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Yonsei University Hwang, Sun-young

Different Substrate Specificity Variations of Sphingomonas yanoikuyae B1 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 by 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.

  3. ◈ BphB 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. ◈ Cloning and purification of BphB

  5. 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

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

  7. ◈ Enzyme assay of B1 and B8/36 BphB Biphenyl-diol Naphthalene-diol Substrate Phenanthrene-diol 3738±608 (14%) 26451±2675 (100%) 4968±984 (18%) BphB (B1) BphB (B8/36) 0 0 0 Table 1. Enzyme assay used pH9 buffer, 0.1mM substrate, 5mM NAD+, purified enzyme (approximately 1.5㎍)

  8. ◈ Site-Directed Mutagenesis of BphB

  9. 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

  10. Substrates 2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene BphB 400.85102(51%) 59180.7(76%) 780.5898(100%) G153N 6.240.6(3.6%) 13.321.2(7.6%) 174.7644.5(100%) 2002/2/26 Table. Enzyme assay of wild-type and mutant G153N BphB Dihydrodiol dehydrogenase activity was measured spectrophotometrecally at 340nm(reduction of NAD+ ). Reaction condition ; 50mM Na-K phosphate buffer(pH7), 5mM NAD+, 0.33mM diol substrate, and purify enzyme)

  11. Substrates 2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene BphB 230.1412.27(37%) 357.8520.1(57.4%) 623.4318.3(100%) G153N 33.196.39(100%) 6.552.2(19.7%) <2.54 2002/2/28 Table. Enzyme assay of wild-type and mutant G153N BphB Dihydrodiol dehydrogenase activity was measured spectrophotometrecally at 340nm(reduction of NAD+ ). Reaction condition ; 50mM Na-K phosphate buffer(pH7), 2.6mM NAD+, 0.33mM diol substrate, and purify enzyme)

  12. Substrates 2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene BphB 460.6316.74(38.7%) 1189.3772.72(100%) 842.8964.08(70%) G153N 127.2423.57(100%) 21.542.87(16.9%) 30.4914.37(24%) 2002/3/15 Table. Enzyme assay of wild-type and mutant G153N BphB Dihydrodiol dehydrogenase activity was measured spectrophotometrecally at 340nm(reduction of NAD+ ). Reaction condition ; 50mM Na-K phosphate buffer(pH7), 2.6mM NAD+, 0.33mM diol substrate, and purify enzyme)

  13. ◈ 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.

  14. ◈ Other works. • XylQ • B. TNF1

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