Molecular and Biochemical Characterization of a Novel Dihydrodiol Dehydregenase

in the Degradation of Aromatic Hydrocarbons by Sphingomonas yanoikuyae B1

Hwang, Sun-young

BphBTodD PhnB

OHOH

OH

OH

HOH

OH

HH

OHOHH

OH

OH

H

H

CH₃

OH

OH

CH₃

cis-biphenyl 2,3-dihydrodiol

2,3-dihydroxybiphenyl

cis- toluene dihydrodiol 3,4-dihydro-3,4-dihydroxyphenanthrene

3,4-dihydroxyphenanthrene3-methylcatechol

ex. P. Putida F1 Burkholderia cepacia LB400

B. sp.Strain RP700

◈ Introduction Dihydrodiol Dehydrogenase

OH

H

NahB

OH

OHOHH

OH

OH

COOH

H

OHOH

BenD

2-hydro-1,2-dihydroxybenzoate

catechol

cis-1,2-dihydroxy-1,2- dihydronaphthalene

1,2-dihydroxynaphthalene

ex. P. putida G7 Acinetobacter calcoaceticus

• 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)

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

102030405060 0

◈ Dendrogram showing the level of homology between the amino acid sequences of different dihydrodiol dehydrogenase

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

KF707-BphB

OU83-BphB

KF715-BphB

B356-BphB

KSS102-BphB

B1-BphB

F199-BphB

LB400-BphB

AN10-NahB

PAK1-PahB

C18-DoxE

OUS82-PahB

U2-NagB

147 214 218

123 153 191 217 221

◈ Alignment of important sequence of different dihydrodiol dehydrogenase

◈ Expression and Purification of BphB in E. coli

30 KDa

C O O ‾

HH OHOH

OH OH

OOHCOO‾

COO‾OH

COO‾TCA

BphA

BphB

BphC

BphD

5′- ATGGCATTGTCTGCAAGG-3′

3′-GTAGCCGTTCGCGGGGCTG -5′

GTGGCATTGTCTGCAAGGCTTGAAGGACAGGTTGCGCTCCTGACTGGCGGCGCAACCGGAATTGGTGCGGCGGTGGTCGCCCGCTATATTGAAGAAGGGGCCAGGGTTGGTGTGCTCGTCCGAGACGACGAGCAGGCCCAGCTGGTGCGTCGCGCCACGGTGACAAGGTCGCCGTGGTCGCGGGCGACGTCCGTTCCTTGCAGACAATGCCCGGGCCGTCGCCGAGACAGTCGGGGCATTCGGCAAGCGGATGTCTTTGTCGGCAATGTCGGGATCTGGGATTTCATGGTGCCCCTTAGCAGCAGGATCCCGAGCTTCTGGAAAAGACATTCGACGAAATCTTTGAGTCAACCTGAAGGGGTATTTCTTCGGTGCGCGCGCCGCGATTCCCGAACGAGGAAAACCAAAGGCAGCATCGTCTTTACTGCCTCGACGTCGAGCTACACACTGGCGGCGGCGGGACACTGTATGTCGCATCGAAGCACGCGGTGCTGGCCTGATCCGGCAGCTGGCCTGGGAACTGACACCCGATATCAGGGTCATGGCGTCGCGCCGGGCGGCACCCGAACTCCGCTCAGCGGCACCAAGACTCGGGTTTTCCCGAAACCAAGATGGAGGAGATGCCTGGCCTGGACGGAATATCGCGGGCATGACGCCACTGGCGCGGATCGCCGAGCCGGACGATCATGAGGTCTCTACGCGCTTTTGGCTTCGCGTCGCGATTCCGCATACATGACCGAACGGTTCTGCTCAGCGACGGCGGCGTCGGCATCGGCAAGCGCCCCGACGCC

◈ Preparation of different aromatic dihydrodiols

A B C

HPLC Data.

A : biphenyl-diol, B : naphthalene-diol, C : phenanthrene-diol

by ethyl acetate extraction

◈ Comparison of dehydrodiol dehydrogenase activities of the wild-type and mutated enzymes

Enzyme assay used pH7 phosphate buffer, 0.33mM substrate, 2.6mM NAD+, purified enzyme (approximately 1.5㎍ )

2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene

WT

G153N

M221V

460.6±16.7 (39%) 1189.4±72.7 (100%) 842.9±64.1 (70%)

127±23.6 (100%) 21.5±2.9 (17%) 30.5±14.4 (24%)

60±19.5 - -

OH

H

OHHHOHOH

HOH

OH

H

B8/36-bphB ATGGCATTGTCTGCAAGGCTTGAAGGACAGGTTGCGCTGCTGACTGGCGGCGCAACCGGA B1-bphB GTGGCATTGTCTGCAAGGCTTGAAGGACAGGTTGCGCTCCTGACTGGCGGCGCAACCGGA

************************************* *********************

B8/36-bphB AAGGGGTATTTCTTCGGTGCGCGCGCCGCGATTCCCGGACTGAGGAAAACCAAAGGCAGC B1-bphB AAGGGGTATTTCTTCGGTGCGCGCGCCGCGATTCCCGAACTGAGGAAAACCAAAGGCAGC

************************************* *********************B8/36-bphB ATCGTCTTTACTGCCTCGACGTCGAGCTACTACACTGGCGGCGGCGGGACACTGTATGTC B1-bphB ATCGTCTTTACTGCCTCGACGTCGAGCTACTACACTGGCGGCGGCGGGACACTGTATGTC

**********************************************************B8/36-bphB GCATCGAAGCACGCGGTGCTTGGCCTGATCCGGCAGCTGGCCTGGGAACTGACACCCGAT B1-bphB GCATCGAAGCACGCGGTGCTTGGCCTGATCCGGCAGCTGGCCTGGGAACTGACACCCGAT

***********************************************************B8/36-bphB ATCAGGGTCAATGGCGTCGCGCCGGGCGGC - - CCGAACTCCGCTCAGCGGCACCAAGACT B1-bphB ATCAGGGTCAATGGCGTCGCGCCGGGCGGCACCCGAACTCCGCTCAGCGGCACCAAGACT

****************************** ***************************B8/36-bphB GGCGGGTTTTCCGAAACCAAGATGGAGGAGATGCCTGGCCTGGACGGAATGATCGCGGGC B1-bphB GCGGGTTTTCCCGAAACCAAGATGGAGGAGATGCCTGGCCTGGACGGAATGATCGCGGGC

* ** *** ************************************************** B8/36-bphB ATGACGCCACTGGCGCGGATCGCCGAGCCGGACGATCATGCAGGTCTCTACGCGCTTTAGB1-bphB ATGACGCCACTGGCGCGGATCGCCGAGCCGGACGATCATGCAGGTCTCTACGCGCTTTTG

********************************************************* * B8/36-bphB GCTTCGCGTCGCGATTTCGCATACATGACCGGAACGGTTCTGCTCAGCGACGGCGGCGTC B1-bphB GCTTCGCGTCGCGATTCCGCATACATGACCGGAACGGTTCTGCTCAGCGACGGCGGCGTC

*************** ****************************************** B8/36-bphB GGCA- - - - - -AGCGCCCGGACGCC- - - B1-bphB GGCATCGGCAAGCGCCCCGACGCCTGA

**** ******* ******

60

301

◈ Nucleotide and amino acid sequence differences between the bphB genes from B1 and B8/36

A

B1

B8/36

B1

B8/36

B1

B8/36

B1

B8/36

B1

B8/36

B

0

Enzyme assay used pH7 phosphate buffer, 0.33mM substrate, 2.6mM NAD+, enzyme (approximately 1.5㎍ )

2,3-DD-Biphenyl 1,2-DD-naphthalene 3,4-DD-Phenanthrene

WT

G133E

addition of T191 single a.a

0 0

0 0 0

0 0 0

OH

H

OHHHOHOH

HOH

OH

H

◈ Comparison of dihydrodiol dehydrogenase activities of the B8/36 and replaced enzyme

◈ Conclusion

B1 의 BphB 를 일반적으로 알려져있는 dihydrodiol dehydrogenase 와 비교해 본 결과 biphenyl-degrader 와 naphthalene-degrader 에서 특징적으로 나타나는 주요 아미조산에서 차이가 있음을 관찰하였다 .

B1 의 BphB 를 E.coli 와 his-tagged vector system 을 이용하여 발현시키고 정제한 결과 약 30 Kda 크기의 dihydrodiol dehydrogenase 를 얻을 수 있었다 . 이들 dehydrogenase 는 naphthalene-diol 에서 약 1189.4±72.7(100%) 으로 가장 높은 활성을 보였고 phenanthrene-diol 에서는 70%, biphenyl-diol 에서는 39%에 해당하는 활성을 나타내였다 . 이는 B1 의 BphB 가 biphenyl-diol dehydrogenase 가 아닌 진정한 의미의 PAH-diol dehydrogenase 임을 제시한다 .

BphB 의 point mutant 인 G153N 은 wild-type 과 비교하여 biphenyl-diol 에서의 활성이 73% 가 소실되었고 phenanthrene-diol 에서는 94% 가 소실되었으며 naphthalene-diol 에서는 98% 의 활성이 소실되었다 . 결과적으로 G153N 은 biphenyl-diol 에서 127±23.6(100%) 으로 가장 높은 활성을 나타내었고 phenanthrene-diol 에서는 24%, naphthalene-diol 에서 는 17% 에 해당하는 활성을 나타내었다 .

BphB 기능이 소실된 B8/36 가 완전한 크기의 효소를 합성하는 것을 확인하여 염기서열을 확인한 결과 두개의 염기결손에 의한 frame-shift mutant ( 구조이동 돌연변이 ) 임을 밝혔다 .

◈ Further study

B1 의 BphB 를 biphenyl 분해자와 더욱 유사해 지도록 디자인된 다른

mutant 들의 제작

T191S, T147N, L214I, I218L, M221L,

B8/36 의 bphB 중에서 두개의 nucleotide 가 deletion 된 부위의

site-specific replacement 제작 .

제작된 mutant 들의 Dihydrodiol dehydrogenase 를 정제하고 그들의 기질

범위 변화 관찰

B8/36 의 BphB 를 wild type 으로 복구시켜 활성측정