L-Amino Acid Oxidases with Specificity for Basic L-Amino Acids in Cyanobacteria Achim E. Gaua, Achim Heindlb, Anke Nodopb, Uwe Kahmannb, and Elfriede K. Pistoriusb,* a InstitutfürBotanik,GottfriedWilhelmLeibnizUniversitätHannover, D-30419Hannover,Germany b BiologieVIII:MolekulareZellphysiologie,UniversitätBielefeld,D-33501Bielefeld, Germany.E-Mail:[email protected] * Authorforcorrespondenceandreprintrequests Z.Naturforsch.62c,273Ð284(2007);receivedOctober23/November24,2006 ThetwocloselyrelatedfreshwatercyanobacteriaSynechococcuselongatusPCC6301and Synechococcus elongatus PCC 7942 have previously been shown to constitutively express a FAD-containingl-aminoacidoxidasewithhighspecificityforbasicl-aminoacids(l-arginine being the best substrate). In this paper we show that such an enzyme is also present in the fresh water cyanobacterium Synechococcus cedrorum PCC 6908. In addition, an improved evaluationofthenucleotide/aminoacidsequenceofthel-aminoacidoxidaseofSynechococ- cus elongatus PCC 6301 (encoded by the aoxA gene) with respect to the FAD-binding site andatranslocationpathwaysignalsequencewillbegiven.Moreover,thegenomesequences of 24 cyanobacteria will be evaluated for the occurrence of an aoxA-similar gene. In the evaluated cyanobacteria 15 genes encoding an l-amino acid oxidase-similar protein will be found. Key words:Cyanobacteria, l-AminoAcid Oxidase, Synechococcuselongatus PCC6301 and PCC7942 Introduction Thel-Aoxencodinggene,calledaoxA,haspre- The two closely related cyanobacteria Synecho- viouslybeenidentifiedinSynechococcuselongatus coccus elongatus PCC 6301 and Synechococcus PCC6301.Apreliminarynucleotidesequencewas elongatus PCC 7942 have previously been shown publishedbyBockholtetal.(1995)andacorrected topossessaFAD-containingl-aminoacidoxidase nucleotide sequence and the derived amino acid (l-Aox) catalyzing the oxidative deamination of sequence were later submitted to the data base basic l-amino acids (l-Arg (cid:2) l-Lys (cid:2) l-Orn (cid:2) l- undertheaccessionnumberZ48565withanentry His) by utilizing O as electron acceptor resulting date of July 1998. 2 in the formation of the corresponding ketoacid, Construction of an l-Aox-free Synechococcus ammonium, and hydrogen peroxide (Pistorius elongatus PCC 7942 mutant and subsequent par- etal., 1979; Pistorius and Voss, 1980; Engels etal., tial characterization of this mutant revealed that 1992). Another rather unusual property, besides in contrast to the wild-type the mutant could not havingahighspecificityforbasicl-aminoacids,is grow on l-arginine as sole nitrogen source sug- itsstronginhibitionbycations(M3+(cid:2)M2+(cid:2)M+) gesting that this l-Aox is the only enzyme that and less strong inhibition by anions. In the group enables the cells to utilize extracellularly added of divalent cations, transition metal ions (like l-arginine as N-source (Bockholt et al., 1996). Mn2+) inhibit more strongly than alkaline-earth However, since Synechococcus elongatus PCC metal ions (like Ca2+). The l-Aox in Synechococ- 7942 can not take up l-arginine effectively (Mon- cus elongatus PCC 6301 and PCC 7942 is in part tesinos etal., 1997), this result does not exclude located in the soluble protein fraction of the peri- that other l-arginine-metabolizing enzymes are plasm and in part in the spheroplast fraction, present intracellularly. mainly precipitating with the membrane fraction Besides being a constituent of proteins, l-argi- (Bockholt et al., 1996 and unpublished results). ninehasanadditionalroleinsomecyanobacteria, which have an alternative CO fixation pathway 2 Abbreviations: l-Aox, l-amino acid oxidase; aoxA, l- via carbamoyl phosphate leading to citrulline and aminoacidoxidaseencodinggene. subsequently to arginine (Linko et al., 1957; Ta- 0939Ð5075/2007/0300Ð0273$06.00 ”2007VerlagderZeitschriftfürNaturforschung,Tübingen·http://www.znaturforsch.com·D 274 A.E.Gauetal.·l-AminoAcidOxidasesinCyanobacteria bita, 1994) in addition to the major CO fixation Inthispaper,wereportonanadditionalcyano- 2 reaction via Rubisco in the Calvin cycle. More- bacterium, besides Synechococcus elongatus PCC over,itiswelldocumentedthatanumberofcyano- 6301 and PCC 7942, which has such an l-Aox be- bacteria areable tosynthesizethepolymer cyano- ing readily detectable by a stimulation of the O 2 phycin, which consists of an aspartic acid uptake by intact cells when l-arginine is added as backbone with l-arginineresidues attached to the substrate. In addition an extended characteriza- (cid:2)-carboxy group of aspartate by isopeptide bonds tionofthel-AoxsequenceofSynechococcuselon- (Simon, 1971, 1987; Allen, 1984). The peptide as gatus PCC 6301 with respect to the FAD-binding wellastheisopeptidebondsaresynthesizedbythe site and a translocation pathway signal sequence cyanophycinsynthetaseencodedbythecphAgene are performed. Moreover, on the basis of the l- (Ziegler et al., 1998; Hai et al., 1999; Berg et al., Aox sequence of Synechococcus elongatus PCC 2000; Aboulmagd et al., 2000). In the early litera- 6301 (encoded by the aoxA gene) the genome se- tureithasbeenstatedthatthetwocloselyrelated quences of 24 cyanobacteria are evaluated for the SynechococcuselongatusPCC6301andPCC7942 presenceofageneencodingsuchaputativel-Aox are not able to synthesize cyanophycin (Simon, with specificity for basic l-amino acids. 1987), and later it has been shown that these two cyanobacteria do not possessthe cphA gene (Zie- Materials and Methods gler et al., 1998). Thus, they are unable to store Origin of cyanobacteria, growth, French press l-arginine in the cyanophycin polymer. However, extract preparation,l-Aox activity determination, they constitutively express the above described l- and immunocytochemical investigation Aox, which converts l-arginine to 2-ketoarginine beingfurthermetabolizedtoguanidinobutyrateby Synechococcus elongatus PCC 6301 was ob- a nonenzymatic reactionwhen hydrogen peroxide tainedfromtheSammlungvonAlgenkulturender is not removed by hydrogen peroxide decompos- Universität Göttingen, Göttingen, Germany, and ing enzymes. SynechococcuselongatusPCC7942aswellasSyn- TableI.Theoriginoftheevaluatedcyanobacterialgenomesequences. Cyanobacterialstrain Originofgenomesequencea ProchlorococcusmarinusSS120 EuropeanUnion/Genoscope ProchlorococcusmarinusMIT9211 CraigVenterInstitute ProchlorococcusmarinusMIT9312 JGI/MIT/DOE ProchlorococcusmarinusMIT9313 JGI/DOE ProchlorococcusmarinusMED4 JGI/DOE ProchlorococcusmarinusNATL2A JGI/MIT/DOE Synechococcussp.WH8102 JGI/DOE Synechococcussp.CC9902 JGI/DOE Synechococcussp.RS9917 CraigVenterInstitute Synechococcussp.CC9605 JGI/DOE Synechococcussp.WH5701 CraigVenterInstitute Synechococcussp.WH7805 CraigVenterInstitute TrichodesmiumerythraeumIMS101 WHOI/JGI/DOE CrocosphaerawatsoniiWH8501 WHOI/JGI/DOE SynechococcuselongatusPCC6301 NagoyaUniversity SynechococcuselongatusPCC7942 JGI/TexasA&MUniversity/DOE Synechocystissp.PCC6803 KazusaDNAResearchInstitute GloeobacterviolaceusPCC7421 KazusaDNAResearchInstitute Nostocsp.PCC7120 KazusaDNAResearchInstitute NostocpunctiformePCC73102 JGI/DOE AnabaenavariabilisATCC29413 MissouriStateUniversity/JGI/DOE ThermosynechococcuselongatusBP-1 KazusaDNAResearchInstitute SynechococcusYellowstoneA(JA-3Ð3Ab) TIGR SynechococcusYellowstoneB(JA-2Ð3B’a(2Ð13)) TIGR a JGI,JointGenomeResearchInstitute;DOE,DepartmentofEnergyUSA;MIT,MassachusettsInstituteofTech- nology;WHOI,WoodsHoleOceanographicInstitute;TIGR,TheInstituteforGenomicResearch. A.E.Gauetal.·l-AminoAcidOxidasesinCyanobacteria 275 echococcus cedrorum PCC 6908 were obtained the Blast algorithm by Altschul et al. (1990) in fromthePasteurCultureCollectionofCyanobac- Swiss-PROT, EMBL, PIR, GenBank, and Cyano- terial Strains, Paris, France. Growth and French Base databases. Sequence alignments were per- press extract preparation were as described in formed with ClustalW (Thompson et al., 1994), Yousefetal.(2003).Thel-Aoxactivitywasdeter- and calculations of identities and similarities were minedasdescribedinEngelsetal.(1992),andthe performed according to Tatusova and Madden immunocytochemical investigation was done as (1999). Prediction of putative signal sequences describedinBockholtetal.(1996)utilizingthean- were done with SMART (Ponting et al., 1999; tiserum raised against the isolated l-Aox from Schultzetal.,1998).Theoriginofthecyanobacte- Synechococcus elongatus PCC 6301. rial genome sequences are listed in Table I. DNA isolation, cloning, and nucleotide Results and Discussion sequencing Presence of anl-Aox with specificity for basic For a re-evaluation of the aoxA sequence, the l-amino acids in Synechococcus cedrorum PCC previouslyobtained10kbEcoRI/BamHIchromo- 6908 somalDNAfragment(pDL100)fromSynechococ- Intheearlyliteratureithasbeenstatedthatbe- cus elongatus PCC 6301, which carries the aoxA sides the two mesophilic cyanobacteria, Synecho- gene (Bockholt et al., 1995), was digested into a coccus elongatus PCC 6301 and Synechococcus 1.9kbPstIfragment(pDV18)anda1.5BglIIfrag- elongatus PCC 7942, the mesophilic cyanobacte- ment (pDL14). For sequencing of these two over- rium Synechococcus cedrorum PCC 6908 also is lapping strands the fragments were cloned into not ableto synthesizecyanophycin (Simon,1987). pUC19 and pSVB30, respectively. Sinceallthreecyanobacteriaarefreshwatercyano- bacteria growing only under photoautotrophic Genome sequence evaluation and analyses conditions, we investigated whether Synechococ- Nucleic acid and the derived protein sequences cus cedrorum PCC 6908 also contains an l-Aox were analyzed using the Staden software package with high specificity for basic l-amino acids. As (Staden,1986).Thededucedaminoacidsequences theresultsofTableIIshow,thisisindeedthecase. were analyzed using two different programs: PC/ The activity of this enzyme was readily detectable Gene (IntelliGenetics Release 6.8, Camino Real incellsuspensionsorcellextractsbyastimulation Mountain View, California, 1993) and XPIP (Sta- of the O uptake when l-arginine was added. The 2 den, 1986). Homology searches were done with properties of the l-Aox in Synechococcus cedro- Table II.Comparison of thel-amino acidoxidase in Synechococcuselongatus PCC 6301,Synechococcus elongatus PCC 7942, and Synechococcus cedrorum PCC 6908. The l-Aox activity was determined in French press extracts (FPE)preparedfromacellsuspensioncontaining100μlcells/ml.GrowthofcellswasfortwodaysinBG11medium. ActivitymeasurementswereperformedasdescribedinMaterialsandMethods. S.elongatus S.elongatus S.cedrorum PCC6301 PCC7942 PCC6908 l-AoxactivityinFrenchpressextracts [μmolO takenup/mlFPE¥h] 60Ð80 10Ð15 45Ð70 2 (l-arginineassubstrate) K valueforl-arginine[mm] 5 3 4 M CaCl inhibition:I [mm] 1.6 1.1 1.1 2 50 Relativesubstratespecifity(%) l-Arginine=100%activity 100 100 100 l-Lysine 73 82 81 l-Ornithine 46 28 35 l-Histidine (cid:3)2 (cid:3)2 (cid:3)2 Thel-Aox,whichischaracterizedinthistable,isencodedinSynechococcuselongatusPCC6301andPCC7942by the aoxA gene (see Table IV). The gene sequence of the l-Aox of Synechococcus cedrorum PCC 6908 is not yetavailable. 276 A.E.Gauetal.·l-AminoAcidOxidasesinCyanobacteria rum PCC 6908 are very similar to the properties 6301 and PCC 7942, the l-Aox in Synechococcus of the l-Aox in Synechococcus elongatus PCC cedrorum PCC 6908 is in part located in the solu- 6301andPCC7942withrespecttosubstratespeci- ble protein fraction of the periplasm and in part ficity, K value for l-arginine, and inhibition by inthespheroplastfraction.Onactivitybasisabout M cations(shownforCaCl inhibition).Moreover,in 40to 60%ofthe totalactivitywasdetected inthe 2 all three Synechococcus species the l-Aox is ex- soluble protein fraction of the periplasm (not pressed constitutively when cells are grown with shown). nitrate as N-source. The amount of the l-Aox ac- Immunocytochemical investigations have con- tivity detectable in cell extracts was somewhat firmed the partially extracellular localization of variable (a range is given in Table II), but in all thel-AoxintheinvestigatedmesophilicSynecho- tested preparations the highest activity was ob- coccus species as previously shown for Synecho- served in Synechococcus elongatus PCC 6301 and coccus elongatus PCC 6301 (Bockholt et al., 1996) the lowest activity in Synechococcus elongatus and as shown in Fig.1 for the l-Aox of Synecho- PCC 7942. As in Synechococcus elongatus PCC coccus elongatus PCC 7942. Nucleotide/amino acid sequence analysis of the aoxA gene of Synechococcus elongatus PCC 6301 encoding anl-Aox with specificity for basic l-amino acids Previouslya10kb EcoRI/BamHIchromosomal DNA fragment (pDL100) from Synechococcus elongatusPCC6301wasshowntocarrythel-Aox encoding gene, which was called aoxA (Bockholt et al., 1995). The sequence of part of this DNA fragment,afterdigestionwithBglIIorPstIresult- ing in two overlapping DNA fragments named pDL14 and pDV18, was re-evaluated. The re-se- quenced DNA region (consisting of 2993 bp with a GC content of 53.5% Ð data base accession number Z48565 Ð entry date July 1998) contains the complete aoxA gene (orf2: bp 1062 to 2549) andtwo incompleteORFstranscribed inopposite direction to the aoxA: orf1 (bp 727 to 1) located upstream of the aoxA and orf3 (bp 2993 to 2568) located downstream of the aoxA. TheaoxA se- quence given underthe accession number Z48565 is 100% identical with the sequence of gene YP_171306ofthewholegenomesequenceofSyn- echococcus elongatus PCC 6301 (Sugita: Nagoya University Ð CanoBase entry in December 2004) andalso100%identicalwiththesequenceofgene ZP_00164087 of the recently available whole ge- nome sequence of Synechococcus elongatus PCC 7942(JGI/TexasA&MUniversity/DOEÐ2005). Synechococcus elongatus PCC 6301 and Synecho- Fig.1. Immunocytochemical detection of the l-amino coccuselongatusPCC7942aretwocyanobacteria, acidoxidaseinSynechococcuselongatusPCC7942.Im- which are very closely related (Golden et al., munocytochemical detection of the l-Aox (encoded by 1989). the aoxA) with the anti-l-Aox antiserum (Engels et al., Evaluation of the re-sequenced nucleotide se- 1992) in combination with gold-labeled anti-rabbit IgG. The cells were grown in BG11 medium for 3 days. A, quence of the aoxA-carrying DNA region led to Controlserum;B,anti-l-Aoxantiserum. theconclusionthattheaoxAgeneconsistsof1488 A.E.Gauetal.·l-AminoAcidOxidasesinCyanobacteria 277 nucleotidesincludingthestopcodonTGAatposi- analyzed with respect to additional functional re- tion bp 2547 to 2549 (GC content of 57.5%). Of gions for FAD binding (see review: Abell and the three possible start codons: ATG starting at Kwan,2001),ledtotheidentificationofthefinger- bp1062,TTGstartingatbp1134,andGTGstart- print site being most likely involved in binding of ing at bp 1188, GTG can be excluded, since it is the ribityl chain of FAD and of a C-terminal re- locatedintheFAD-bindingsite(seebelow).Since gion being part of the active site (marked by A ATG is the most frequently used start codon in and B in Fig.2, respectively). cyanobacteria and since 11 bp upstream of this Oneadditionalaspectshouldbementioned.We ATG a putative Shine-Dalgarno core sequence observed substantial modifications of FAD lead- (1047-AGGA-1050) is located, the most likely ing to modified absorbance spectra of FAD in start of the aoxA is at bp 1062. So far no typical some of the purified enzyme fractions and as a promoter region(s) could be identified. Based on consequence to substantial inactivation of the en- thisassumptionthederivedl-Aoxproteinconsists zyme (Wälzlein et al., 1988). Since a methionine of 495 amino acids with a calculated molecular residue is present in the FAD-binding site (see mass of 54131 Da (the native enzyme is a homo- Fig.2) and since methionine can readily become dimer)andanisoelectricpointof8.52(PCGene). oxidizedbyhydrogenperoxide(beingaproductof 17 amino acids have been verified by partially se- the l-Aox reaction) to methionine sulfoxide and quencingoftheisolatedl-AoxproteinfromSyne- further to methionine sulfone (Kachurin et al., chococcus elongatus PCC 6301 (amino acid resi- 1995), it is likely that in some enzyme molecules dues 101 to 117) (Bockholt et al., 1995). FADbecamemodifiedbyinteractionwiththeoxi- dized methionine residue in the active centre of the enzyme. Characteristics of the FAD-binding motif Previously, we have shown that the l-Aox from SynechococcuselongatusPCC6301aswellasfrom Prediction of a possible translocation pathway PCC 7942 contain noncovalentlybound FAD as a signal prosthetic group(Pistorius and Voss,1980; Engels Bacterial proteins, which contain a redox factor etal.,1992).Thepeptidefragmentbetweenamino of seven different typesincluding FAD and which acid residues 42 to 70 in Synechococcus elongatus are transported into the periplasm, frequently PCC 6301 contains the highly conserved motif of have an unusual long signal sequence bearing the a dinucleotide binding site according to Wierenga consensusS/T-R-R-x-F-L-Kmotifimmediatelybe- et al. (1986) (Fig.2). This binding site (consisting fore the hydrophobic region. According to Berks of 29 to 31 residues in a number of investigated (1996)thetwin-argininesarecompletelyinvariant, FAD-containing enzymes) can be recognized by a and the frequency of occurrence of each of the common fingerprint which consists of a set of 11 other amino acids exceeds 50%. The l-Aox of rulesdescribingthetypeofaminoacidthatshould Synechococcus elongatus PCC 6301 has such a occurinaspecificposition.Whilethethreeglycine twin-arginine translocation pathway signal se- residues as well as the acid residue at the end of quenceof32aminoacidresidueswithapredicted the fingerprint are strictly conserved, the others cleavage site behind the glutamine in position 32 can be changed according to the rules (Wierenga (accordingtoprogramSMART).Thecorrespond- et al., 1986; Eggink et al., 1990). The l-Aox frag- ing motif in Synechococcus elongatus PCC 6301 is ment consists of 29 residues (amino acids 42 to -S-R-R-R-F-L-Q-(aminoacidresidues4to10,see 70) and contains the three conserved glycines Fig.2)withonly oneaminoacidnot followingthe (GxGxxGatposition47to52),theconservedglu- rule (Q instead of K). Cleavage of the l-Aox be- tamic acid residue at the end of the motif as well hindglutamine32wouldresultinamatureprotein as the other seven residues of the fingerprint re- of 50896 Da having an isoelectric point of 6.2. sulting in the maximum fingerprint score of 11. Thus, the cleaved protein is an acidic protein in Secondary structure analysis for the l-Aox pre- contrast to the uncleaved protein with a pI of 8.5. dicts the expected (cid:2)α(cid:2)-fold with the loop posi- The presence of two forms of the l-Aox protein tionedbetweentheα-helixandthesecond(cid:2)-sheet inthecellissupportedbytheobservationthatthe (Fig.2). Comparison of the Synechococcus l-Aox uncleaved protein binds tightly to CM Sepharose sequence with the sequence of FAD-containing at pH 5.5, while the cleaved form does not. Both monoamine oxidases, which have recently been forms catalyze the oxidative deamination of basic 278 A.E.Gauetal.·l-AminoAcidOxidasesinCyanobacteria Fig.2.Alignment oftheAoxA(accession numberYP_171306)and the l-AoxB(accession numberYP_171854)of Synechococcus elongatus PCC 6301 with the monoamine oxidase B from human (MAO B Ð Swiss Prot accession numberP27338).Theasterisksmarkidenticalresidues,doublepointsmarkconservedsubstitutions,andsinglepoints mark semiconserved substitutions. The fingerprint of the dinucleotide-binding site of FAD according to Wierenga et al. (1986) is shown together with the prediction of the two (cid:2)-sheets and the α-helix. Bold letters, dinucleotide- binding site; Δ, basic or hydrophilic amino acid residue; (cid:2), small and hydrophobic amino acid residue; (cid:3), highly conserved glycine residue; (cid:4), highly conserved acidic amino acid residue; -(cid:2)-, (cid:2)-sheets; -α-, α-helix; L, loop; -A-, additionalaminoacidregioncontributingtoFAD-binding;-B-,partofthepresumedactivesite.Moreover,atwin- argininetranslocationpathwaysignalismarkedwithgreycolour. A.E.Gauetal.·l-AminoAcidOxidasesinCyanobacteria 279 l-amino acids with O as electron acceptor (not Since the l-Aox in Synechococcus elongatus 2 shown). PCC 6301 (and also in Synechococcus elongatus It is also well documented in the literature (see PCC 7942) has been shown to be in part located e.g. Wexler et al., 1998; Brink et al., 1998) that a in the soluble protein fraction of the periplasm, twin-arginine motif immediately prior to a hydro- the presence of a putative translocation pathway phobicregionisalsoanessentialfeaturefortrans- signalisinagreementwiththislocation.However, location of proteins across the thylakoid mem- a substantial part of the enzyme remains intracel- brane in a Sec-independent transport system in lular. This finding can possibly be best explained plant chloroplasts. Another protein that we inves- by a partial translocation of the l-Aox into the tigate in our group is a protein called IdiA whose periplasm (via the cytoplasmic membrane) and a expression greatly increases under iron limitation partial transport of the l-Aox to the thylakoid in Synechococcus elongatus PCC 6301 and PCC membrane being there most likely loose associ- 7942 (Michel and Pistorius, 2004). This protein ated with the cytoplasmic side of this membrane. also carries a putative twin-arginine translocation pathway signal in the N-terminal region being -S- R-R-D-F-L-L- with also only one amino acid not Evaluating of 24 cyanobacterial genome followingtherule(LinsteadofK).Thisproteinis sequences for the presence of an aoxA-similar intracellularly located and is mainly attached to gene the thylakoid membrane. As suggested in the lit- erature (Wan et al., 1996; Kouranov and Schnell, We evaluated the genomes of 24 cyanobacteria, 1996), this implies that the passenger protein be- which are listed in Table III, for the presence of hindsuchasignalpeptidealsohasaninfluenceon a gene encoding a protein with similarity to the the localisation of the protein. l-Aox(encodedbytheaoxAgene)withspecificity Table III. Evaluation of 24 Cyanobacterium Presence(+)orabsence(Ð)ofa cyanobacterial genome se- genewithsimilarityto quencesforthepresenceofa gene encoding an l-amino aoxA cphA acidoxidasewithsimilarityto the AoxA in Synechococcus Marinecyanobacteria: elongatus PCC 6301 and for ProchlorococcusmarinusSS120 Ð Ð the presence of a cphA gene ProchlorococcusmarinusMIT9211 Ð Ð encoding a cyanophycin syn- ProchlorococcusmarinusMIT9312 Ð Ð thetase. ProchlorococcusmarinusMIT9313 Ð Ð ProchlorococcusmarinusMED4 Ð Ð ProchlorococcusmarinusNATL2A Ð Ð Synechococcussp.WH8102 Ð Ð Synechococcussp.CC9902 Ð Ð Synechococcussp.RS9917 Ð Ð Synechococcussp.CC9605 + + Ð Synechococcussp.WH5701 + Ð Synechococcussp.WH7805 + Ð TrichodesmiumerythraeumIMS101 + + CrocosphaerawatsoniiWH8501 Ð + Freshwatercyanobacteria: SynechococcuselongatusPCC6301 + + Ð SynechococcuselongatusPCC7942 + + Ð Synechocystissp.PCC6803 + + + GloeobacterviolaceusPCC7421 + + + Nostocsp.PCC7120 + + NostocpunctiformePCC73102 + + AnabaenavariabilisATCC29413 Ð + ThermosynechococcuselongatusBP-1 Ð + SynechococcusYellowstoneA(JA-3-3Ab) Ð Ð SynechococcusYellowstoneB(JA-2-3B(cid:4)a(2-13)) Ð + Two(+)indicatethepresence oftwoaox-similargenes. 280 A.E.Gauetal.·l-AminoAcidOxidasesinCyanobacteria for basic l-amino acids as being present in Syne- so far sequenced Prochlorococcus species an aox- chococcus elongatus PCC 6301 and PCC 7942. type gene was found. Atthebeginningofthisevaluationarathersur- Three of the Aox-type proteins show a higher prisingandunexpectedresultwasobtainedforthe similarity to AoxA than to AoxB of Synechococ- cyanobacteria Synechococcus elongatus PCC 6301 cus elongatus PCC 6301 and PCC 7942 being the and PCC 7942. The recently available complete Aox of Nostoc sp. PCC 7120, Nostoc punctiforme genomesequenceofthesetwocyanobacteriagave PCC 73102, and one of Aox-similar proteins of evidence that Synechococcus elongatus PCC 6301 Gloeobacter violaceus PCC 7421, while all the and PCC 7942 contain besides the previously in- otherAox-similarproteinslistedinTableIVhave vestigated aoxA (accession numbers YP_171306 ahighersimilaritytoAoxBoradistinctioncannot andZP_00164087,respectively)anadditionalgene be made. The molecular masses, pI and identity/ withsimilaritytoaoxAbeingthegeneYP_171854 similarity values of these Aox-type proteins are in Synechococcus elongatus PCC 6301 and showninTableIV,andaphylogenetictreeisgiven ZP_399388 in Synechococcus elongatus PCC 7942 in Fig.3. (Table IV). These genes have 25% identity and The proteins encoded by the aox-type genes 37%similaritytotheaoxAgene.Thenewlyidenti- havesimilaritytoeachother(TableIV)aswellas fied gene was named aoxB. Since the genome se- to several l-amino acid oxidases from snakes [as quence of Synechococcus cedrorum PCC 6908 is e.g. to the l-amino acid oxidase of Calloselasma not yet available, it can not be decided whether rhodostoma (CAB71136) or Crotalus adamanteus this cyanobacterium also possesses an aoxB-type (AAC32267)], and also to the l-amino acid oxi- gene in addition to the aoxA-type gene encoding dase of Neurospora crassa (A38314; Niedermann an l-Aox catalyzing the oxidative deamination of andLerch,1990).Thereisnosignificantsimilarity basic l-amino acids with l-arginine as the best to the l-amino acid oxidase of Chlamydomonas substrate (see Table II). rheinhardtii (Piedras et al., 1992; Vallon et al., BesidesSynechococcuselongatusPCC6301and 1993). A slightly higher similarity than to the PCC7942,thegenomesof22additionalcyanobac- snake l-amino acid oxidases exists to various mo- teria were evaluated for the presence of an aoxA- noamine oxidases (MAO), e.g. to MAO of Myxo- similar gene (Table III). The derived protein se- coccus xanthus DK 1622 (XP_633086), MAO of quences were classified either as A or B depend- Bacillus anthracis str. Ames (YP_018569), MAO ing on whether the similarity was higher to the of Nocardia farcinica IFM 10152 (YP_119289), AoxA or AoxB protein of Synechococcus elonga- MAO of Bacillus cereus (CAA72047), MAO of tus PCC 6301 (Table IV). Besides the two fresh human (P27338) or MAO of Mus musculus water cyanobacteria Synechococcus elongatus (NP_034345), and to the murine interleukin-4-in- PCC 6301 and PCC 7942, both possessing an ducibleFig1proteinpredictedtobeaflavoprotein aoxA- and aoxB-type gene, eight further cyano- with similaritiy to MAOs (Raibekas and Massey, bacteriacontainoneortwogene(s)withsimilarity 1998; Pawelek et al., 2000). to the aoxA gene. In the fresh water cyanobacte- Initially,wethoughtthatacorrelationmightex- rial group these are Synechocystis sp. PCC 6803 (twoaoxA-similargenesÐalthoughonegenewith ist between the presence of an aoxA gene encod- only veryminor similarity), Gloeobacter violaceus inganl-Aoxcatalyzingtheoxidativedeamination PCC 7421 (two aoxA-similar genes), Nostoc sp. of basic l-amino acids and the absence of the cy- PCC7120,andNostocpunctiformePCC73102.No anophycin synthetase-encoding cphA gene (Zieg- aoxA-similar gene is present in the three investi- ler et al., 1998) in Synechococcus species, as being gated thermophilic cyanobacteria (Thermosyne- the case for the fresh water cyanobacteria Syne- chococcus elongatus BP-1 and the two Synecho- chococcus elongatus PCC 6301 and PCC 7942 as coccus Yellowstone species) and also not in the well as Synechococcus cedrorum PCC 6908 (Si- mesophilic N -fixing Anabaena variabilis ATCC mon,1987andTableII).ThepresenceofanaoxA- 2 29413. In the group of the marine cyanobacteria type gene in combination with the absence of a an aoxA-similar gene is present in Synechococcus cphA gene was not found in any of the other sp. CC 9605 (two aoxA-similar genes), WH 5701 cyanobacteria.Thisseemstobeacharacteristicfor and WH 7805 as well as Trichodesmium ery- these three cyanobacteria Ð at least among the thraeum IMS 101 (low similarity). In none of the 24cyanobacteria investigated. A.E.Gauetal.·l-AminoAcidOxidasesinCyanobacteria 281 ofthetwo62matrix, ersimilari-AoxA(A)AoxB(B) ABABBBBA/BBBAAA/BAA/B lpeirsryteIetnshdetrnhatieenugimnrToaubtIhplMeeoSfINItIh12-e0fa1imxicanaprgnhindAeTrcCgiycreahonnoceoodbseipassmchtoaeiuenrrmilaay gnmentBlossom565. Hightytoor twchhaeltosgroronociuoipcWcouHfst8oh5re0Sf1ry,enbsehucthwionactonecorcncueysaonsfpotebhcaeicetPse.rrIoina- aliP,48 to ue 882 3 1 thepresenceofacphAgeneiswidespread. btainedbytheC6301(BlastsionnumberZ ntity/similarityAoxB (%)e-Val 23/361e-500/100023/361e-599/99031/471e-331/464e-332/493e-424/390.232/502e-523/441e-425/403e-124/405e-524/373e-524/411e-722/418e-2 OtaS3hnAynednbley)PcehvCSdoayoClcnuoen7acco9tchi4tuoo2snccooanscoYctfweauliselntolhlewealaosstcn2optg4nhhaeeAtcuytshagAenePrnoCmebC(.oaJcApFt6hre-3oi3rl0miÐia1c valueswereoelongatusPCAoxAofacces aritytoIde e-Value 02e-11102e-119e-112e-73e-80.112e-11Ð7e-186e-197e-54e-148e-5 fogaaoilnfvlugeanpenodonx:isen-sptsiybTropelaersebeplcaneronceImedsIeIboa,nirinccteaapctbhoaiosAnren-nabstbcyeewspceeioonthnfcgecebrlnuoeoedstfhpecoedantcnyettphbetoaoestf sandthee-nechococcusalwiththe dentity/similAoxA (%) 100/10023/37100/10023/3723/3922/4124/4024/3821/38Ð27/4227/4521/3527/4523/40 tplyhrzeoTesvhdeiedgPeeesrnvoeaecvlshu.idlaoetrinoocnceoctghcivuaetsnisnpinneociTneaesb,olwefhtIhiIceIhaanalsraoe- eyc I valuofSdenti 848483749625318 mnoinniemoaflisthtsewanitahlyazesdmtahlelrgmeonpohmileic, acynadnoin- ntity/similarityoxAorAoxBY_171306isi edprotein Mol.masspI[Da] 541317.8526088.8541317.8525998.8528786.2508245.8508665.5484815.0514045.1512754.7477845.1478179.3520795.0303596.6594665.4 TebibnvaahecceTrttree,aerrtbfiihoaaleerae(m1n,I0IataIhjoo)oexur-ctifttouyyonnpftocea1ftigi5notehncneayenoarwnfeomaatbohsaaxefiocn-stetuiyennpregdiena.czyHlgyiaesmontnewoeeds--. a.TheideseitherAne/protein Deriv Length[aa] 495484495484487469473445471458435426473278523 ibunancktthneeoriwaonvi.serIoatflliisnmtweeertelalsbtdoaolincsdummsiseonpftreetdsheenthtclayytasntthoile-l eriwage isolated l-Aox (encoded by the aoxA) of xidasesincyanobactreferencesequence1,extension1).The AccessionNo. 1YP_1713061YP_1718542ZP_001640872ZP_399388YP_382202YP_381069ZP_01083514ZP_01124606NP_442072NP_942249ZP_00110437NP_490275NP_924069NP_92393701ZP_00675074 SaVSPrduriyeCongmnasCicmesne,ci7ihPnn1t9hoeC9a4ec8tC2oia0oecs;anncs6Euzbo9wysne0fmseg8bteleelallossscaiuniasscebgtitSaslantly-tayruanplazms.etae,cersi1hPnt9oC(olt9PcohC2aoices;ccatictoTd6eour3asdxsi0buwi1cdleseieaxtdhaattIirrnnIvoal)dde---. cidoThence1 C630C630C794C794 02 421421MS1 caerglliunlianrely,aasrNol-esoinuructeiliizningnaetxutrraalceelnluvliarronl-- TableIV.Putativel-aminoacomparedproteinsequences.wordsize3,gapcosts:existe Cyanobacterium SynechococcuselongatusPCSynechococcuselongatusPCSynechococcuselongatusPCSynechococcuselongatusPCSynechococcussp.CC9605Synechococcussp.CC9605Synechococcussp.WH5701Synechococcussp.WH7805Synechocystissp.PCC6803Synechocystissp.PCC6803NostocpunctiformePCC731Nostocsp.PCC7120GloeobacterviolaceusPCC7GloeobacterviolaceusPCC7TrichodesmiumerythraeumI mnbs5sas1aituccaenrmμeiictacddmtnihmthoeste,oesnafatsxochnpsuiooprdedhcffueuhalyeclnsa-esdaianeixcfosrszitbtbcgliyrcoroewiiameotngncyhlaaiilee,ncis-dclssa(elhhhuusulsaeaarmlfpaavfensuaererednecacdTceila.KfatsK-iilHubavcoM-MgirlenaoteggrymwiyeovvInIsaefaifni)ntolnvlte.auuohreedreeilrrassl,-cotcoA-ldoawiabfd-nemmAheas3olaunmiiooxnnvbtoxwiioooe--t-, 282 A.E.Gauetal.·l-AminoAcidOxidasesinCyanobacteria Fig.3. Phylogenetic tree of 15 putative l-amino acid oxidases being present in the cyanobacteria shown in Tables III and IV. The phylogenetic tree was constructed on the basis of the program T-COFFE 4.45 using the Blossom 62matrix. dases in some marine phytoplankton genera as PCC6803,whichonlycontainsanAoxB-typepro- Pleurochrysis species (Palenik and Morel, 1990a, tein (and not an AoxA-type protein), we were b). Therefore,it seemsmore likelythat thel-Aox neverabletodetectanl-aminoacidoxidaseactiv- in Synechococcus elongatus PCC 6301 and PCC itybymeasuringtheO -uptakeinthepresenceof 2 7942 has a role in degrading l-arginine intracellu- l-arginine with cells grown with nitrate or with l- larly, since excess l-arginine can not be stored in arginine, and this holds also true for Nostoc sp. cyanophycin due to the absence of the CphA en- PCC7120,whichhasanAoxwithhighersimilarity zyme. to the AoxA-type protein. This would imply that The function as well as the activity of all the these enzymes do not interact with oxygen and other aoxgene products (AoxA andAoxB) listed therefore might be l-amino acid dehydrogenases, possibly interacting with the respiratory electron in Table IV are so far completely uncertain. On chain. In E. coli it has been shown that an amino the basis of similarity evaluations the Aox-type acid dehydrogenase can interact with the respira- proteins could be an l-amino acid oxidase or a tory electron chain (Anraku and Gennis, 1987). monoamine oxidase. The enzymes contain a The true activity of the Aox-type proteins, except GxGxxG motif for dinucleotide binding, suggest- for the three l-Aox proteins isolated from Syne- ing that AoxA as well as AoxB are FAD-binding chococcus elongatus PCC 6301, Synechococcus proteins. In Synechococcus elongatus PCC 6301 elongatusPCC7942,andSynechococcuscedrorum andPCC7942bothenzymeshaveaputativetrans- PCC 6908 (Table II), and their function are still locationpathwaysignalsequencewithatwin-argi- uncertain.Thisaspectispresentlyunderinvestiga- nine motif (Berks, 1996). However, in case of tionwithrespect tothepropertiesand functionof AoxB this sequence deviates from the rule in two the enzyme in Synechocystis sp. PCC 6803. amino acids (the motif being -S-R-R-A-L-L-G- havingLinsteadofFandGinsteadofK)(Fig.2). Acknowledgement Thus, the relative distribution between intra- and extra-cellular location might be different between The financial support of the Deutsche For- the AoxA and AoxB protein. In Synechocystis sp. schungsgemeinschaft is gratefully acknowledged.