Yonsei University Hwang, Sun-young
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Transcript of Yonsei University Hwang, Sun-young
Different Substrate Specificity Variations of Sphingomonas yanoikuyae B1 BphB by S
ite-Directed Mutagenesis
Yonsei UniversityHwang, Sun-young
◈ 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 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)
◈ Cloning and purification of BphB
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
◈ Sustrate preparation of BphB
A B C
Fig. 3. HPLC Data. A : biphenyl-diol, B : naphthalene-diol, C : phenanthrene-diol by ethyl acetate extraction
◈ Enzyme assay of B1 and B8/36 BphB
Biphenyl-diol Naphthalene-diol Phenanthrene-diolSubstrate
BphB (B1) 3738±608 (14%)
26451±2675 (100%)
4968±984 (18%)
BphB (B8/36) 0 0 0
Table 1. Enzyme assay used pH9 buffer, 0.1mM substrate, 5mM NAD+, purified enzyme (approximately 1.5㎍ )
◈ Site-Directed Mutagenesis of BphB
- 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
2002/2/26Table. Enzyme assay of wild-type and mutant G153N BphB
Substrates 2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene
BphB 400.85102(51%)
59180.7(76%) 780.5898(100%)
G153N 6.240.6(3.6%) 13.321.2(7.6%)
174.7644.5(100%)
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)
2002/2/28Table. Enzyme assay of wild-type and mutant G153N BphB
Substrates 2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene
BphB 230.1412.27(37%)
357.8520.1(57.4%)
623.4318.3(100%)
G153N 33.196.39(100%) 6.552.2(19.7%) <2.54
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)
2002/3/15Table. Enzyme assay of wild-type and mutant G153N BphB
Substrates 2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene
BphB 460.6316.74(38.7%)
1189.3772.72(100%)
842.8964.08(70%)
G153N 127.2423.57(100%)
21.542.87(16.9%)
30.4914.37(24%)
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)
◈ Conclusion - BphB – 30kDa catalyzed the dehydrogenation of biphenyl-, naphthalene- and phenanthrene-diol.- The substrate range of G153N is different from that of BphB. And specif
ic activities of G153N are reduced. - A number of enzyme assays- Other mutant constructions.- Various substrate preparation.
◈ Other works.
- XylQ
- B. TNF1