTACTGG~TTATT~TT~~AAAAQ~AAAAG~A~GGA~AAC ThrThrA~nLeuThrGlyLeu~leQlyPheAl~Ly~Al~AlaLy~ValThrGl~l~h~
360
370 380 390 400 ~TAAAQT~~~~TAAATTC~T~h~AAATCA~GA~~C Gl~lyLy~VelV~lThrValA~nSerLe~l~A~~PheLY~SerAl~V~lS~rGlYSe~
410
420
430 440 460 460 C~AAAAACTA~OT~~hTCAT~TQA~~~~~QA~hA~T~T Al~LY~ThrIl~V~lV~lLeuQlySer3erLe~y~ThrS~rAl~L~uThrLY~V~lVal
470
480
490 600 610 520 AT~~A~AATAAAA~ATffiT~TC~T~T~TAATQTA~QACCAATAT FheQlyS6rA~nLy~ThrlleV~lGlySerPheG~~IyAl~AanValLeuThrAsnIle
530
540
560 560 670 680 590 TCACCTG~T~TGAQA~AATTCATCTAA~TCATTTT~AGAATCT~TTTTTAAACA HlsLeuAr~AlaG~USerA~nSerSerA~nValIlePheQlnA~nLeuValPheLY6HiS
000
610 620 630 640 660 TGATGTT~ATCAAAGATAATGACGATATCCAGCTGTATTTGAA~A~GTAAAGQGTA A~~V~~A~~I~~L~~A~~A~~A~~A~P~~~Q~~L~~TY~L~~~~TY~G~YLYSG~YTY~
660
670 680 690 700 710 TTGGGTGGATCACTGTTCATGGCCTGGGCATA~T~AG~ATAACGATGGTAGCCT~A TrRVelA~pHi~CySSerTrpProGlyHlsThrTrpSerA~pAsnA~pGlySerLeUAsp
720
730 740 760 760 TAAACTGA~TATAT~AGAAG~ATTACATCA~ATCAGTAACT~TTATTCTC Ly~LeulleTyrII~GlyGluLy~~laA8pTyrlleThrIleSerA~nCy~LeuPheSer
760
770
790 800 810 820 8SO AAACCATAAAThTGGTTGCATTTTCGGCChTCCQQCTQhCJ3hTAAThACAGCUCTTACAA A~nHl~Ly~TyrQl~y~IlePheQlyHl~PraAlaA~pA~pA~nAanSerAlaTyrAan
840
850 860 670 880 890 TGGCThTCCACGTTTl3ACTATTTGU!ATAACTATTACGAAAAThTTCAGGTGCGTGCGCC GlYTy~P~oA~gLe~Th~IleCy~H~~AsnTy~TyrQl~A~nIleOlnValA~gAlaP~o
900
910 920 930 940 960 CGGCCTGAT~GTTATGGCTATTTCCACGTATTCAAThACTACGTCAATAAATTCCAGTT GlYLeU~etArsTyrGl~yrPheHlsV~lPheAsnA~nTyrV~lA~nLy~PheGlnLeu
960
970 980 990 1000 1010 GGCTTTTAC~TCGCGCAAAATGCCAACGTTATTTCTGAACGCAACGTATTTGGCAGCGG AlEPheThrValAlaGlnA~nAlaAsnV~llleSerGluAr~AsnValPheGlySerGly
1020
1030 1040 1060 1060 1070 TGCTGAAAAGAAAGGQAT~TTGATGATAAAGGCAATGGTTCAACCTTTA~GATAATGG Al~GluLy~LysGly~etValA~pAspLy~GlyAs~GlYS~~~~h~PheTh~A6PA6nGly
1080
1090 1100 1110 1120 1130 CAGTTCGCCA~AG~TA~GAGTAAATCG~AGC~GAAATGGA-ATCATCTAA SerSerProAl~~laValAlaSerLy~SerPr~AlaAlaLyaTrpThrAlaSerSerA6n
1140
1150 1160 1170 1160 1x90 CTATTC~TACAGTTTGATGACAACCG~~GCCCAATCCT~GTTGTTTCGAATGCAGG TyrSerTyrSerLeuNetThrThrAlaA~~Al~GlnSerTrpValVelSe~ASnAl~GlY
1200
1210 1220 1230 1240 1260 GGCACAAAATAGT~GCTGAAATTCCCATCATAATCAC~AAGGTTTATTTTATGACGTA AlaGlnAsnSerAlaLeuLysPhePi-aSem**
1260
1270 1260 1290 CCTGCATGCTCACTTTTATTGATAATTGAAA
u Nucleotide sequence of the StuI-EcoRI deduced amino acid sequence of pectin lyase. arranged so that bp 6 is the first nucleotide site for StuI. The strand shown is the 5’ to deduced amino acid sequence is given bellow nucleotide sequence. The predicted ribosomal promoter site are indicated by double underlines boxes, respectively. Arrows represent inverted
323
fragment and the The sequence is in the recognition 3’ direction. The the corresponding binding site and and rectangular repeats.
Vol.
176,
No.
quence a
BIOCHEMICAL
1, 1991
of
PNL
deduced
polypeptide
with
comprising
314
determined
by
deduced the
the
for
methods
The
analysis
revealed
Codon
following
and
GAT
AGG
is
Ile
of
15
all
in
in
the
characteristics in
PLI
Asp
18
gene.
protein, the
and
in
PLIII
PNL. and
Lys
bias residues
Neither
addition,
none
of
AGA
nor
although them
is
(10,ll)
(9).
strong
21
(b)
usage
the
computed,
of
In
codon
to
the
out
was
hydropathy
was
residues.
the
in
used
by
indica-
PNL
and
concerning
used
no
15
used
very
codon
similar
except
in
to
the
case
Asp. Amino
acid
fyngal
PLa
homology
-Fy--L-. The
sequence
of
(12)
and
determined.
the (15). our regions
nucleotide
the
PLI
PLb
(13,14)
niger,
sequence
and
Amino PNL
acid and
of relatively
the
among
and
for
the
A.niger
homology short,
et a
al.
PLD, among they
of
them.
the D
acid
in
well.
324
were
pelD
from
(PLD)
1, the
gene and
reported
of
the
lyase
revealed
be
gene (PL), several
homologous
should
and
carotovora
sequence
Table
It
acid Er
pectate
Although
aligned
amino
subsp.
bacterial
shown
and
E.carotovora
lyase
amino between
deduced
cloned
pectin
predicted
homology
(11)
Ecarotovora
Gysler coding
and
PLIII
from
Erwinia species
PNL, PL from sequence
(10)
Recently,
Aspergillus
are
at
43.0%
pnl gene
the
with
Vanderkooi
segment
made
is
13 out
present
codon
for
AAA
Arg
are
of
usage
that
according
hydrophobic
were
by
for
residues
those
codon
with
of
and
was
kDa,
residues
was
sequence
Capaldi
PNL
significant
Codon
used
used
These
of
(a)
is
ATA.
of
33.7
acid
There
polarity
and
to
corresponded
(4).
The
(8)
amino
exactly PNL
of
coincides 18
overall
Doolittle
weight
first
N-terminus.
observations
usage.
The
COMMUNICATIONS
corresponds
which
purified
polarity
The
molecular
and
no
usage.
the
the
and
respectively.
in
of
RESEARCH
sequence
sequence
of
Kyte
DNA
(4,7).
hydropathy
of
the
residues,
nucleotide
processing
analysed
the
acid
ones
BIOPHYSICAL
apparent
SDS-PAGE
N-terminal of
an
amino
from
tion
from
AND
regions noted
that
Vol.
176,
No.
BIOCHEMICAL
1, 1991
Table 1. carwtovox~
Comparison Er PNL,
Protein
a
of deduced Aspergillus pectate Region
E.s.Er
AND
b
BIOPHYSICAL
RESEARCH
amino acid sequences niger PLD and Erwinia lya3ez.3
sequence
COMMUNICATIONS
of E. species
c
PNL
A.*
PLO
E.G.
Er PLI
E.G.
EC Ph
&e.EC
PLb
&&.Er &=.Er c.m.
Er
E.=.EC F.e.EC 5.e.
SCRIl93 PLb
~The deduced amino acid sequence of the E.carotovora Er(E.carot. Er) PNL, of the A.niger pectin lyase D (PLD), of the E.carotovom Er pectate lyases PL I and III, of the E.carotovora EC (E.carot. EC)pectate lyases PL a and b, and of E.carotovora SCRI193 (E.carot.SCRIl93) pectate lyase PLb are compared. b Regions (I-VIII) with putative amino acid similarities are indicated, starting with the comparison from the N-terminal of the protein. The position of the first amino acid residue of a region in each compared sequence is indicated between brackets. identical residues at corresponding posic Within region I-VIII, tions in the compared sequences are boxed. 325
Vol.
176,
No.
BIOCHEMICAL
1, 1991
the
similar
(amino the
motif
acid case
The
the
IV
nucleotide
from
the
codon
sequence
pnl
4 out
of
6 bases
the
Pribnow
(18,19).
The
sequences
III
(10,
11).
We
probable
presence
pnl
translational
function) 12
No
bp
existed regulation
the
the
of
pnl
The
regulation
inverted
and
the
site
transcription
pnl
transcription
repeat
which
is
sequence
was
found
codon
the
upstream
sequenced.
326
with
the (17).
pel
I and
was
not
Lex
were
151
A
pnl
binding perfect
and
might
9
170
respectively.
of
of
the
two
bp, The
be an
related unknown
sequences
currently
being of
stop
the site
palindromic
the
and
site
of a
characteristic
between
pelB
promoter
binding
is
TTG
consen-
gene
the
site,
these
se-
suggesting
which
by of
the
of
within
promoter
significance of
last
of
the
a
in
pnl
there
StuI
the
sequence
those
resembling
from
to
contains
pTN2159,
present
bp
former
homology
to
bp
-11
from
recognition
290
sequences
-10
5/7
although
243
The
with
near
to
TATTGAA
bp
latter
that
sequence
found,
and
in
it.
termination gene
was
,212
to
no
palindromic
between
protein in
site,
and
sequence
site
whithin
-7
consensus
resemble
A binding
However,
at
-161
homology
harboring
Lex
the
polymerase also
in
A
correspond
to
the
observed
JMlO9
start
to
shows
RNA
previously
of
gene.
(SOS
the here
E.coli
in
-155
promoter
also
PLD)
regio+
TTTTAT
at
same
of
sequence at
may
while
the
in
found
respectively.
(17),
spacing
145-160
was
homology
site
presented
expressed
to
-35
TGTTGAC
The
former
the
similar
3’-flankins
sequences and
COMMUNICATIONS
PLD.
which
gene,
exactly
and
and
pnl,
bp
box
exhibits of
consensus
-136
PNL, and
The
the
sequence
and
(16).
with
(AAGGA)
the
of
and
and
of
codon
in
pelC
site
to
TATAAT
bp
&ht.-5’-
-131
shares
site
of
at
quence
the
sequence
sequence
initiation
sus
PNL
initiation
found
in
the
binding
Shine-Dalgarno were
86-101
among
ribosome
RESEARCH
occurred
position
region
presumed
BIOPHYSICAL
LR--S--SNVIFQNLV
at
of
AND
codon
tested.
transcription of
the
pnl
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
We are grateful to Dr.E.Nakano and Mr.Y.Harada of Kikkoman Corporation for their kind help for the DNA sequence. We are also expressed our acknowlegmentto Mr.A.Rahman -in our laboratory for his critical reading of the manuscript.
Acknowledgments:
REFERENCES
1. Tomizawa, S. and Takahashi, H. (1971) Agric. Biol. Chem. 35, 191-200. 2. Itoh, Y., Izaki, K. and Takahashi, H. (1980) Agric. Biol. Chem. 44, 1135-1140. 3. Zink, R. T., Engwall, J. K., McEvoy, J. L. and Chatterjee, A. K. (1985) J. Bacterial. 164, 390-396. 4. Nishida, T., Suzuki, T., Ito, K., Kamio, Y. and Izaki, K.(l990) Biochem. Biophys. Res. Commun. 168, 801-808. 5. Henikoff, S. (1984) Gene 28, 351-359. 6. Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Froc. Natl. Acad. Sci. U.S.A. 74, 5463-5467. 7. Kamimiya, S., Nishiya, T., Izaki. K. and Takahashi. H. (1974) Agric. Biol. Chem. 38, 1071-1078. F. R. (1982) J. Mol. Biol. 157, 1058. Kyte, ,J. and Doolittle, 132. 9. Capaldi, A. R. and Vanderkooi, G. (1972) Proc. Nat]. Acad. Sci, l!.S.A. 69, 930-932. 10. Ito, K., Kobayashi, R., Nikaido, N. and Izaki, K.(l988) Agric. Biol. Chem. 52, 479-487. 11. Yoshida, A., Izuta, M., Ito, K., Kamio, Y. and Izaki, K. (1991) Agric. Biol. Chem. 55, in press. 1.2. Lei, S. -P., Lin, H. -C., Wang, S. -S. and Wilcox, G. (1988) Gene 62, 159-164. 13. Lei, S. -P., Lin, H. -C., Wang, S. -S., Callaway, J. and Wilcox, G. (1987) J.BacterioI. (1987) 169, 4379-4383. 14. Hinton, J. C. D., Sidebotham, ,J. M., Gill, D. R. and Salmond, G. P. C. (1989) Mol. Microbial. 3, 1785-1795. 15. Gysler, C., Harmsen, J. A. M., Kester, H. C. M., Visser, J. and Heim, J. (1990) Gene 89, 101-108. 16. Shine, .J. and Dalgarno, L. (1975) Nature 254, 34-38. 17. Rosenberg, M. and Court, D. (1979) Ann. Rev. Genetics 13, 319-353. 18. Tamaki, S. J., Gold, S., Robeson, M., Manulis, S. and Keen, N. T. (1988) J. Bacterial. 170, 3468-3478. 19. Keen, N. T. and Tamaki, S. J. (1986) *J. Bacterial. 168, 595606.
327
176,
No.
April
15,
1991
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
321-327
Pages
Nucleotide Hiroki
sequence
Ohnishi,
Tamotsu
Department Tohoku
Received
of pnl
of
March
from
Nishida, and Kazuo
Agricultural University,
1,
gene
Erwinia
Aruto Izaki
Chemistry, Amamiya-machi,
Er
mmtovora
Yoshida,
Yoshiyuki
Kamio
Faculty Sendai,
of Agriculture 981 Japan
1991
SUMMARY:
The nucleotide sequence of pnl gene encoding pectin lyase (PNL; EC4.Z.Z.lO)from Erwinia carotovora Er was determined. The structural gene of pnl consisted of 942 base pairs. An open reading frame that could encode a 33,700 dalton polypeptide consisting 314 amino acids was assigned. The molecular size of the polypeptide predicted from the amino acid composition was close to the value of PNL determined in E.carotovora Er. The nucleotide sequence of the 5’-flanking region showed the presence of the consensus sequence of ribosome binding site, Pribnow box and the RNA polymerase recognition site in E.carotovora and Escberichia coli. Between the presumed Pribnow box and the ribosome binding site, two pairs of inverted repeats were found. By comparing the predicted amino acid sequences of pnl, several reported bacterial pectate lyases and Aspergillus niger pectin lyase, short regions of homology were found despite the different 0 1991 Academic B-e**, Inc. substrate specificities of these enzymes.
Many
vora (PL; ing
Er EC the
produce ing
secrete
the
4.2.2.2)
which
soft-rot.
In
pectin agents
most
such
are
as
of
Exarotovora
a
We
have
and
expressed
(4).
In
the
the
response
mitomycin
study,
pnl
gene
by
In
PNL
the
addition,
using we and
pnl
gene
tic
promoter
determined its
321
flanking
enzymes
caus-
Exarotovora DNA-damag-
UV cell
in
In
lysis
recA
and in gene
E.carotovora
Er
Escherichia
coli
complete region
light(l).
production
functional from
the
lyase
to
C or
a
pectate
of
accompanied
requires
caroto-
the
in
carotovora cloned
of
4.2.2.10)
is
by
one
strains
(2).
gene
be
Erwinia as
some
acid,
bacteriocin
present of
EC
such
PL,
production
recently the
to
to
nalidixic
subsp.
(3).
thought
(PNL;
including
enzymes
addition
PNL
production
species
pectolytic
lyase
strains,
sequence
Erwinia
phytopathogenic
nucleotide of
Copyright f3 I991 All rights of reproduction
E.cartovora 0006-291X/91 $1.50 by Academic Press* Inc. in any form reserved.
Vol.
176,
No.
BIOCHEMICAL
1, 1991
Er to study chemical
the
mechanism
structure
AND
of the
strains
and
A(lac-proAL?), x, [F’,traD36, proAB,lacP
expression
plasm!&
endAl,
gyrA96,
Escherichia thi, hsdRl7,
and
gene
and
the
Ml3KO7 were for preparation
coli
JMlO9
irecAl,
relA1, supE44, was used as a host strain for pUCll8 and pUCll9 were used as {ara,A (lac-pro), ), [F’,traD36,
A(.srl-recA)306::Tn10(tetr
respectively
of pnl
COMMUNICATIONS
AND METHODS
Bm5]} recombinant plasmids. Plasmid cloning vectors. E.coli MVll84 lac,ZWfl5), Akfls]},
RESEARCH
of PNL. MATERIALS
Hacterial
BIOPHYSICAL
strA, proA&
thi, (@80 la& Z-
used as a host and a helper of single stranded DNA.
phage,
DNA sequencing. The 2.1 kb of StuI-EcoRI fragment of pTN2159 (4) was subcloned into Smal site of pUCll9 and pUCl18 after EcoRI site of the fragment was treated with Sl nuclease. A series of deletion derivatives of each subclones were obtained by exonuclease III and mung bean nuclease digestion, according to the procedures described by Henikoff (5). DNA sequencing was performed by the dideoxy chain termination method of Sanger et al.
(61. RESULTS Nucleotide-ence
AND DISCUSSION
of the*@
gene
fragment
in pTN2159
contains
We have
determined
the
nucleotide
pd
gene
which
contained
the
following
the
sequencing
sequence
of
the
the
was
its
this
sequence,
frame
which
begins
with
an
ATG
codon
at
position
and
sequence
w EcoRI
with
Physical fragment.
TAA
map
we
of
can
1,286 identify
codon
strategy
the
bp
in
regions
1. The
nucleotide
as shown open
in
reading 290
amino
1286
(4).
fragment
position
The
of
the
an at
1232.
Er
3’-flanking
in Fig.
comprising
EcoRI-StuI
E.cartovora
S’- and
shown
2. Within
The
of
sequence
Fig.
terminates
gene
pnl
and
strategy
fragment
from~~~NZl59.
acid
bp
and se-
StuI-
Lines and arrows indicate the direction and extent of a portion of DNA fragment of which the nucleotide sequence have been cIetermined and are aligned in the 5’ to 3’ direction. Restriction sites: D, DraI; E, EcoRV; P, PvaII. 322
Vol.
176,
No.
1,
1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
-70 80 90 100 110 GGTAAAAGGCTGGTGATGATAATCGTAGCQCTGCCATTTTACTAAAAQATGG~TAT
190 200 210 230 220 ATGAAAT~TTTCTATACAATTTTTAATTGTC~A~GTATTATTT~OTCTCAATTAAA
COMMUNICATIONS
120
240 -
260 260 270 280 290 300 TAATA~CT~AAGACATTATTATTCACTCATTGTAAA~AAACTTAT~CTTAT~ MetAlaTyrF’ro 310 320 330 340 350 AACAACAAATCTTACTGG~TTATT~TT~~AAAAQ~AAAAG~A~GGA~AAC ThrThrA~nLeuThrGlyLeu~leQlyPheAl~Ly~Al~AlaLy~ValThrGl~l~h~
360
370 380 390 400 ~TAAAQT~~~~TAAATTC~T~h~AAATCA~GA~~C Gl~lyLy~VelV~lThrValA~nSerLe~l~A~~PheLY~SerAl~V~lS~rGlYSe~
410
420
430 440 460 460 C~AAAAACTA~OT~~hTCAT~TQA~~~~~QA~hA~T~T Al~LY~ThrIl~V~lV~lLeuQlySer3erLe~y~ThrS~rAl~L~uThrLY~V~lVal
470
480
490 600 610 520 AT~~A~AATAAAA~ATffiT~TC~T~T~TAATQTA~QACCAATAT FheQlyS6rA~nLy~ThrlleV~lGlySerPheG~~IyAl~AanValLeuThrAsnIle
530
540
560 560 670 680 590 TCACCTG~T~TGAQA~AATTCATCTAA~TCATTTT~AGAATCT~TTTTTAAACA HlsLeuAr~AlaG~USerA~nSerSerA~nValIlePheQlnA~nLeuValPheLY6HiS
000
610 620 630 640 660 TGATGTT~ATCAAAGATAATGACGATATCCAGCTGTATTTGAA~A~GTAAAGQGTA A~~V~~A~~I~~L~~A~~A~~A~~A~P~~~Q~~L~~TY~L~~~~TY~G~YLYSG~YTY~
660
670 680 690 700 710 TTGGGTGGATCACTGTTCATGGCCTGGGCATA~T~AG~ATAACGATGGTAGCCT~A TrRVelA~pHi~CySSerTrpProGlyHlsThrTrpSerA~pAsnA~pGlySerLeUAsp
720
730 740 760 760 TAAACTGA~TATAT~AGAAG~ATTACATCA~ATCAGTAACT~TTATTCTC Ly~LeulleTyrII~GlyGluLy~~laA8pTyrlleThrIleSerA~nCy~LeuPheSer
760
770
790 800 810 820 8SO AAACCATAAAThTGGTTGCATTTTCGGCChTCCQQCTQhCJ3hTAAThACAGCUCTTACAA A~nHl~Ly~TyrQl~y~IlePheQlyHl~PraAlaA~pA~pA~nAanSerAlaTyrAan
840
850 860 670 880 890 TGGCThTCCACGTTTl3ACTATTTGU!ATAACTATTACGAAAAThTTCAGGTGCGTGCGCC GlYTy~P~oA~gLe~Th~IleCy~H~~AsnTy~TyrQl~A~nIleOlnValA~gAlaP~o
900
910 920 930 940 960 CGGCCTGAT~GTTATGGCTATTTCCACGTATTCAAThACTACGTCAATAAATTCCAGTT GlYLeU~etArsTyrGl~yrPheHlsV~lPheAsnA~nTyrV~lA~nLy~PheGlnLeu
960
970 980 990 1000 1010 GGCTTTTAC~TCGCGCAAAATGCCAACGTTATTTCTGAACGCAACGTATTTGGCAGCGG AlEPheThrValAlaGlnA~nAlaAsnV~llleSerGluAr~AsnValPheGlySerGly
1020
1030 1040 1060 1060 1070 TGCTGAAAAGAAAGGQAT~TTGATGATAAAGGCAATGGTTCAACCTTTA~GATAATGG Al~GluLy~LysGly~etValA~pAspLy~GlyAs~GlYS~~~~h~PheTh~A6PA6nGly
1080
1090 1100 1110 1120 1130 CAGTTCGCCA~AG~TA~GAGTAAATCG~AGC~GAAATGGA-ATCATCTAA SerSerProAl~~laValAlaSerLy~SerPr~AlaAlaLyaTrpThrAlaSerSerA6n
1140
1150 1160 1170 1160 1x90 CTATTC~TACAGTTTGATGACAACCG~~GCCCAATCCT~GTTGTTTCGAATGCAGG TyrSerTyrSerLeuNetThrThrAlaA~~Al~GlnSerTrpValVelSe~ASnAl~GlY
1200
1210 1220 1230 1240 1260 GGCACAAAATAGT~GCTGAAATTCCCATCATAATCAC~AAGGTTTATTTTATGACGTA AlaGlnAsnSerAlaLeuLysPhePi-aSem**
1260
1270 1260 1290 CCTGCATGCTCACTTTTATTGATAATTGAAA
u Nucleotide sequence of the StuI-EcoRI deduced amino acid sequence of pectin lyase. arranged so that bp 6 is the first nucleotide site for StuI. The strand shown is the 5’ to deduced amino acid sequence is given bellow nucleotide sequence. The predicted ribosomal promoter site are indicated by double underlines boxes, respectively. Arrows represent inverted
323
fragment and the The sequence is in the recognition 3’ direction. The the corresponding binding site and and rectangular repeats.
Vol.
176,
No.
quence a
BIOCHEMICAL
1, 1991
of
PNL
deduced
polypeptide
with
comprising
314
determined
by
deduced the
the
for
methods
The
analysis
revealed
Codon
following
and
GAT
AGG
is
Ile
of
15
all
in
in
the
characteristics in
PLI
Asp
18
gene.
protein, the
and
in
PLIII
PNL. and
Lys
bias residues
Neither
addition,
none
of
AGA
nor
although them
is
(10,ll)
(9).
strong
21
(b)
usage
the
computed,
of
In
codon
to
the
out
was
hydropathy
was
residues.
the
in
used
by
indica-
PNL
and
concerning
used
no
15
used
very
codon
similar
except
in
to
the
case
Asp. Amino
acid
fyngal
PLa
homology
-Fy--L-. The
sequence
of
(12)
and
determined.
the (15). our regions
nucleotide
the
PLI
PLb
(13,14)
niger,
sequence
and
Amino PNL
acid and
of relatively
the
among
and
for
the
A.niger
homology short,
et a
al.
PLD, among they
of
them.
the D
acid
in
well.
324
were
pelD
from
(PLD)
1, the
gene and
reported
of
the
lyase
revealed
be
gene (PL), several
homologous
should
and
carotovora
sequence
Table
It
acid Er
pectate
Although
aligned
amino
subsp.
bacterial
shown
and
E.carotovora
lyase
amino between
deduced
cloned
pectin
predicted
homology
(11)
Ecarotovora
Gysler coding
and
PLIII
from
Erwinia species
PNL, PL from sequence
(10)
Recently,
Aspergillus
are
at
43.0%
pnl gene
the
with
Vanderkooi
segment
made
is
13 out
present
codon
for
AAA
Arg
are
of
usage
that
according
hydrophobic
were
by
for
residues
those
codon
with
of
and
was
kDa,
residues
was
sequence
Capaldi
PNL
significant
Codon
used
used
These
of
(a)
is
ATA.
of
33.7
acid
There
polarity
and
to
corresponded
(4).
The
(8)
amino
exactly PNL
of
coincides 18
overall
Doolittle
weight
first
N-terminus.
observations
usage.
The
COMMUNICATIONS
corresponds
which
purified
polarity
The
molecular
and
no
usage.
the
the
and
respectively.
in
of
RESEARCH
sequence
sequence
of
Kyte
DNA
(4,7).
hydropathy
of
the
residues,
nucleotide
processing
analysed
the
acid
ones
BIOPHYSICAL
apparent
SDS-PAGE
N-terminal of
an
amino
from
tion
from
AND
regions noted
that
Vol.
176,
No.
BIOCHEMICAL
1, 1991
Table 1. carwtovox~
Comparison Er PNL,
Protein
a
of deduced Aspergillus pectate Region
E.s.Er
AND
b
BIOPHYSICAL
RESEARCH
amino acid sequences niger PLD and Erwinia lya3ez.3
sequence
COMMUNICATIONS
of E. species
c
PNL
A.*
PLO
E.G.
Er PLI
E.G.
EC Ph
&e.EC
PLb
&&.Er &=.Er c.m.
Er
E.=.EC F.e.EC 5.e.
SCRIl93 PLb
~The deduced amino acid sequence of the E.carotovora Er(E.carot. Er) PNL, of the A.niger pectin lyase D (PLD), of the E.carotovom Er pectate lyases PL I and III, of the E.carotovora EC (E.carot. EC)pectate lyases PL a and b, and of E.carotovora SCRI193 (E.carot.SCRIl93) pectate lyase PLb are compared. b Regions (I-VIII) with putative amino acid similarities are indicated, starting with the comparison from the N-terminal of the protein. The position of the first amino acid residue of a region in each compared sequence is indicated between brackets. identical residues at corresponding posic Within region I-VIII, tions in the compared sequences are boxed. 325
Vol.
176,
No.
BIOCHEMICAL
1, 1991
the
similar
(amino the
motif
acid case
The
the
IV
nucleotide
from
the
codon
sequence
pnl
4 out
of
6 bases
the
Pribnow
(18,19).
The
sequences
III
(10,
11).
We
probable
presence
pnl
translational
function) 12
No
bp
existed regulation
the
the
of
pnl
The
regulation
inverted
and
the
site
transcription
pnl
transcription
repeat
which
is
sequence
was
found
codon
the
upstream
sequenced.
326
with
the (17).
pel
I and
was
not
Lex
were
151
A
pnl
binding perfect
and
might
9
170
respectively.
of
of
the
two
bp, The
be an
related unknown
sequences
currently
being of
stop
the site
palindromic
the
and
site
of a
characteristic
between
pelB
promoter
binding
is
TTG
consen-
gene
the
site,
these
se-
suggesting
which
by of
the
of
within
promoter
significance of
last
of
the
a
in
pnl
there
StuI
the
sequence
those
resembling
from
to
contains
pTN2159,
present
bp
former
homology
to
bp
-11
from
recognition
290
sequences
-10
5/7
although
243
The
with
near
to
TATTGAA
bp
latter
that
sequence
found,
and
in
it.
termination gene
was
,212
to
no
palindromic
between
protein in
site,
and
sequence
site
whithin
-7
consensus
resemble
A binding
However,
at
-161
homology
harboring
Lex
the
polymerase also
in
A
correspond
to
the
observed
JMlO9
start
to
shows
RNA
previously
of
gene.
(SOS
the here
E.coli
in
-155
promoter
also
PLD)
regio+
TTTTAT
at
same
of
sequence at
may
while
the
in
found
respectively.
(17),
spacing
145-160
was
homology
site
presented
expressed
to
-35
TGTTGAC
The
former
the
similar
3’-flankins
sequences and
COMMUNICATIONS
PLD.
which
gene,
exactly
and
and
pnl,
bp
box
exhibits of
consensus
-136
PNL, and
The
the
sequence
and
(16).
with
(AAGGA)
the
of
and
and
of
codon
in
pelC
site
to
TATAAT
bp
&ht.-5’-
-131
shares
site
of
at
quence
the
sequence
sequence
initiation
sus
PNL
initiation
found
in
the
binding
Shine-Dalgarno were
86-101
among
ribosome
RESEARCH
occurred
position
region
presumed
BIOPHYSICAL
LR--S--SNVIFQNLV
at
of
AND
codon
tested.
transcription of
the
pnl
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
We are grateful to Dr.E.Nakano and Mr.Y.Harada of Kikkoman Corporation for their kind help for the DNA sequence. We are also expressed our acknowlegmentto Mr.A.Rahman -in our laboratory for his critical reading of the manuscript.
Acknowledgments:
REFERENCES
1. Tomizawa, S. and Takahashi, H. (1971) Agric. Biol. Chem. 35, 191-200. 2. Itoh, Y., Izaki, K. and Takahashi, H. (1980) Agric. Biol. Chem. 44, 1135-1140. 3. Zink, R. T., Engwall, J. K., McEvoy, J. L. and Chatterjee, A. K. (1985) J. Bacterial. 164, 390-396. 4. Nishida, T., Suzuki, T., Ito, K., Kamio, Y. and Izaki, K.(l990) Biochem. Biophys. Res. Commun. 168, 801-808. 5. Henikoff, S. (1984) Gene 28, 351-359. 6. Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Froc. Natl. Acad. Sci. U.S.A. 74, 5463-5467. 7. Kamimiya, S., Nishiya, T., Izaki. K. and Takahashi. H. (1974) Agric. Biol. Chem. 38, 1071-1078. F. R. (1982) J. Mol. Biol. 157, 1058. Kyte, ,J. and Doolittle, 132. 9. Capaldi, A. R. and Vanderkooi, G. (1972) Proc. Nat]. Acad. Sci, l!.S.A. 69, 930-932. 10. Ito, K., Kobayashi, R., Nikaido, N. and Izaki, K.(l988) Agric. Biol. Chem. 52, 479-487. 11. Yoshida, A., Izuta, M., Ito, K., Kamio, Y. and Izaki, K. (1991) Agric. Biol. Chem. 55, in press. 1.2. Lei, S. -P., Lin, H. -C., Wang, S. -S. and Wilcox, G. (1988) Gene 62, 159-164. 13. Lei, S. -P., Lin, H. -C., Wang, S. -S., Callaway, J. and Wilcox, G. (1987) J.BacterioI. (1987) 169, 4379-4383. 14. Hinton, J. C. D., Sidebotham, ,J. M., Gill, D. R. and Salmond, G. P. C. (1989) Mol. Microbial. 3, 1785-1795. 15. Gysler, C., Harmsen, J. A. M., Kester, H. C. M., Visser, J. and Heim, J. (1990) Gene 89, 101-108. 16. Shine, .J. and Dalgarno, L. (1975) Nature 254, 34-38. 17. Rosenberg, M. and Court, D. (1979) Ann. Rev. Genetics 13, 319-353. 18. Tamaki, S. J., Gold, S., Robeson, M., Manulis, S. and Keen, N. T. (1988) J. Bacterial. 170, 3468-3478. 19. Keen, N. T. and Tamaki, S. J. (1986) *J. Bacterial. 168, 595606.
327
