Download Analysis of the Nitrous Oxide Reduction Genes, nosZDFYL, of

DNA
RESEARCH
5, 365-371 (1998)
Short Communication
Analysis of the Nitrous Oxide Reduction Genes,
of Achromobacter cycloclastes
nosZDFYL,
Ken-ichi INATOMI*
Advanced Technology R & D Center, Mitsubishi Electric Corp., 811, Tsukaguchi, Amagasaki,
Hyogo 661-0001, Japan
(Received 22 July 1998; revised 12 October 1998)
Abstract
The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from
the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid
residues and the deduced amino acid sequence showed homology to the previously derived sequences for the
dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist
of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The
gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and
the nosYgene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting
the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly
hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues
and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The
expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the
nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.
Key words: multi copper protein; N2O reductase; nosZ; nosD; nosF; nosY; nosL; signal peptide;
lipoprotein
1.
Introduction
Nitrous oxide reductase is one of the enzymes involved
in bacterial denitrification. The reductase catalyzes the
reduction of N2O to N2, which is the final step in the denitrification process.1 N2O reductase is a homodimeric or
monomeric multi-copper enzyme located in the periplasmic space. The reductase has two distinct copper-binding
sites of Cu A and Cu z , and they are assigned to an electron transfer and a catalytic sites, respectively.2 The
dimeric N2O reductases have been isolated and their
structural gene (nosZ) and relevant genes have been
cloned and sequenced from Pseudomonas stutzeri3'4'5
Paracoccus denitrificans,6 Rhizobium meliloti7 and Alcaligenes eutrophus.3 The dimeric N2O reductases typically exist in either a high-active form I (violet form) or
a less active form II (pink form).8 These forms are obtained from an anaerobic or aerobic purification procedure, respectively. The dimeric reductase is presumably
damaged by oxygen under the aerobic condition.8
Communicated by Masahiro Sugiura
To whom correspondence should be addressed. Tel. +81-6497-7067, Fax. +81-6-497-7294, E-mail: [email protected].
melco.co.jp
t EMBL nucleotide sequence database (accession number:
Y15161).
X Abbreviations: CoxII; cytochrome c oxidase subunit II.
*
A monomeric N2O reductase was isolated from Achromobacter cycloclastes by Hulse and Averill,9 and interestingly the enzyme is stable as a high active pink form
(form II), in spite of an aerobic preparation process. The
monomeric N2O reductases were isolated from several
denitrifying bacteria, 10 however their nosZ genes have
not yet been cloned. As an initial effort to investigate the
structure of monomeric N2O reductases and to characterize the pink form with a high specific activity, I cloned
and analyzed the monomeric N2O reductase gene (nosZ)
from A. cycloclastes. In addition, I also identified the
relevant genes of nosDFYL downstream from the nosZ
gene. The sequences and organization of N2O reduction
genes were found to be homologous between monomeric
and dimeric enzymes, but sequence similarity searches
of the translated products did not show significant homology with other proteins, suggesting that a common
Cu-transport or processing system is unlikely in bacteria.
As shown in Fig. 1, the genomic library of A. cycloclastes (IAM1013) was screened with a probe for the nosZ
gene (see Fig. 1 legend), and three positive clones, pAC-1,
pAC-2 and pAC-3 carrying inserts of 4.4, 1.1 and 6.5 kbp,
respectively, were obtained. Analysis of the plasmids
in the region which hybridized to the probe showed a
1929-nucleotide open reading frame (ORF) which is proceeded by a potential ribosome-binding site, AGGAA.
t&fi
A. cycloclastes nosZDFYL Genes
nosR
nosZ
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AlGTnGCGGCMGCCCGGGCGGCGTGCCGACCCATCMCCCGCAGCCAMGTTTMTCGMaCMGGTTCMGCAAAMGGAACCAGATCATGGAATCAAAGGMCACAAGGGACTAA
120
M E S K E H K G L S
240
GCCGGCGAGCACTTTTCAGCGCGAC^CAGGCAGCGCCATTCTGGCGGGCACTGTAGGGCCGGaGCACTCAGaTCGaGCTGCAG«nGGCGACACCGGCCCGTGCGGCCACM
360
51
D G S V A P G K L D D Y Y G F W S
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480
91
F N R C S A T G W G O T N E S I R I H O R T M T E K T K K Q L A A N G K K I H D
131
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171
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600
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291
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960
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331
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CGGCCAATCCTGGCGTCTACTGGTACTATTGCCAATGGTTCTGCCATGCCCTGCACATGGAAATGCGCGGCCGCATGTTCGTGGAACCGAAGGGCGCCTGATCGATGCGGCTGTCCGTTC
A N P G V Y W Y Y C Q W F C H A L H M E M R G R M F V E P K G A *
M R L S V L
2040
2160
7
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2280
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K. Inatomi
No. 6]
367
2520
127
M H G I D V H G G R D T I V S G N E I
I G T R S A R M N E R G N G I Y V W N S P
CCGGTACGCTGCnCAAGACMTATTATCCGCTACGGTCGCGACGGCATCTTTTCGAACGCCAGCGCCGACAGCATCTATCGCCGCAACATCATGCGCGACCTGCGCTTTGCCGTGCACT
G T L L Q D N I
I R Y G R D G I F S N A S A D S I Y R R N I M R D L R F A V H F
2640
167
TCATGTACACCCGCMCACCGAGGTncCGACMCATCTCGATCGGAMCCATCTGGGCTTTGCGATCATGTTCTCCAACCGCGCGAAGATCCTGAACAATCTCAGCCTCGGCGACCGCG
U Y T R N T E V S D N I S I G N H L G F A I M F S N R A K I L N N L S L G D R E
2760
207
247
H G L M L N Y A N N A D V S G N L V R G G T K K C L F I Y N A H K N L V W G N R
2880
3000
287
I G I H F T A G S E K N V L T G N A F
F E S C G
I A N R E Q V K Y V G T R N M E
3120
327
N S H E G R G N F W S D H P A F D L N G D G V A D S F Y R P N D L M D O I L H S
3240
CGCA«CCGCCX£MGCCTGCTCACCGGCTCGCC(mGT«AGATCGTTCGCT^^
Q P A A S L L T G S P A V Q I V R H I S
367
Q
R
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F
P
A
T
L
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G
G
V
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D
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A
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L
M
R
P
nosF
3360
407
L T I P V P L E I L A Y E A E A A G R W T E G N Y D D T D A D N L Q A H
3480
1 5 Q S V E A L K S V S L A L E P G R R A A L L G H N G A G K S T M M K
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P F D S G E V S V C G S A P G S P A A R T
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295
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R R D G Q *
M S R
I L A T A V S E F R
I A L R N R « V S
I
A T G M M V L F A L V L A
CCGCCaCGGCTCTGCCCCGACGGGCGATGTCGGCGTCGACCGKTCTCCGTMCffiTCGCCTCGCTCACnCGCnGCGGTCTATCTGGTTCCGCTTnGGCATTGCTGATGAGTrTCG
A A G S A P T G D V G V D R L S V T V A S L T S L A V Y L V P L L A I L M S F D
4320
4440
PMl
4560
77
A V A G E V E R G T L P L L L T Y P V S R L Q
I L L G K L L A H L A I
L G L A V
TGACaTTGGCTACGGCGCGGCGaGCTCGCAGaGTCTGGTTCBATCCTGGAGCMCCGCAGGGCnGGCGCCCTATGGCGCXrrGATCTGGTCCTCTGTGCTGCTGGGTGCGACCTT^^
T L G Y G A A A L A A V V I F D P G A T A G L G A L W R L
I
W S S V L L G A T F L
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117
TTGGCACCGGCTATGCGCTTTCGGCTTTGGCGCGACGGCCGTCGGGGaGGCGGGCnGGCCGTCGCGCTCTGGCTGGTGaGGTGGTGCTCTACGACCTCGCCCTGnGaGTTM
G T G Y A L S A L A R R P S G A A G L A V A L W L V A V V L Y D L A L L A L I
4800
157
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4920
197
T D G G G A F T T H A L P V A L L A N P A D A F R V F N L S A A Q A V S A A G G
237
L G G A A G T I P L W Q S A A S L L A W P L A A I A L A A A A F R K V T P
5040
5160
4
R L R F V L V A A A L A L L S A C K E D V A Q S
I V P Q D H T P E T L G H Y C Q
5280
44
H N L L E H P G P K A Q I F L E G S P A P L F F S O V R D A I A Y A R G P E Q I
84
A P I L V I Y V N D M G A A G A T N D Q P G D G N W I A A D K A F Y V V G S A R
5400
GCGAGGCGGCATGGGTGCGCCCGMGCCGTGCCGTTTTCMGCCGCGACGAGGCTGCGGCCnCGTTCTTGCCGAGGGCGGCCAGGTGCTTGCGCTCGCCGATATTACCGATGCCATGGT
124
R
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M
G
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A
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164
L T P V E T G S E P R A D D E D Y L G R L R A L P H P A G G
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9
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5640
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368
A. cycloclastes nosZDFYL Genes
The ORF encodes a precursor protein of 642 amino acids,
and the deduced amino acid sequence is 55%, 87%, 60%
and 47% identical with those of dimeric N2O reductases
from P. stutzeri,4 P. denitrificans,6 R. meliloti7 and A.
eutrophus,3 respectively.
The N-terminal amino acid sequence of the mature enzyme (Fig. 1) indicated that the precursor protein has a relatively long signal sequence of 46 amino
acids residues. 12 The signal peptide showed N-terminally
located positive charges, a hydrophobic potentially
membrane-spanning segment and the cleavage site following the -1, -3 rule. 13 Thus, the N2O reductase
of A. cycloclastes (lacking signal peptide) consisted of
596 amino acid residues with calculated molecular mass
of 66,503 Da. This value is consistent with that estimated from the mobility of the purified enzyme on polyacrylamide gel electrophoresis in the presence of SDS.9
Alignment of the amino acid sequences of N2O reductases (Fig. 2A) shows' key residues (Cys, His, Met and
Trp) for ligands of the CUA or Cuz site which has been
proposed by Zumft et al. 3 ' 5 In this study, the number of
conserved histidine and cysteine residues decreased from
14 to 11 and from 3 to 2, respectively (full alignment
not shown). Two histidine residues at positions 78 and
467, and one cysteine residue at position 165 (numbering
of P. stutzeri N2O reductase) are now known not to be
conserved. Since Cysl65 is the only conserved residues
out side the CUA region (Fig. 2A, boxed by a line), it is
a possible candidate ligand for the Cuz catalytic site. 3 ' 5
A recent mutational study (Cysl65 to Gly in P. stutzeri
N2O reductase) also demonstrated that Cysl65 appears
not to be part of a cysteine coordination of the Cuz- 14
It is an interesting idea that the Cys 165 may be involved in an intersubunit disulfide bridge of dimeric N2O
reductases, because the A. cycloclastes N2O reductase
which has Thrl71 (Fig. 2A, reverse type letter) instead
of Cysl65, is a monomeric reductase. However the mutation of P. stutzeri N2O reductase (Cysl65 to Gly) did
[Vol. 5,
not generate a monomeric enzyme.14
At the C-terminal end of the N2O reductase sequence
there is significant similarity with the sequence found
around the CUA site of cytochrome c oxidase subunit
II (CoxII), as first reported by Zumft et al. 5 As shown
in Fig. 2A, when aligned to the CuA-binding site sequence of CoxII, the sequence of A. cycloclastes N2O reductase positionally matched with two cysteines (Cys620
and Cys624, numbering of A. cycloclastes enzyme), two
histidines (His585 and His628) and methionine (Met631)
residues (indicated by arrows). These five amino acids
residues are consistent with those recently confirmed as
the CUA ligands of the bovine heart CoxII by X-ray crystallographic analysis. 15 Thus there was no significant difference in the primary sequences between the monomeric
and dimeric N2O reductases, except for the Thrl71 in A.
cycloclastes.
For the expression of A. cycloclastes nosZ gene in
E. coli cells, the nosZ gene was ligated with a pGEX-3
plasmid (Amersham) and transferred to E. coli strain
BL21. The N2O reductase was isolated by a GTS-column
(Amersham) and purified to homogeneity by digestion
with Factor Xa protease (data not shown). The purified protein had the same molecular mass as the native
enzyme from A. cycloclastes on poly aery lamide gel electrophoresis in the presence of SDS. Although the N2O
reductases of E. coli and A. cycloclastes were immunologically indistinguishable, the characteristic absorbance of
the form II (pink) and the N2O reducing activity were absent. A similar result was also observed in the expression
of dimeric N2O reductase {P. stutzeri) in E. coli.5 Relevant genes nosDFYL have been identified in the flanking
regions of nosZ genes in R. meliloti and P. stutzeri, and
they seem to have the role of complete Cu incorporation
into the active N2O reductase. 4 ' 7 Therefore I investigated
the flanking regions of the A. cycloclastes nosZ gene.
The 5' end of an ORF (1329 bp) was found downstream from the A. cycloclastes nosZ gene, and the DNA
Figure 1. Nucleotide and deduced amino acid sequences of N2O reductase structural gene (nosZ) and relevant genes of nosDFYL
from A. cycloclastes. (A) Organization of the nosZDFYL genes. The location and order of the genes are shown by arrows and the
shaded box shows the restriction map. Three clones (pAC-1, 2, and 3) including the nos region are indicated at the bottom. (B)
Nucleotide and deduced amino acid sequences of the nosZDFYL genes. Nucleotides and amino acids are numbered at the right
and left margin, respectively. The termination codon is indicated by the asterisk. The processing site of a signal peptide is shown
by the arrow at the position between Ala46 and Ala47 in nosZ gene. The mature N2O reductase starts from the Ala47 residue.
A. cycloclastes (IAM1013) was grown in a medium containing potassium nitrate (2g/l). Cells were suspended in 50 mM Tris-HCl
containing 2% sodium dodecyl sulfate (SDS), and chromosomal DNA was prepared by phenol extraction. The DNA was digested
with restriction endonuclease Pst I or Hindlll and the fragments were ligated into the Pst I or Hindlll site of pUC18, respectively.
The recombinant plasmids obtained were introduced into E. coli JM109. A. cycloclastes N2O reductase was purified according to
the published method.9 The enzyme was digested with trypsin and the fragments obtained were separated and sequenced from the
amino terminus with a peptide sequencer ABI 473A (underline). Two primers were synthesized on the basis of the partial amino
acid sequences determined as above, and a polymerase chain reaction (PCR) was performed to amplify a probe for the cloning of
nosZ gene; forward primer (20 mer) corresponding to the amino acids at positions 61 to 67; 5'ACTACTACGGCTTCTGGTCC3',
reverse primer (20 mer) corresponding to the amino acids at positions 487 to 493; 5'CGGTTCGGCAAAGGTCGGGC3'. The
amplified DNAs were sequenced to confirm their identity and then they were labeled by the ECL direct nucleic acid labelling kit
(RPN 3000, Amersham) for the screening of nosZ gene in the A. cycloclastes DNA library. Hybridization was carried out in the ECL
gold hybridization buffer (RPN 3006, Amersham) for 13-15 hr at 37-40°C. After washing replica filters (Amersham Hybond N+),
hybridization signals were detected by the enhanced chemiluminescence method (ECL, Amersham). Genomic DNA and plasmid
isolation, endonuclease digestion, electrophoresis, ligation and transformation were carried out as described in reference.11 The
nucleotide sequences were determined on both strands using the ABI PRISM 310 genetic analyzer. The sequences of nosZDFYL
genes from another clone of A. cycloclastes are also available in the data base (AF047429) which was opened at June 1998.
No. 6]
K. Inatomi
*
A
NosZ
A. cycloclastes
*
*
cycloclastes
denitrificans
me IiIo t i
stutzeri
eutrophus
c
NosL
*
*
*
STMTDVATYVN 222
** * * ***
******* ** * ** **** * ** *
TNLDEIDDLTHGFTMGNHGVAMEVGPQQTSSVTFVAANPGVYHYYCQWFCHALHMEMRGR 634
TNU5EIDDLTHGFTWGNYGVAMEIGPOMTSSVTFVAANPGVYWYYCOWFCHALHMEMRGR 644
TNIDEVEDLTHGFCIVNYGINMEVAPQATASVTFKASRPGVYWYYCTWFCHAMHMEMKGR 631
TNIDQIEDVSHGFWVNHGVSMEISPQQTSSITFVADKPGLHWYYCSWFCHALHMEMVGR 632
TNLDKIEDLTHGFAIPKYNVNFIVNPQETASVTFVADKPGVFWCYCTHFCHALHLEMRTR 638
t
NosF
*
VMKBDAILEIPNAKGIHG-MRPQKWPRSNYV-FCNGEDEAPLVNDG
P. deni t r i f icans VMKCbAiLEIPNAKGIHG-LRPOKWPRSNYV-FCNGEDETPLVNDG
TNMEDVANYVN 232
P. meliloti
VMKCDKIIQLPNQHTVHG-LRVQKYPKTGYV-FCNGEDAVPVPNDG-K—TMGDKNSYQA 223
P. s t u t ze r i
IMK C 3KMITVPNVQAIHG-LRLQKVPHTKYV-FANAEF11PHPNDG-KVFDLQDENSY-T 217
A. eutrophus
YFigXITELPNVQGFHGIFPDKRDPVDTKINYTTRVFCGGEFGIPLPSAPTEDAGKYRS 230
A.
P.
R.
P.
A.
B
*
369
r r t r
A. cycloclastes
P. stutzeri
R. me Iilot i
* GHNGAGKS
*******rMMKIVLGLIPFDSGE
*
MTPTLTISRLTKRFQSVEALKSVSLALEPGRRAALL
60
MN-AVEIQGVSQRYGSMTVLHDLNLNLGEGEVLGLF GHNGAGKT TSMKLILGLLSPSEGQ 59
MSGTVEIAGVSKCYGDSTWRDISFGLGAAETVALV GHNGAGKT FLIKLMLGLIRPTXGL 60
A. cyoloclastes
P. stutzeri
R. meliloti
********* **** ***** ***** ****
LG-HAARRRIGT fSKGMRQRVGLAQTLIGRPRLLVLDEPTSGLD'VSRRDFYDLLDGLAA 177
LAH-AADRRVKT CSKGMRQRLGLAQALLGEPRLLLLDEPTVGLD»IATQOLYLLIDRLRQ 176
LAQEAVDRPVRT CSKGMRQRLGLAQALLGMPRILLLDEPTSGLD 'ALRRNFYELITELRA 180
A. c y c l o c l a s t e s
P. s t u t z e r i
R. m e l i l o t i
M—RTRLRFVLVAAALA-LLSAffl-KEDVAQSIVPOOMTPETLGHYCOMNLLEHPGPKA 54
MNALHRIGAGTLLAVLLAFGLTGffiEKEEVQQSLEPVAFHDSDECHVCGMIITDFPGPKG 60
M—KLTVTAIL-AATL—FLAGffl-QKEEDTTMPSPYSLTADAMGRYCGMNVLEHPGPKG 53
Figure 2. Alignment of the derived amino acid sequences of the nosZ, nosF and nosL genes from A. cycloclastes and several denitrifiers.
The multiple alignment was performed by the program MULTI ALIGNMENT (SDC, Japan). Asterisks indicate the positions where
a residue is conserved in all five (A) or three sequences (B and C). (A) Alignment of N2O reductases from A. cycloclastes, P.
denitrificans,6 R. meliloti,7 P. stutzeri4 and A. eutrophus.3 The cysteine residues conserved in four bacteria are boxed by a line. The
reversed letter shows the Thrl71 in A. cycloclastes N2O reductase. In the C-termini, conserved cysteine, histidine and methionine
residues, which correspond to the ligands for CUA site of cytochrome c oxidase subunit II, 15 are indicated by arrows. (B) Alignment
of two regions of NosF proteins having a consensus sequence for ABC transporters.16 The ATP-binding sequences are boxed by a
line. (C) Alignment of the N-terminal of NosL proteins to compare their consensus sequence of lipoproteins. Precursor signal peptides
in the N-terminal are presumably cleaved by a specific lipoprotein signal peptidase upstream the cysteine residue indicated by the
reverse type letter.
sequence showed homology to those of nosD genes identified in P. stutzeri4 and R. meliloti.7 The termination
codon of nosZ was very close (3 bp) to the ATG codon
of nosD, and a potential ribosome-binding site, GAAGG,
was observed 12 bp upstream of the ATG codon (Fig. 1).
The A. cycloclastes nosD gene coded for 442 amino acids,
and 34% and 46% identical with those of nosD genes from
P. stutzeri4 and R. meliloti,7 respectively. According to
the program SignalP,13 the cleavage site of the export signal was predicted to be between the two Ala residues at
positions 18 and 19, suggesting the periplasmic location
of the mature NosD protein (Table 1).
Overlapping the end of nosD was another ORF
(900 bp), a nosF gene and a potential ribosome-binding
site, GAAGG, was observed 9 bp upstream of the
ATG codon of nosF. The nosF gene encodes 299 amino
acid residues and the translated amino acid sequences
were 43% identical to those of NosF from P. stutzeri4
and R. meliloti7 A DNA motif analysis clearly showed
that A. cycloclastes NosF has a consensus sequence,
GXXXGK(T/S), for ATP-binding sites found in ATPbinding/hydrolysis proteins 16 (Fig. 2B), which was first
reported in P. stutzeri by Zumft et al. 4 Sequence similarity searches revealed around 25% matches of NosF
with ATP-binding transport proteins such as the Fe 3 +
dicitrate transport protein FecE 17 and the ferrichrome
transport protein FhuC. 18 From the program PSORT, 19
NosF was predicted to belong to the ATP-binding cassette (ABC) superfamily of transporters that mediate transport and channel functions in prokaryotes and
eukaryotes. 16 In addition to the ATP-binding sequence
of GXXXGK(T/S), the ABC proteins share a second
nucleotide binding motif with significant homology.16
These nucleotide binding sites were also conserved in
NosF proteins of three dinitrifiers as shown in Fig. 2B
(boxed part). The hydropathy profile of A. cycloclastes
NosF indicates the absence of both a signal peptide sequence and transmembrane segment, 20 suggesting that
NosF is a peripheral protein and located in cytoplasm or
at the surface of inner membranes (Table 1).
370
A. cycloclastes nosZDFYL Genes
[Vol. 5,
Table 1. Properties of the predicted gene products involved in N2O reductase biosynthesis of A. cycloclastes.
Distinctive
Gene
Calculated
Amino acid
Transmembrane Predicted signal Predicted cellular
feature0'
product residues molecular mass (Da)
locationc'
segments'1'
sequence'3'
642
none
46 residues '
periplasm
N2O reductase
70921
NosZ
—
442
none
MRLSVLLIGFL
periplasm
47823
NosD
LASPLLA-AERA or outer membrane
299
cytoplasm
ABC transporter
NosF
none
none
31741
273
inner membrane integral membrane
none
5 or 6
NosY
27649
protein
193
lipoprotein
outer or inner
MRTRLRFVLVA
none
20449
NosL
AALALLSA-CK0'
membrane
a)
b)
c)
d)
e)
Transmembrane segments were predicated by PSORT program19 or hydropathy profiles.20
Signal sequence was calculated by the SignalP program10 and the presumed N-terminal is shown in bold-face letters.
Classification of the proteins were predicted by PSORT program19 or Motif analysis (GENETYX DB, SDC, Japan).
The signal sequence was determined by the N-terminal analysis of the mature protein with a peptide sequencer.
Modification site of the lipoprotein was determined by PSORT program.19
Partially overlapping the nosF termination codon
TGA is an ORF (822 nucleotides) of the nosYgene. The
sequence of GGAGG was observed 12 bp upstream of the
nosY initiation codon as a potential ribosome-binding
site. The nosY gene encodes 273 amino acid residues
and the product was found to be highly hydrophobic
protein; 74% of the amino acids are hydrophobic. The
NosY protein has at least five transmembrane segments
with no predicted prokaryotic export signal, suggesting
that NosY is a integral membrane protein and located
in the inner membrane (Table 1). The amino acid sequence was 41% and 45% identical with those of the
NosY proteins of P. stutzeri4 and R. meliloti,7 respectively. Sequence similarity searches showed no significant matches of NosY protein with other entry proteins.
The PSORT analysis 19 also did not give any distinctive
motif in the sequence. ABC transporter proteins in eukaryotes have two typical domains, a hydrophobic membrane domain consisting of six transmembrane segments
and a cytoplasmic domain with ATP-binding/hydrolysis
sites, and they seem to serve as a substrate specificity
determination and an energy coupling to transport substrate, respectively.16 The combination of hydrophobic
NosY and NosF with ATP-binding sequences may function as a Cu-transport in the A. cycloclastes membranes,
like the eukaryotic ABC transporters for inorganic ion
transport. 16
Partially overlapping the termination codon of nos Y is
an ORF (582 bp), nosL gene, and a potential ribosomebinding site of AGGTG was seen 6 bp upstream of the initiation codon of ATG. The ORF encodes 193 amino acid
residues and showed 31% and 45% identical with those
of NosL proteins of P. stutzeri (AC number: Z69589)
and R. meliloti (AC number: U94899), respectively. The
sequences of NosL have been deposited in data banks,
but characterization of the sequence has not yet been reported in detail. The hydropathy profile of the A. cyclo-
clastes NosL protein indicates a hydrophilic nature and
the absence of transmembrane segments.20 The PSORT
analysis showed a typical consensus sequence of L- (A/S)(G/A)-C in the N-terminal which is consistent with the
modification and processing site of prokaryotic membrane lipoproteins (Fig. 2C). 21 The precursor signal peptide of 19 amino acid residues is presumably cleaved by a
specific lipoprotein signal peptidase upstream the Cys20
residue to which a glyceride-fatty acid lipid might be attached. The N-terminal lipid moieties of lipoproteins are
usually integrated into membranes, 21 suggesting that the
NosL protein is first exported to a periplasmic space and
then associated with inner or outer membranes after the
modification by the signal peptidase. Sequence similarity
searches revealed no significant matches with other proteins. The copper transport and homeostasis in E. coli
is mediated by cutABCDEF genes, and the CutF protein
is known as a lipoprotein with the consensus lipoprotein
modification sequence.22 The molecular weight of NosL
protein is similar to that of CutF, however there was no
significant sequence homology expect of the N-terminal
region.
In conclusion, the primary sequence of monomeric
N2O reductase of A. cycloclastes was found to be similar to those of dimeric N2O reductase, in spite of the
clear difference in their optical spectra. In addition,
relevant DNA regions of nosDFYL genes, which is suggested to be involved in the periplasmic biosynthesis of
Cu center of N2O reductase in P. stutzeri,4 were also
conserved downstream of the A. cycloclastes N2O reductase gene (nosZ). Although the biosynthesis of the Cu
chromatophore of N2O reductase may require another additional gene such as a proposed Cu ion channel (nosA),4
the essential genes and their organization seem to be
conserved in at least the three denitrifiers, P. stutzeri,
R. meliloti and A. cycloclastes. But sequence similarity
searches of the translated amino acid sequences of the
No. 6]
K. Inatomi
371
nosDFYL genes did not show significant homology with
from Achromobacter cycloclastes, Biochem. Biophys. Res.
Commun., 166, 729-735.
other entry proteins, in particular bacterial transport systems of inorganic ions except of NosF, suggesting that a 10. Michalski, W. P., Hein, D. H., and Nicholas, D. J. D.,
1986, Purification and characterization of nitrous oxide
common Cu-transport, insertion or processing system is
reductase
from Rhodopseudomonas sphaeroides f. sp. denunlikely in bacteria.
itrificans, Biochem. Biophys. Acta, 872, 50-60.
For further understanding the biosynthesis of N2O re11. Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989,
ductase, the expression of nosZ and relevant genes in
Molecular cloning: A Laboratory Manual, 2nd Ed., Cold
E. coli cells are in progress in my laboratory. The A.
Spring Harbor Laboratory, Cold Spring Harbor, NY.
cycloclastes N2O reductase must be advantageous to in- 12. Dreusch, A., Burgisser, D. M., Heizmann, C. W., and
vestigate the transport process of the N2O reductase into
Zumft, W. G. 1997, Lack of copper insertion into unperiplasm, because it consists of a single subunit.
processed cytoplasmic nitrous oxide reductase generated
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