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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 r Pad BimiH\ nosD HnMW BmuHl I I [Vol. 5, nosF nosY nosL nosX aiRI Pxt\ I 2Kb 1Kb 3Kb I 4Kb 5Kb Psi\ Psit I pAC-l Wrallil tfmdlll I pAC-2 1 Hindlll Him -pAC-3- B 1 nosZ Hindlll 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 S G Q T G E H R I L G I P S M R E L M R V PV 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 N G O L H H V H U S F T D G K Y D G R Y L F M N D K A N T R V A R V R C D V M K 171 T B A I L E I P N A K G I H G M R P Q K W P R S N Y V F C N G E D E A P L V N D 600 720 840 211 I G S T M T D V A T Y V N F T A V D A D K W E V A W Q V K V S G N L D N C D A D 251 Y E G K W A F S T S Y N S E M G M T L E E M T K S E M D H V V V F N I A E I E K 291 A I K A G Q Y E E I N G V K V V D G R K E A K S L F T R Y 1 P I A N N P H G C N 960 1080 ACATGGCaCGGACAGGMGCATCTGTGCGnGCCGGCAl(G^fnCGCCMCCGTCACCGTGCTGGACGTGACGMGTTCGATGCCCTGnCTACGACMTGCCGAGCCa^ 331 M A P D 371 V A E P 411 R A 451 C L 491 A E C P D I R E Y K H K L G A F H G S A E W C L E K D L G K D V A P I L N R 1 A G K H P L T I K S A D P F K T D L V G D T R V V G Q L N Y D A V Y Q P T T G K S H F L F D A L F L D S Q V M G L K T I D I Y D N A E P V V K W N E T L D A R S I A 1200 N A V D E A I D V W L F L N V G P L K P E N D Q L P T F V S P S I L P N I R S V W D R K D P L W A E T R K Q A E A D E V G N K V R T S V A P S F S Q P S F T V K E G D E V S G D K M V L V H D G 1320 1440 1560 1680 1800 531 O T E A V I R D V Y H 1920 611 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 L I G F L L G A Y D G P T L A S P L L A A E R A V M P G A G S L A A A I A G A Q P V V T R V E G D V L V L T 2280 47 D V I T D R P L T R I N I T G P R G A V V N G L K Q G S I A A S D V 2400 87 T G F T V T G S G Q D L D A G V K I V O G A D R A K L L V T D N 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 D F P A T L P G G V R D S A P 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 5 5 9 5 I V L G L 3600 CCCnCGACAGCGGCGAGCmCGGmGCGGCTCGGCGCCCGGTTCGCCCKCGCGC^ P F D S G E V S V C G S A P G S P A A R T O V A Y L P E N V A F H P A L T G E E 3720 Q L R H Y L A L R G E N P K Q A T E L L A G R P R L L V L R V G L G H A A R R R I G T Y S K G M 3840 1 3 5 R Q R V G L A Q T L I D E P T S G L D P V S R R D F Y D L L D G L V A E G T L A 3960 1 7 5 L A A E G T A 2 1 2 5 I L L S S H V L T E V E A R GATCnCGCACCCGCGCGGCACnCCGGTCGCCTmCCGTTCGCCC(^ACC(^ 5 D L R T R A A L P V A F S V R P A P G H T D S I L I L S G G E 4080 A P A L I A D A A T F P E G V E G E D G L L R I P P S L E D I Y S H F S 4200 5 L R G S O S E K L P L L A R I nosY 295 37 A A L G S A L D V EcoRl C(»AG(»ACGaK;MTGA(KCGCATCCnGCaCCGCCGTCAGCGMnCCGTATCaTTTGCGCMTCGTTGGGTCTCTATCGCCACCGGCATGATGGTGCTGTTCGCCCTGGTGCTGG 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 4680 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 V 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 G G M G A P E A V P F S S R D E A A A F V L A E G G Q V L A L A D I T D A M nosX 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 Psll 9 TGATTGCAATTTCTGCAG I A I S A 5658 M L 5520 V 5640 L T R R R L 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 References 1. Hochstein, L. I. and Tomlinson, G. A. 1988. The enzymes associated with denitrification, Ann. Rev. Microbwl, 42, 231-261. 2. Farrar, J. A., Thomson, A. J., Cheesman, M. R., Dooley, D. M., and Zumft, W. G. 1991, A model of the copper centres of nitrous oxide reductase, FEBS Lett., 294, 1115. 3. Zumft, W. G., Dreusch, A., Lochelt, S., Cuypers, H., Friedrich, B., and Schneider, B. 1992, Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copperbinding residues, Eur. J. Biochem., 208, 31-40. 4. Zumft, W. G., Viebrock-Sambale, A., and Braun, C. 1990, Nitrous oxide reductase from denitrifying Pseudomonas stutzeri, Eur. J. Biochem., 192, 591-599. 5. Viebrock, A. and Zumft, W. G. 1988, Molecular cloning, heterologous expression, and primary structure of the structural gene for the copper enzyme nitrous oxide reductase from denitrifying Pseudomonas stutzeri, J. Bacteriol., 170, 4658-4668. 6. Hoeren, F. U., Berks, B. C , Ferguson, S. J., and McCarthy, E. G. 1993, Sequence and expression of the gene encoding the respiratory nitrous-oxide reductase from Paracoccus denitrificans, Eur. J. Biochem., 218, 49-57. 7. Holloway, P., Mccormick, W., Watson, R. 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