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Molecular and Biochemical Characterization of a Novel Dihydrodiol Dehydregenase

Molecular and Biochemical Characterization of a Novel Dihydrodiol Dehydregenase in the Degradation of Aromatic Hydrocarbons by Sphingomonas yanoikuyae B1. Hwang, Sun-young. H. OH. H. OH. CH₃. OH. OH. H. H. H. OH. OH. OH. H. OH. CH₃. OH. OH. OH. OH. ◈ Introduction

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Molecular and Biochemical Characterization of a Novel Dihydrodiol Dehydregenase

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  1. Molecular and Biochemical Characterization of a Novel Dihydrodiol Dehydregenase in the Degradation of Aromatic Hydrocarbons by Sphingomonas yanoikuyae B1 Hwang, Sun-young

  2. H OH H OH CH₃ OH OH H H H OH OH OH H OH CH₃ OH OH OH OH ◈ Introduction Dihydrodiol Dehydrogenase 3,4-dihydro-3,4-dihydroxyphenanthrene cis-biphenyl 2,3-dihydrodiol cis- toluene dihydrodiol TodD BphB PhnB 2,3-dihydroxybiphenyl 3-methylcatechol 3,4-dihydroxyphenanthrene ex.P. Putida F1 Burkholderia cepacia LB400 B. sp.Strain RP700

  3. H OH COOH OH OH OH H H cis-1,2-dihydroxy-1,2- dihydronaphthalene 2-hydro-1,2-dihydroxybenzoate BenD NahB OH OH OH OH 1,2-dihydroxynaphthalene catechol ex. P. putida G7 Acinetobacter calcoaceticus

  4. Crystal structure : Burkholderia cepacia sp. LB400 (2,3-dihydro- • 2,3-dihydroxybiphenyl dehydrogenase) • (Hulsmeyer M. et al, 1998, Protein Science (7):1286-1293) • Rat liver (3-alpha-hydroxysteroid/dihydrodiol • dehydregenase) • (Bennett MJ. et al, 1996, Biochemistry 20;35(33):10702-10711) • Requires NAD+ as coenzyme • Commonly tetramer • SDR(Short-Chain Dehydrogenase/Reductase)family • - Different enzyme belong to this family identify only at the • 15~30% • - Tetrameric members, dimeric members • - Coenzyme binding fold (GXXXGXG) • - Functionally important (Tyr152 and Lys156)

  5. KF707-BphB OU83-BphB KF715-BphB B356-BphB KSS102-BphB F1-TodD RHA1-BphB RP700-PhnB B1-BphB F199-BphB M5-BpdD P51-TcbB LB400-BphB TA421-BphB AN10-NahB PAK1-PahB C18-DoxE OUS82-PahB U2-NagB MT2-XylL 63.2 0 60 50 40 30 20 10 ◈ Dendrogram showing the level of homology between the amino acid sequences of different dihydrodiol dehydrogenase

  6. 147 214 218 123 153 191 217 221 G G G Y T G Y K T L M I M G G G Y T G Y K T L M I M G G G Y V G Y K T I M I L G G G Y V G Y K T I M I L G G G Y V G Y K T I M I L G G G Y V G Y K T I M I L G G G Y V G Y K T I M I L G G G Y N N Y K S L M L V G G G Y N N Y K S L M L V Y N N Y K S L M L V G G G Y N N Y K S L M L V G G G Y N N Y K T L M L V G G G Y N N Y K T L M L V B1-BphB F199-BphB AN10-NahB PAK1-PahB C18-DoxE OUS82-PahB U2-NagB KF707-BphB LB400-BphB KF715-BphB OU83-BphB B356-BphB KSS102-BphB ◈ Alignment of important sequence of different dihydrodiol dehydrogenase

  7. O 5′- ATGGCATTGTCTGCAAGG-3′ OH OH GTGGCATTGTCTGCAAGGCTTGAAGGACAGGTTGCGCTCCTGACTGGCGGCGCAACCGGAATTGGTGCGGCGGTGGTCGCCCGCTATATTGAAGAAGGGGCCAGGGTTGGTGTGCTCGTCCGAGACGACGAGCAGGCCCAGCTGGTGCGTCGCGCCACGGTGACAAGGTCGCCGTGGTCGCGGGCGACGTCCGTTCCTTGCAGACAATGCCCGGGCCGTCGCCGAGACAGTCGGGGCATTCGGCAAGCGGATGTCTTTGTCGGCAATGTCGGGATCTGGGATTTCATGGTGCCCCTTAGCAGCAGGATCCCGAGCTTCTGGAAAAGACATTCGACGAAATCTTTGAGTCAACCTGAAGGGGTATTTCTTCGGTGCGCGCGCCGCGATTCCCGAACGAGGAAAACCAAAGGCAGCATCGTCTTTACTGCCTCGACGTCGAGCTACACACTGGCGGCGGCGGGACACTGTATGTCGCATCGAAGCACGCGGTGCTGGCCTGATCCGGCAGCTGGCCTGGGAACTGACACCCGATATCAGGGTCATGGCGTCGCGCCGGGCGGCACCCGAACTCCGCTCAGCGGCACCAAGACTCGGGTTTTCCCGAAACCAAGATGGAGGAGATGCCTGGCCTGGACGGAATATCGCGGGCATGACGCCACTGGCGCGGATCGCCGAGCCGGACGATCATGAGGTCTCTACGCGCTTTTGGCTTCGCGTCGCGATTCCGCATACATGACCGAACGGTTCTGCTCAGCGACGGCGGCGTCGGCATCGGCAAGCGCCCCGACGCC H H OH OH OH 3′-GTAGCCGTTCGCGGGGCTG -5′ COO‾ OH O COO‾ TCA COO‾ COO‾ OH ◈ Expression and Purification of BphB in E. coli BphA BphB BphC BphD 30 KDa

  8. ◈ Preparation of different aromatic dihydrodiols C A B HPLC Data. A : biphenyl-diol, B : naphthalene-diol, C : phenanthrene-diol by ethyl acetate extraction

  9. 2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene 1189.4±72.7 (100%) 842.9±64.1 (70%) 460.6±16.7 (39%) WT H OH OH G153N 127±23.6 (100%) 21.5±2.9 (17%) 30.5±14.4 (24%) H M221V 60±19.5 - - Enzyme assay used pH7 phosphate buffer, 0.33mM substrate, 2.6mM NAD+, purified enzyme (approximately 1.5㎍) H OH OH OH OH H H ◈ Comparison of dehydrodiol dehydrogenase activities of the wild-type and mutated enzymes

  10. ◈ Nucleotide and amino acid sequence differences between the bphB genes from B1 and B8/36 • B8/36-bphB ATGGCATTGTCTGCAAGGCTTGAAGGACAGGTTGCGCTGCTGACTGGCGGCGCAACCGGA • B1-bphB GTGGCATTGTCTGCAAGGCTTGAAGGACAGGTTGCGCTCCTGACTGGCGGCGCAACCGGA • ********************************************************** • B8/36-bphBAAGGGGTATTTCTTCGGTGCGCGCGCCGCGATTCCCGGACTGAGGAAAACCAAAGGCAGC • B1-bphBAAGGGGTATTTCTTCGGTGCGCGCGCCGCGATTCCCGAACTGAGGAAAACCAAAGGCAGC • ********************************************************** • B8/36-bphBATCGTCTTTACTGCCTCGACGTCGAGCTACTACACTGGCGGCGGCGGGACACTGTATGTC • B1-bphBATCGTCTTTACTGCCTCGACGTCGAGCTACTACACTGGCGGCGGCGGGACACTGTATGTC • ********************************************************** • B8/36-bphBGCATCGAAGCACGCGGTGCTTGGCCTGATCCGGCAGCTGGCCTGGGAACTGACACCCGAT • B1-bphBGCATCGAAGCACGCGGTGCTTGGCCTGATCCGGCAGCTGGCCTGGGAACTGACACCCGAT • *********************************************************** • B8/36-bphBATCAGGGTCAATGGCGTCGCGCCGGGCGGC - - CCGAACTCCGCTCAGCGGCACCAAGACT • B1-bphBATCAGGGTCAATGGCGTCGCGCCGGGCGGCACCCGAACTCCGCTCAGCGGCACCAAGACT • ****************************** *************************** • B8/36-bphBGGCGGGTTTTCCGAAACCAAGATGGAGGAGATGCCTGGCCTGGACGGAATGATCGCGGGC • B1-bphBGCGGGTTTTCCCGAAACCAAGATGGAGGAGATGCCTGGCCTGGACGGAATGATCGCGGGC • * ** ***************************************************** • B8/36-bphBATGACGCCACTGGCGCGGATCGCCGAGCCGGACGATCATGCAGGTCTCTACGCGCTTTAG • B1-bphBATGACGCCACTGGCGCGGATCGCCGAGCCGGACGATCATGCAGGTCTCTACGCGCTTTTG • ********************************************************* * • B8/36-bphBGCTTCGCGTCGCGATTTCGCATACATGACCGGAACGGTTCTGCTCAGCGACGGCGGCGTC • B1-bphBGCTTCGCGTCGCGATTCCGCATACATGACCGGAACGGTTCTGCTCAGCGACGGCGGCGTC • *************** ****************************************** • B8/36-bphBGGCA- - - - - -AGCGCCCGGACGCC- - - • B1-bphBGGCATCGGCAAGCGCCCCGACGCCTGA • **** ************* A 60 301

  11.   B1 B8/36 B1 B8/36      B1 B8/36      B1 B8/36 B1 B8/36 B

  12. H OH OH H H OH OH OH OH H H ◈ Comparison of dihydrodiol dehydrogenase activities of the B8/36 and replaced enzyme 2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene 0 0 0 WT G133E 0 0 0 addition of T191 single a.a 0 0 0 Enzyme assay used pH7 phosphate buffer, 0.33mM substrate, 2.6mM NAD+, enzyme (approximately 1.5㎍)

  13. ◈ Conclusion • B1의 BphB를 일반적으로 알려져있는 dihydrodiol dehydrogenase와 비교해 본 결과 biphenyl-degrader와 naphthalene-degrader에서 특징적으로 나타나는 주요 아미조산에서 차이가 있음을 관찰하였다. • B1의 BphB를E.coli와 his-tagged vector system을 이용하여 발현시키고 정제한 결과 약 30 Kda 크기의 dihydrodiol dehydrogenase 를 얻을 수 있었다. 이들 dehydrogenase는 naphthalene-diol에서 약 1189.4±72.7(100%) 으로 가장 높은 활성을 보였고 phenanthrene-diol에서는 70%, biphenyl-diol에서는 39%에 해당하는 활성을 나타내였다. 이는 B1의 BphB가 biphenyl-diol dehydrogenase가 아닌 진정한 의미의 PAH-diol dehydrogenase임을 제시한다. • BphB의 point mutant인 G153N은 wild-type과 비교하여 biphenyl-diol 에서의 활성이 73%가 소실되었고 phenanthrene-diol 에서는 94%가 소실되었으며 naphthalene-diol 에서는 98%의 활성이 소실되었다. 결과적으로 G153N은 biphenyl-diol에서 127±23.6(100%)으로 가장 높은 활성을 나타내었고 phenanthrene-diol에서는 24%, naphthalene-diol에서 는 17%에 해당하는 활성을 나타내었다. • BphB기능이 소실된 B8/36가 완전한 크기의 효소를 합성하는 것을 확인하여 염기서열을 확인한 결과 두개의 염기결손에 의한 frame-shift mutant (구조이동 돌연변이)임을 밝혔다.

  14. ◈ Further study • B1의 BphB를 biphenyl 분해자와 더욱 유사해 지도록 디자인된 다른 mutant들의 제작 T191S, T147N, L214I, I218L, M221L, B8/36의 bphB중에서 두개의 nucleotide가 deletion된 부위의 site-specific replacement 제작. • 제작된 mutant들의 Dihydrodiol dehydrogenase를 정제하고 그들의 기질 범위 변화 관찰 • B8/36의 BphB를 wild type으로 복구시켜 활성측정

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