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