Enzyme Evolution
Home Up Introduction Structural Characterization Catalytic Mechanism Enzyme Evolution References

 

Enzyme Evolution:

   Recent scientific studies provide strong evidence that nucleic acid polymerases share a common ancestor.  Structural analyses support the notion that the similarities between polymerases go beyond the arbitrary assignment into classes on the basis of the use of DNA or RNA templates and deoxyribonucleoside or ribonucleoside triphosphates (Figure 18).  In fact, it has been postulated that most of the nucleic acid polymerizes belong to a polymerase superfamily containing closely related active sites that are similarly positioned within the polymerase active cleft (Figure 19).  Thus, it appears that there is a genetic polymerase module that provide the active site the architecture to carry out the phosphoryl transfer reaction (Joyce, 1997).   Only subtle modifications to this module achieve the substrate specificity that is unique for each polymerase class.  We see over and over in the course of evolution the tendency of nature to copy and paste particular structural motifs.  

 

Figure 18.jpg (65987 bytes)

Figure 18. Schematic representation illustrating the division into classes of nucleic acid polymerases in terms of sugar and template  specificities.

 

   As Joyce C. (1997) has stated   "nucleic acid polymerases are suffering from an identify crisis."   Recent published papers have brought us,  a DNA  polymerase as a reverse trancriptase (Ricchetti et. al., 1993), the T7 RNA polymerase as a DNA polymerase (Sousa el. al., 1995), and a DNA polymerase as an RNA polymerase (Gao et. al., 1997).  This switch in specificity was done by single amino acid substitution.  As illustrated in Figure 19, the polymerase domains of the Klenow fragment, the HIV-1 RT, and that for T7 RNA polymerase are structurally similar.  The yellow spheres shown in the palm domain represent the catalytic aspartate residues.  The relative positions of the residues involved in sugar discrimination are indicated in each polymerase structure.

 

  

Figure 19. Illustration of the polymerase domains of Klenow fragment, HIV-1 RT, and T7 RNA polymerase (Joyce, 1997).  

 

    DNA polymerases discriminates against ribonucleotide through a "steric gate" mechanism.  For example, the Klenow fragment and the HIV-RT polymerase discriminate against ribose sugar because bulky amino acid residues (Phe762 and Tyr155, respectively) make steric interaction with the 2'-OH group of the incoming nucleotide (see Figure 19).  The Moloney murine leukemia virus (MoMLV) polymerase contains Phe155, which is homolog to Tyr155 in HIV-RT.  Figure 20 shows how Phe155 in MoMLV clashes with the 2'-OH group of the incoming ribonucleotide (Gao et. al., 1997).  Thus, mutation of this bulky residue to alanine, as it was found, will switch the sugar specificity of the polymerase.

 

        Figure 20.jpg (90970 bytes)

Figure 20. In MoMLV RT, Phe155 acts as a "steric gate" to prevent incorporation of r-NTPs (Gao et. al., 1997).

 

On the other hand, T7 RNA polymerase uses a totally different mechanism to discriminate against deoxyribose nucleotides.  Studies have shown that, in  T7 RNA polymerase, Tyr639 makes hydrogen-bonding interaction with the 2'-OH group of the incoming ribonucleotide.  Mutation in this position from Tyr to Phe renders the polymerase specific for deoxynucleotides.  (Sousa et. al., 1995). 

    Bacteriophage T7 RNA polymerase shares some amino acid sequence similarities with other RNA polymerases. Table 2 illustrates the result obtained when the FASTA program was used for sequence alignment.  As it is shown, only bacteriophage T3 RNA polymerase has the highest amino acid identity (82 %) to T7 RNA polymerase, while the other RNA polymerases share only about a 29 % amino acid identity.

 

Table 2. Amino acid identity between T7 RNA polymerases and other RNA polymerases.

            Polymerases    % of identity           amino    acid        number        amino acid overlap

 
Bacteriophage T7 RNA polymerase      100     883             1- 883:1883
Bacteriophage T3 RNA polymerase       81.9     884             1-883:1-884
Bacteriophage SP6 RNA polymerase      32.3     874            36-883:8-874
ARATH mitochondrial RNA polymerase      28.8     976          16-882:115-975
Yeast mitochondrial RNA polymerase      27.9    1351         36-875:414-1249
ARATH chloroplastic RNA polymerase      27.8     993         30-882:160:992
Arabidopsis RNA polymerase      29.0    1011         40-882:163-1010
Zea mays RNA polymerase      28.3     975          27-882:133-974

 

Figure 21. illustrates in more details the amino acid sequence similarities among some RNA polymerases.  The alignment was generated using the BLAST program.  When the complete amino acid sequence of bacteriophage T7 RNA polymerase was used, BLAST pulled out a consensus sequence that is common to some RNA polymerases.  As it is expected, this sequence belongs to the polymerase domain where the active site is located. 

 

 

 

   B                     10        20        30        40        50        60
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus     1 GKPLG-PSGLKWLKIYGANLF--GLDKKSFDERIA--WVEENLDNIIDSANNPLTGE-WW 54
1QLN_A      449 GKPIG-KEGYYWLKIHGANCA--GVDKVPFPERIK--FIEENHENIMACAKSPLENT-WW 502
gi 133451   440 GRPVNgVEALKWFCINGANLW--GWDKKTFDVRVSnvLDEEFQDMCRDIAADPLTFT-QW 496
gi 3914832  584 GKPLG-KSGLRWLKIHIANLYagGVDKLAYEDRIA--FTESHLEDIFDSSDRPLEGKrWW 640
gi 3914826  601 GRPLG-KSGLHWLKIHLANLYagGVEKLSHDARLA--FVENHLDDIMDSAENPIHGKrWW 657
gi 3914825  696 GKPLG-PKGLNWLKVHLANLF--GISKKDFATRQA--FVDDNMQEVFDSADRPLDGNkWW 750
gi 133457   854 GKKLG-PSGLKWLKIHLSNLF--GFDKLPLKDRVA--FTESHLQDIKDSAEKSLTGDrWW 908
gi 730615   809 GKPLG-ESGLRWLKVHLANVY--GFDKASLQERQD--FADENIENIRDSVNNPLNGNqWW 863
gi 3914823  830 GRPLG-PHGLDWLKIHLVNLTg-LKKREPLRKRLA--FAEEVMDDILDSADQPLTGRkWW 885                                                                 
                        70        80        90       100       110       120
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus    55 DKADNPFQFLAFCFELAKYLDNPd---pESFISHLPIHQDASCNGLQHYAALLRDSVLAK 111
1QLN_A      503 AEQDSPFCFLAFCFEYAGVQHHG-----LSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGR 557
gi 133451   497 AKADAPYEFLAWCFEYAQYLDLVdegraDEFRTHLPVHQDGSCSGIQHYSAMLRDEVGAK 556
gi 3914832  641 LNAEDPFQCLAACINLSEALRSPf---pEAAISHIPIHQDGSCNGLQHYAALGRDKLGAD 697
gi 3914826  658 LKAEDPFQCLAACVILTQALKSPs---pYSVISHLPIHQDGSCNGLQHYAALGRDSFEAA 714
gi 3914825  751 SKADDPFQALAACFEIAEAVRSGd---hESYISHIPIQQDGTCNGLQHYAALGGDIEGAK 807
gi 133457   909 TTADKPWQALATCFELNEVMKMDn---pEEFISHQPVHQDGTCNGLQHYAALGGDVEGAT 965
gi 730615   864 LQAEDPWQCLATCFELAAALELEd---pTKYVSHLPIHQDGTCNGLQHYAALGGDTWGAQ 920
gi 3914823  886 MGAEEPWQTLACCMEVANAVRASd---pAAYVSHLPVHQDGSCNGLQHYAALGRDSVGAA 942                                                                 
                       130       140       150       160       170       180
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus   112 AVNLLPStDSPRDFYSFVAPCVIKFLKADAEKK------------T-------------- 145
1QLN_A      558 AVNLLPS-ETVQDIYGIVAKKVNEILQADAINGtdnevvtvtdenTgeise--------- 607
gi 133451   557 AVNLKPS-DAPQDIYGAVAQVVIKKNALYMDADdattftsgsvtlSg------------- 602
gi 3914832  698 AVNLVTG-EKPADVYTEIAARVLKIMQQDAEEdp----------eT-------------- 732
gi 3914826  715 AVNLVAG-EKPADVYSEISRRVHEIMKKDSSKdp----------eS-------------- 749
gi 3914825  808 QVNLWPS-DHPSDVYEAVAEIVRGFLKKDAEAG--------------------------- 839
gi 133457   966 QVNLVPS-DKPQDVYAHVARLVQKRLEIAAEKG--------------------------- 997
gi 730615   921 QVNLVPG-DRPADVYSAVAKLVIKGIEDDLAK---------------------------- 951
gi 3914823  943 SVNLEPS-DVPQDVYSGVAAQVEVFRRQDAQR---------------------------- 973                                                                 
                       190       200       210       220       230       240
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus   146 -------KDIESAARLLDIGITRKVVKKTVMTIPYGVTYVGLRKQIEEKLEEKSDDEDEK 198
1QLN_A      608 ---kvklGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKG 664
gi 133451   603 ------tELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQK 656
gi 3914832  733 -------FPNATYAKLMLDQVDRKLVKQTVMTSVYGVTYSGARDQIKKRLKERGTFEDDS 785
gi 3914826  750 -------NPTAALAKILITQVDRKLVKQTVMTSVYGVTYVGAREQIKRRLEEKGVITDER 802
gi 3914825  840 ----------DEMANFLKDKVTRSVVKPTVMTNVYGVTYVGARKQISEKLENIDGMEKLK 889
gi 133457   998 ----------DENAKILKDKITRKVVKQTVMTNVYGVTYVGATFQIAKQLSPIFDDRKES 1047
gi 730615   952 ---------DNEFAKAMHGKITRKVVKQTVMTNVYGVTYVGARKQVLKQIEAAYPNITAE 1002
gi 3914823  974 --------GMRVAQVLES-FITRKVVKQTVMTVVYGVTRYGGRLQIEKRLRELSDFPQEF 1024                                                                 
                       250       260       270       280       290       300
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus   199 LIF---VG---AATYLAKKVFKALREMFLLAKAIYNWLGACAKKLASLLTyyNSIVSIKL 252
1QLN_A      665 LMF---TQpnqAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVK--DKKTGEIL 719
gi 133451   657 AVA---EG----RTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVkaPIVAMKMI 709
gi 3914832  786 LTF---H----ASCYAAKITLKALEEMFEAARAIKSWFGDCAKIIASENN---------- 828
gi 3914826  803 MLF---A----AACYSAKVTLAALGEIFEAARAIMSWLGDCAKIIASDNH---------- 845
gi 3914825  890 VAD---Y-----ANYLTKKVFEALRSLFTQAHEIQDWLSACCNLITHSLPadYIKEGIK- 940
gi 133457  1048 LDF---S------KYLTKHVFSAIRELFHSAHLIQDWLGESAKRISKSIRldVDEKSFKN 1098
gi 730615  1003 SGI---EAa-lLASYVTQHIFRAMSTMFKGAHDIQNWLGEIGGRVCRALTpeQLDEFERS 1058
gi 3914823 1025 VWE---A-----SHYLVRQVFKSLQEMFSGTRAIQHWLTESARLISHMGS---------- 1066                                                                 
                       310       320       330       340       350       360
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus   253 ----------------------------------KLDIPVIWVTPLGLPVVQPYRKSKKK 278
1QLN_A      720 ----------------------------------RKRCAVHWVTPDGFPVWQEYKKPIQT 745
gi 133451   710 rqla--------------------------rfaaKRNEGLMYTLPTGFILEQKIMATEML 743
gi 3914832  829 --------------------------------------AVCWTTPLGLPVVQPYRKPGRH 850
gi 3914826  846 --------------------------------------PVRWITPLGLPVVQPYCRSERH 867
gi 3914825  941 ----------------------------------DELTPVVWTTLLNLPIVQPYRNYKSR 966
gi 133457  1099 gn------------------------------kpDFMSSVIWTTPLGLPIVQPYREESKK 1128
gi 730615  1059 ersphgdgtasgenitlagnprkssahkndeilnNFQSTIIWTTPLRMPVVQPYRKHGTK 1118
gi 3914823 1067 --------------------------------------VVEWVTPLGVPVIQPYRLDSKV 1088                                                                 
                       370       380       390       400       410       420
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus   279 K-----TVPTNLQDTTLTLK-ETDKVDVR--KQKTAFMPNFIHSLDASHLILLADSCNKK 330
1QLN_A      746 RlnlmfLGQFRLQPTINTN--KDSEIDAH--KQESGIAPNFVHSQDGSHLRKTVVWAHEK 801
gi 133451   744 R-----VRTCLMGDIKMSLqvETDIVDEA--AMMGAAAPNFVHGHDASHLILTVCELVDK 796
gi 3914832  851 ------LVKTTLQVLTLSR--ETDKVMAR--RQMTAFAPNFIHSLDGSHMMMTAVACNRA 900
gi 3914826  868 ------LIRTSLQVLALQR--EGNTVDVR--KQRTAFPPNFVHSLDGTHMMMTAVACREA 917
gi 3914825  967 ------QIRTNLQTVFIEE--RDRTATVQphKQATAFPPNFIHSLDATHMFMTCLKCSEQ 1018
gi 133457  1129 ------QVETNLQTVFISDpfAVNPVNAR--RQKAGLPPNFIHSLDASHMLLSAAECGKQ 1180
gi 730615  1119 ------TVSTCMQDLVMTIpeRSDPVNRR--KQLQAFPPNFIHSLDASHMILSALHCDEL 1170
gi 3914823 1089 Kqi--gGGIQSITYTHNGD--ISRKPNTR--KQKNGFPPNFIHSLDSSHMMLTALHCYRK 1142                                                                 
                       430       440       450       460       470       480
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus   331 GG-L-NFASVHDCFGTHANDMDTLNKLLRETFIRLYSNDDYLLKLDDDFQNRIKNALDey 388
1QLN_A      802 YGiE-SFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQL-- 858
gi 133451   797 Gv-T-SIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQKLLEEHEVRWMV----- 849
gi 3914832  901 G--L-SFAGVHDSFWTHACDVDVMNTILREKFVELYEK-PILENLLESFQKSFPD----- 951
gi 3914826  918 G--L-NFAGVHDSYWTHACDVDTMNRILREKFVELYNT-PILEDLLQSFQESYPN----- 968
gi 3914825 1019 N--I-NFAAVHDSYWTHACDVDQMNSLLREAFVLLHSN-NIMERLKQEFEERYKGFLVsk 1074
gi 133457  1181 G--L-DFASVHDSYWTHASDIDTMNVVLREQFIKLHEV-DLVLRLKEEFDQRYKNYVKig 1236
gi 730615  1171 G--L-TFAAVHDSFWTHASDIDSMNAVLRDAFIRIHSE-DVIGRLAAEFQARYKNSLYla 1226
gi 3914823 1143 G--L-TFVSVHDCYWTHAADVSVMNQVCREQFVRLHSE-PILQDLSRFLVKRFCSEPQki 1198                                                                 
                       490       500       510       520       530       540
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus   389 kenektde---------------------------------------------------- 396
1QLN_A          ------------------------------------------------------------
gi 133451       ------------------------------------------------------------
gi 3914832      ------------------------------------------------------------
gi 3914826      ------------------------------------------------------------
gi 3914825 1075 kaikandedl-------------------------------------------------- 1084
gi 133457  1237 klkrstdlaqkiirirkdlsrklgrsttladeiyfekkrqelln---------------- 1280
gi 730615  1227 kietgtkvaqeiqrwrvrnklgprkelllekerqellrssnpedvergkkmispaslyel 1286
gi 3914823 1199 lea--------------------------------------------------------- 1201                                                                 
                       550       560       570       580       590       600
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus       ------------------------------------------------------------
1QLN_A          ------------------------------------------------------------
gi 133451       ------------------------------------------------------------
gi 3914832      ------------------------------------------------------------
gi 3914826      ------------------------------------------------------------
gi 3914825      ------------------------------------------------------------
gi 133457       ------------------------------------------------------------
gi 730615  1287 yssaedltvpedlkevtignlagveetkvrrgremdeegevdgseeavehedgmhedeml 1346
gi 3914823      ------------------------------------------------------------                                                                 
                       610       620       630       640       650       660
                ....*....|....*....|....*....|....*....|....*....|....*....|
consensus   397 ----------------------------------------eaitglsnydILKFPDLPKV 416
1QLN_A      859 ---------------------------------------------------DKMPALPAK 867
gi 133451   850 ---------------------------------------------------DTGIEVPEQ 858
gi 3914832  952 ---------------------------------------------------ISFPPLPER 960
gi 3914826  969 ---------------------------------------------------LVFPPVPKR 977
gi 3914825 1085 ----------------------------------------kakfgnksyipLEFPPLPAR 1104
gi 133457  1281 -----spliedrnvgekmvttvslfeditdldalelenggdensgmsvllpLRLPEIPPK 1335
gi 730615  1347 adeprdmdgnsgldelselrntnhfalsqkrakasiasggkqkhyldiwlpLVFPPIPEK 1406
gi 3914823 1202 -----------------------------------------------sqlkETLQAVPKP 1214                                                                 
                       670
                ....*....|....*...
consensus   417 GDnTFDLKEILNSDYFFN 434
1QLN_A      868 GN--LNLRDILESDFAFA 883
gi 133451   859 GE--FDLNEIMDSEYVFA 874
gi 3914832  961 GD--FDLRKVLESTYFFN 976
gi 3914826  978 GD--FDLKEVLKSQYFFN 993
gi 3914825 1105 GA--LDLKKVLESKYFFS 1120
gi 133457  1336 GD--FDVTVLRNSQYFFS 1351
gi 730615  1407 GD--FDVRSLKDSTYFFS 1422
gi 3914823 1215 GA--FDLEQVKRSTYFFS 1230

Figure 21.  Multiple alignments correspoding to part of the polymerase domain of T7 RNA polymerase, 1QLN, and other RNA polymerases.   In (A), the concensus domain among RNA polymerases is shown relative to the amino acid sequence of T7 RNA polymerase; in (B), the alignment sequence is displayed.  The polymerases are labeled according to their PID accession number.

 

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