2. PROSPECTING FOR THE ACID PHOSPHATASE GENE FROM THE MYCO-
2.3 RESULTS
2.3.2 Phylogeny relationship with similar proteins from different organisms
57
sp|P20584| eqqhLlcPqrqvhHphhqrhgrqh--- tr|Q5AR94| vVNwefvkgDdgstgDWlTlvkget---ctinv---- sp|Q12546| alkwelirgDdgtvgDslTllkpsH---vAggkKlHs tr|A2R1M4| alkwelirgDdgtvgDslTllkpsH---vAggkKfHa tr|A1D0I1| eVkwefirgDdgsvgDylTl---rk---ektqcKgN- tr|B0Y1M6| eVkwefirgD-gsvgDylTl---rk---ektqsKEk- tr|Q8J255| G---DWaeC---fa--- tr|E3UHI6| G---DWaeC---fa--- tr|Q6J336| G---DWgeC---fa--- tr|O93838| G---DWgeC---fa--- tr|A2QIG7| G---DWgeC---fa--- tr|Q6GYA8| G---DWgeC---fa--- sp|P34752| G---DWaeC---fa--- tr|B3VPB2| G---DWaeC---fa--- tr|Q1KNE3| G---nWgeC---fa--- tr|B0Y655| G---nWgeC---fs--- tr|A1CXB1| G---nWgeC---fs--- tr|B6H7V5| G---DWarC---yk--- tr|D9I0I9| G---DWakCgvssk--- tr|Q6YNE9| G---DWakCgvssk--- tr|Q2HPM1| dVpt-dyPqhLlFFWnynkttdfnYqkdPIgyQanlitwDgkpfKKsk tr|A1CXF7| eIpe-sdPqhLdFWWDysTattdnYR---Detkcd--- tr|D5HQ11| nVSa-syPqhLSFWWnynTttelnYRSSPIACQ---egdamd--- sp|P34755| nVSa-syPqyLSFWWnynTttelnYRSSPIACQ---egdamd--- sp|P52289| eVqqVsnttELTFYWDynev---eY---NgpvsnK-- tr|B3LGS8| dVSkVskekELTFYWDWnTt---rY---NAslvnQ-- sp|P38693| nVSsVsnstELTFFWDWnTk---hY---NdtllKQ-- tr|C7GJR1| nVSsVsnstELTFFWDWnTk---hY---NdtllKQ-- tr|D3UEI8| nVSsVsnstELTFYWDWnTt---hY---NAsllRQ-- sp|P00635| nVSsVsnstELTFYWDWnTt---hY---NAsllRQ-- sp|P24031| nVSsVsnvtELTFYWDWnTt---hY---NdtllKQ-- tr|A3LV80| Glkd-dvPqDVSFYWDyksv---tY---NAtlgnf-- tr|A3LUP9| Gper-gsPlDLTFYWDykTi---kY---DAplidQ--
Figure 2.1: Multiple sequence alignment of 33 selected representatives of the HAP super family of fungal and yeast apases. Abbreviation of the enzyme sources are defined in Table 2.1 and represented in the phylogram in Figure 2.2. Numbers indicate the position of each presented segment within the corresponding sequence. Sequences are grouped together as discussed in the text. Similar residues are coloured as the most conserved one (according to BLOSUM62). Average BLOSUM62 score: Max: 3.0 Mid: 1.5 Low: 0.5
58 Within five initial selected sequences, the sequence alignment revealed that PHO3, PHO5 and secreted acid phosphatases had a strong homology to each other, thus these enzymes were grouped together as group B (Figure 2.2). The secreted apases comprised of soil and plant pathogen fungi such as Botryotinia fuckeliana. This group also aligned well with PHO3. All these proteins are controlled by phosphorus concentration in the medium and are secreted glycoproteins.
Due to poor homology displayed by apase genes, few sequences were selected for alignment in order to design degenerate primers (Figure 2.3). Strong homology was found in the active sites and HD residues. These regions were used to design the primers (Figure 2.3).
59 Figure 2.2: A phylogenic tree (N-J with branched length) demonstrate the order of evolutionary events in three groups of apases used in this study. All taxonomic entities are part of Actonomytes, comprising of 33 protein sequences.(A) indicate the secreted apase known as aph, encoding gene expressed at pH 2.5 (A. niger, phyB); (B) secreted apases (including yeast apases); (C) apase proteins encoding pH 5.0 (A. niger, phyB). The tree was constructed by PhyML 3.0 (www.phylogeny.Fr). Most nodes are on the same path, thus they are related to each other. The scale bar represents 30% of the estimated sequence divergence.
A
B
C
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1 50
Arxula adeninivorans (1) ---MLNAISVLALAALVNGQLYGGGPEKQFSQKIDDGYNLL Aspergillus clavatus (1) ---MLYSNSQLLVLIPTVAASTLLFSQQTLDGNNIF Neosartorya fischeri (1) ---MVRRYAQLLVLIP-ATTATLLFSQQTLDGNNIL Aspergillus niger (1) ----MPRTSLLTLACALATGASAFSYGAAIPQSTQEKQFSQEFRDGYSIL Monascus purpureus (1) MPLFSFLSATPTVMQLIFTVAAIASVAAGFQSVISEKQFSQEFLDNYSIL Consensus (1) A A L EKQFSQ LDGYNIL 51 100 *******
Arxula adeninivorans (39) KHFGHLGPHTDRQSYGISRDTPAQCEVDQVIMVHRHGERYPDASDTYPQL Aspergillus clavatus (34) KHNGAMGPYVDRTSY---GCMVDQVIMIKRHGERYPLPGSEGPKI Neosartorya fischeri (33) KHNGAMGPYVDRSNYGINRAPPAGCSVDQVIMIKRHGERYPLAS-EGPKI Aspergillus niger (47) KHYGGNGPYSERVSYGIARDPPTSCEVDQVIMVKRHGERYPSPS-AGKDI Monascus purpureus (51) KHYGGNGPYSSRRSYGISREPPDSCSVDQVIMIMRHGERYPSPD-LGASI Consensus (51) KH G GPY DR SYGI R PP C VDQVIMIKRHGERYP PS GP I 101 150 Arxula adeninivorans (89) VEALEKLYNYSDQFT---SGSLEFLNTWESFLNDEAMLEQETYSGPYNGL Aspergillus clavatus (76) EDALQKVKDAILDEPPISDGDLAFVKNWTYFVSSDCYYD---AGI Neosartorya fischeri (82) EKALQKVKKAVFDEP-HADGDLDFVKNWTYFVPSSCYYDKETTTGPYNGI Aspergillus niger (96) EEALAKVYSINTTEY---KGDLAFLNDWTYYVPNECYYNAETTSGPYAGL Monascus purpureus (100) EAALAKIKSSNVSTY---QGDLDFLNSWTYYVPNHCAYNAETSTGPYAGL Consensus (101) E AL KVK E GDL FLN WTYFVP CYY ET GPYAGL 151 200 Arxula adeninivorans (136) KTAFDRGVDYRARYGHLWDGEG-VPIFTSGSQRVLDTARRFGEGFFG-YN Aspergillus clavatus (118) QAAYSHGVDARNRYGHLWDEETVIPLFASDTSRIVDTARMFGEGFFGAAD Neosartorya fischeri (131) QDAYKHGMDARNRYGHLWDEETIVPLFASDAGRIVDTARMFGEGFFGDDE Aspergillus niger (143) LDAYNHGNDYKARYGHLWNGETVVPFFSSGYGRVIETARKFGEGFFG-YN Monascus purpureus (147) LEGFKRGSDYRARYGHLWDGESIVPIFAAGYQRIIATSRKFGEGFFG-AN Consensus (151) AY HG DYRARYGHLWDGET VP FASG RI DTAR FGEGFFG N
Figure 2.3: Multiple sequence alignment of apase genes from different fungal and yeast organisms used to design degenerate primers for Cladonia portentosa.
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201 250 Arxula adeninivorans (184) YSSKAYMNIIPETEDQGANSLTPPCFVP-SNVPFSIFFAPPVLASFFDAA Aspergillus clavatus (168) YKTSALN---IVSELSRACRSRDADGQSICDAWPQTLPQLEVAA Neosartorya fischeri (181) YKTKAAINIIPESARQGANALSRTCHARDHHAQRICDAWPQSLPQLELAT Aspergillus niger (192) YSTNAALNIISESEVMGADSLTPTCDT--DNDQTTCDNLTYQLPQFKVAA Monascus purpureus (196) YSTNAAINIISEAKEMGANSLTPTCDH--DNDTSTCNSLTTVWPQFKVAA Consensus (201) YST AA NII E GANSLTPTC N Q CD P LPQF VAA 251 300 Arxula adeninivorans (233) DRLNREYPGLNLTATDVKTLMNLAPYELNTRPYTPWADVFTRDEWIAYRY Aspergillus clavatus (209) QRLNAQYSSLNLTSTDVFWLMTMASYEPSVRGYS---NEWVSLGY Neosartorya fischeri (231) QRLNGQYLGLDLTSTDIFWLMTMSSYEPSVRGHSDWTGVFTMDEWVSFGY Aspergillus niger (240) ARLNSQNPGMNLTASDVYNLMVMASFELNARPFSNWINAFTQDEWVSFGY Monascus purpureus (244) ARLNSQNPGLDLNATDIYYLMSMASFELNARPYSDWINVFTLDEWVTFGY Consensus (251) RLN QYPGLNLTATDV LM MASYELN RPYS W VFT DEWVSFGY 301 350 **
Arxula adeninivorans (283) TFDLAFYYFAGPGSNTSAAVGSVYSNATLALLNQGPEKAGKLHFSFAHDT Aspergillus clavatus (251) IWDLHFYYCAGPGNEKMRSVGAVYVNATLALLNWGPS-SGTLFFNFAHDT Neosartorya fischeri (281) IWDLHFYYCAGPGNKMMRPVGSLYVNASLALLEQGPS-SGTLFFNFAHDT Aspergillus niger (290) VEDLNYYYCAGPGDKNMAAVGAVYANASLTLLNQGPKEAGSLFFNFAHDT Monascus purpureus (294) VQDLNYYYCAGPGDKNMAAVGAVYVNASLTLLNQGPS-AGTLWFNFAHDT Consensus (301) DL FYYCAGPG K MAAVGAVYVNASLALLNQGPS AGTLFFNFAHDT 351 400 **
Arxula adeninivorans (333) NITPILYALGLLVPERPLPKDYIDWTSPYKISDIMPMGGHLVLERLACNA Aspergillus clavatus (300) DITPII---DADLPVDRVAFGSPWSSSELVPMGGHLVMERLSCNA Neosartorya fischeri (330) DITPIIGALGILNPPEDLPTDRVSFGHSWLSSELVPMGGHLTMERLSCNA Aspergillus niger (340) NITPILAALGVLIPNEDLPLDRVAFGNPYSIGNIVPMGGHLTIERLSCQA Monascus purpureus (343) NITPILAALGVLTPERDLPTDRVVFDSKWSSGDIVPQAGHLTIERLNCTS Consensus (351) NITPIL ALG L P DLP DRV FGSPWSSS IVPMGGHLT ERLSCNA
Figure 2.3 (Continued) Multiple sequence alignment of apase genes from different fungal and yeast organisms used to design degenerate primers for Cladonia portentosa
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401 450 Arxula adeninivorans (383) TAKYPKGSYARAVLNEAVVPFNECQNGPGFSCPLSNYTELVGGRAQALDY Aspergillus clavatus (342) TAVSPAGPYVRLVLNEAVVAFRACQSGPGY---SILSGDLPDF Neosartorya fischeri (380) TAIVPAGTYVRVVLNEAVVPFRACQSGPGYSCPLQEY--ASIVRQDLPDY Aspergillus niger (390) TALSDEGTYVRLVLNEAVLPFNDCTSGPGYSCPLANY--TSILNKNLPDY Monascus purpureus (393) TAASPAGVYVRLVQNEAVIPVEACQSGPGYSCSLADF--TEIMSKQLPDF Consensus (401) TA SPAG YVRLVLNEAVVPF ACQSGPGYSCPL Y SI LPDY 451 500 Arxula adeninivorans (433) VSTCDVPTDYPQHLLFFWNYNKTTDFNYQKDPIGYQANLITWDGKPFKKS Aspergillus clavatus (382) VSECEIPESLPQYLDLWWNYSSSFVNLQMESR--- Neosartorya fischeri (428) VLECEIPESDPQHLDFWWDYSTATTDNYRDETKCD--- Aspergillus niger (438) TTTCNVSASYPQYLSFWWNYNTTTELNYRSSPIACQEGDAMD--- Monascus purpureus (441) VSTCSIRSSYPQYLDFWWNYNTTTDLNYPKGPVPCAEGVATS--- Consensus (451) VSTC IP SYPQYLDFWWNYNTTT NY P 501
Arxula adeninivorans (483) K Aspergillus clavatus (414) - Neosartorya fischeri (463) - Aspergillus niger (480) - Monascus purpureus (483) - Consensus (501)
Figure 2.3: (Continued) Multiple sequence alignment of apase genes from different fungal and yeast organisms used to design degenerate primers for Cladonia portentosa. Numbers indicate the position of each presented segment within the corresponding sequence. Sequences are grouped together as discussed in the text according to physiological function. Sequence similarities were graded from light green (low similarity) to yellow (highest similarity). Yellow indicates identical residues, green and blue indicate similar residues and a white letter (no colour) are regions of non- homology. Functionally important residues involved in the catalytic mechanism are marked with squares. The figure was constructed using Vector NTI (Invitrogen). Gaps are indicated by dashes. The serine and arginine residues forming the catalytic sites (phosphorylation site) and phosphate binding site of fungal apases are indicated by *
63