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Yonsei University Hwang, Sun-young

Different Substrate Specificity Variations of Sphingomonas yanoikuyae B1 BphB and B8/36 BphB by Site-Directed Mutagenesis. Yonsei University Hwang, Sun-young. ◈ Introduction.

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Yonsei University Hwang, Sun-young

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  1. Different Substrate Specificity Variations of Sphingomonas yanoikuyae B1 BphB and B8/36 BphBby Site-Directed Mutagenesis Yonsei University Hwang, Sun-young

  2. ◈Introduction • S.yanoikyae B1 is able to utilize biphenyl, naphthalene, phenanthrene, anthracene, toluene, m-xylene, and p-xylene as sole sources of carbon and energy for growth. • BphB(cis-Biphenyl Dihydrodiol Dehydrogenase) catalyzes the second step in the biphenyl degradation pathway. • BphB of B8/36 is mutant of B1 BphB and does not catalyze the second step in biphenyl degradation pathway.

  3. ◈ BphB of B1 and B8/36 is… • cis-biphenyl Dihydrodiol Dehydrogenase • Composed of 806bp • 28~30KDa, tetramer in other strains this case, about 30KDa • Requires NAD+ • SDR(Short-Chain Dehydrogenase/Reductase)family - Different enzyme belong to this family identify only at the 15~30% - Coenzyme binding fold (GXXXGXG) - Functionally important (Tyr152 and Lys156)

  4. Lane 1 : marker Lane 2 : uninducer of B1 BphB Lane 3 : inducer of B1 BphB Lane 4 : purify of B1 BphB Lane 5 : purify of B8/36 BphB Lane 6 : inducer of B8/36 BphB Lane 7 : uninducer of b8/36 BphB Fig 2. SDS-PAGE of his-tagged B1 BphB and B8/36 BphB

  5. ◈ Sustrate preparation of BphB C B A Fig. 3. HPLC Data. A : biphenyl-diol, B : naphthalene-diol, C : phenanthrene-diol by ethyl acetate extraction

  6. ◈ Site-Directed Mutagenesis of BphB

  7. Substrate binding site (Protein science ;1998(7), 1286-1293.) • Burkholderia cepacia sp. LB400. • Asn149, Gly150, Leu209, Met212, Leu213 and Val216. • B1 Gly153Asn construction • Screened by direct sequencing • - IPTG 0.5mM induction

  8. Biphenyl-diol Naphthalene-diol Substrate 1349±61 (51%) 2645±27 (100%) BphB (B1) BphB (B8/36) 0 0 Biphenyl-diol Naphthalene-diol Substrate 460.6316.74 (38.7%) 1189.3772.72 (100%) BphB (B1) - - G153N Table 1. Enzyme assay used pH9 buffer, 0.1mM substrate, 5mM NAD+, enzyme (approximately 1.5㎍)

  9. ◈ Conclusion - BphB – 30kDa catalyzed the dehydrogenation of biphenyl-, naphthalene- and phenanthrene-diol. • The substrate range of G153N is different from that of BphB. And specific activities of G153N are reduced. • A number of enzyme assays • Other mutant constructions. • Various substrate preparation.

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