Enzyme–substrate interaction and characterization of a 2,3-dihydroxybiphenyl 1,2-dioxygenase from Dyella ginsengisoli LA-4

R E S E A R C H L E T T E R

Enzyme^substrate interactionand characterizationofa2,3-dihydroxybiphenyl1,2-dioxygenasefromDyellaginsengisoli LA-4Ang Li1, Yuanyuan Qu1, Jiti Zhou1 & Fang Ma2

1Key Laboratory of Industrial Ecology and Environmental Engineering, School of Environmental and Biological Science and Technology, MOE, Dalian

University of Technology, Dalian, China; and 2School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin, China

Correspondence: Yuanyuan Qu, Key

Laboratory of Industrial Ecology and

Environmental Engineering, School of

Environmental and Biological Science and

Technology, MOE, Dalian University of

Technology, Dalian 116024, China.

Tel.: 186 411 8470 6251; fax: 186 411 8470

6252; e-mail: [email protected]

Received 9 July 2008; accepted 16 December

2008.

First published online 29 January 2009.

DOI:10.1111/j.1574-6968.2009.01487.x

Editor: Hans-Peter Kohler

Keywords

Dyella ginsengisoli ; 2,3-dihydroxybiphenyl

1,2-dioxygenase; kinetic parameters;

enzyme–substrate complex.

Abstract

A bphC gene (915 bp) encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)

was amplified by PCR from Dyella ginsengisoli LA-4, which was heterologously

expressed in Escherichia coli. The purified His-Tag BphC was able to catalyze

the meta-cleavage reaction of the dihydroxylated aromatic rings. According to

the specificity constant (Kcat/Km) of BphC_LA-4, the specificity of BphC_LA-4

was determined in the following order: 2,3-dihydroxybiphenyl4 3-methyl-

catechol4 catechol4 4-chlorocatechol4 4-methylcatechol. The experimental

data were consistent with the prediction of enzyme–substrate complexes.

The highest specific activity of BphC_LA-4 was 118.3 U mg�1 for 2,3-dihydrox-

ybiphenyl.

Introduction

Polychlorinated biphenyls (PCBs) have been of public and

scientific concern for several decades because of their

persistence in the environment, their bioaccumulation and

their potential carcinogenicity (McKay et al., 2003). Nowa-

days, a number of PCB-degrading microorganisms have

been isolated and characterized, and the major biodegrada-

tion pathway of biphenyl has been reported (Furukawa et al.,

2004; Pieper, 2005). Most biphenyl-degrading bacteria can

metabolize biphenyl to benzoate and 2-hydroxy-penta-2,4-

dienoate via the so-called upper pathway, which consists of

four enzymes: biphenyl 2,3-dioxygenase (BphA), dihydro-

diol dehydrogenase (BphB), 2,3-dihydroxybiphenyl dioxy-

genase (BphC) and 2-hydroxyl-6-oxo-6-phenylhexa-2,4-

dienoic acid hydrolase (BphD).

Among the enzymes mentioned above, BphCs catalyze

the meta-cleavage reaction of 2,3-dihydroxybiphenyl, which

belongs to a class of extradiol dioxygenases (Eltis & Bolin,

1996). Ring cleavage is an important step during the

degradation of aromatic compounds including biphenyl.

These extradiol dioxygenases can catalyze the meta-cleavage

of the dihydroxylated aromatic rings; therefore, they play a

key role in biphenyl mineralization or biodegradation of

other aromatic compounds (Vaillancourt et al., 2002a, b; Lee

et al., 2003).

Up to now, many bphC genes have been cloned and

heterologously expressed in the metabolic pathways for

biphenyl/PCBs (Eltis & Bolin, 1996; Kim et al., 1996; Hatta

et al., 2003; Lee et al., 2003; McKay et al., 2003). Most reports

on BphCs mainly focused on functional characterization, but

research on structural properties has been limited, especially

on the enzyme–substrate complexes (Han et al., 1995; Senda

et al., 1996). Recently, the complete 12.1-kb bph gene cluster

(GenBank accession no. EU258607) was amplified from

strain LA-4 by the genome walking method. The deduced

amino acid sequence of BphC_LA-4 shared the highest

similarity with BphC of Burkholderia xenovorans LB400 with

only 74% identity. This is the first report that the bphC gene

is amplified from the genus Dyella and expressed in

FEMS Microbiol Lett 292 (2009) 231–239 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Escherichia coli. Meanwhile, the specificity of BphC_LA-4 for

dihydroxylated substrates was predicted according to the

enzyme–substrate complexes. Also, the characterization of

purified BphC_LA-4 was investigated in detail.

Materials and methods

Bacterial strains, plasmids and growthconditions

A novel biphenyl-degrading bacterium, Dyella ginsengisoli LA-

4, was grown at 30 1C on defined basal salts medium (Qu

et al., 2005) containing 0.5 g L�1 biphenyl and 2 g L�1 bacto-

tryptone. Escherichia coli JM109 (TaKaRa, Dalian, China) and

E. coli BL21 (DE3) (Novagen) were used for plasmid con-

struction and gene expression, respectively. Escherichia coli

were grown on Luria–Bertani (LB) media at 37 1C with

100mg mL�1 ampicillin or 30mg mL�1 kanamycin. The plas-

mids used in this study were pMD18-T simple vector (cloning

vector, Ampr) and pET28(a) (expression vector, Kmr).

Construction of plasmids and overexpression

DNA was manipulated using standard protocols (Ausubel

et al., 2000). Genome DNA was extracted from strain LA-4

using a Bacterial Genomic DNA Extraction Kit (TaKaRa).

According to the ORF of bphC gene (EU258607), the

primers were designated: bphC-F, 50-GAATTCATGAGCGT

CAAGAACTTGGG-30; bphC-R, 50-AAGCTTTCATGCGG

TCTCTCTTGTAG-30, where the EcoRI and HindIII sites are

underlined, respectively. The entire bphC gene was amplified

by PCR using PrimeSTAR HS DNA Polymerase (TaKaRa).

The PCR was run for 30 cycles with an annealing tempera-

ture of 55 1C. The PCR product was ligated into pMD18-T

simple vector before sequencing. DNA sequence was deter-

mined by TaKaRa Biotechnology. Then the gene was sub-

cloned into the pET28(a) vector, yielding pET-bphC.

Subsequently, the resulting plasmids (pET-bphC) were in-

troduced into E. coli BL21 (DE3) for overexpression.

For overexpression, E. coli BL21 (DE3) harboring pET-

bphC was cultured in 3 mL LB medium containing

30mg mL�1 kanamycin at 37 1C for 8 h, and then 100mL

was inoculated into 100 mL LB with 30mg mL�1 kanamycin.

After cells were allowed to grow at 37 1C to an OD600 nm of

0.6, the cultures were induced with 1 mM IPTG and

reincubated at 37 1C for 3 h. Escherichia coli was harvested

by centrifugation at 9820 g for 5 min, and then washed twice

with cold 20 mM sodium phosphate buffer (pH 7.4). Finally,

the pellets were frozen at � 80 1C until use.

Sequence analysis and molecular modeling

Sequence analysis, database searches and sequence compar-

ison were performed using the programs from NCBI. Amino

acid sequences of BphCs were aligned using CLUSTAL W, and

the aligned sequences were used to construct a phylogenetic

tree using the neighbor-joining method (Saitou & Nei,

1987).

The automated protein structure homology-modeling

server, SWISS-MODEL, was used to generate the three-dimen-

sional (3D) model (Schwede et al., 2003). In addition,

PYMOL (version 0.99; DeLano Scientific, San Carlos, CA)

was used to view the graphic. The enzyme–substrate com-

plexes were generated by superposition using DEEPVIEW, and

the substrates used in this study were drawn from the

template model [Protein Data Bank (PDB) code 1kndA,

BphC_LB400 complexed with catechol] to replace catechol.

Preparation of the crude extracts

Escherichia coli (pET-bphC) was suspended in 20 mM so-

dium phosphate buffer (pH 7.4) and lysed by freezing and

thawing followed by sonication (225 W at 4 1C for 30 min,

Ultrasonic processor CPX 750). After centrifugation

(58 545 g, 20 min, 4 1C), the supernatants were used as the

crude extracts.

Purification of His-Tag BphC

Protein was purified at room temperature by AKTA Explorer

100 (Amersham Biosciences, Montreal, QC, Canada). The

crude extracts were loaded onto a HisTrap FF crude column

(1 mL, Amersham Biosciences) equilibrated with buffer A

(20 mM sodium phosphate, 500 mM NaCl, 20 mM imida-

zole, pH 7.4). The column was operated at the flow rate of

1.0 mL min�1, and the enzyme was eluted with a 20-mL

linear gradient from 0% to 100% buffer B (20 mM sodium

phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4).

Active fractions were applied to a Hitrip Desalting column

(Amersham Biosciences) with buffer C (50 mM Tris-HCl,

pH 8.0) for desalting. Finally, the purified BphC was

obtained and concentrated to about 0.6 mg mL�1.

Purified enzymes were analysed by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as

described by Laemmli (1970). The acrylamide concentra-

tions for the concentrating and separating gels were 5% and

15%, respectively. The gel was stained with Coomassie

brilliant blue R250. Protein concentrations were determined

using the Bradford (1976) method.

Determination of enzyme activity with thepurified enzyme

2,3-Dihydroxybiphenyl 1,2-dioxygenase activity was mea-

sured by monitoring the reaction products HOPDA at

434 nm with a scanning spectrophotometer (V-560, JASCO,

Tokyo, Japan). Activity assays were performed in 50 mM

Tris-HCl (pH 8.0) containing 125mM 2,3-dihydroxybiphenyl

FEMS Microbiol Lett 292 (2009) 231–239c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

232 A. Li et al.

at 20 1C. The total volume of the reaction mixture was

2.0 mL. One unit of enzyme activity was defined as the

amount of enzyme that catalyzed the formation of 1 mmol of

product per minute at 20 1C. The molar extinction coeffi-

cient of the product was e434 = 17.9 cm�1 mM�1 at pH 8.0

(Eltis et al., 1993). Specific activities were expressed as

micromoles of product formed per minute per milligram of

protein at 20 1C. The other substrates used in this study had

the following ring fission product absorbances and molar

extinction coefficients at pH 8.0: catechol, e375 = 67.8 cm�1

mM�1; 4-chlorocatechol, e379 = 40 cm�1 mM�1; 3-methylca-

techol, e388 = 73.6 cm�1 mM�1; 4-methylcatechol, e382 =

73.9 cm�1 mM�1 (Eltis et al., 1993; Hatta et al., 2003).

Apparent kinetic parameters were determined at substrate

ranges from 50 to 500 mM 2,3-dihyroxybiphenyl, 125 to

600 mM catechol, 125 to 500 mM 3-methylcatechol and 4-

methylcatechol, and 25 to 175mM 4-chlorocatechol. Kinetic

data were calculated from the initial velocities with the

Michaelis–Menten equation by ORIGIN 7.5. All the experi-

ments were carried out at least three times.

Effects of pH and temperature on enzymeactivity

The optimal pH of enzymatic cleavage was determined at

different pH values ranging from 6.0 to 9.0 using 50 mM

K2HPO4/NaOH buffer (pH 6.0–7.0) and Tris-HCl buffer

(pH 7.0–9.0). The molar extinction coefficients of the

products were: e434 = 8.0 cm�1 mM�1 at pH 7.0;

e434 = 13.2 cm�1 mM�1 at pH 7.5; e434 = 17.9 cm�1 mM�1 at

pH 8.0; e434 = 20.6 cm�1 mM�1 at pH 8.5; e434 =

22.0 cm�1 mM�1 at pH 9.0 (Eltis et al., 1993). The optimal

temperature for the enzyme activity was determined by

measuring the reactions at different temperatures ranging

from 20 to 80 1C.

Effects of metal ions and chemical compoundson enzyme activity

The effects of different metal ions on the enzyme activity

were tested by incubating 12 mg of purified BphC in 50 mM

Tris-HCl buffer (pH 8.0) for 15 min at 20 1C in the presence

of 1 mM different metal ions before initiating the reaction.

The effects of various compounds were investigated by

incubating 12 mg purified BphC with the different concen-

trations of chemical compounds.

Results

Cloning of the bphC gene and sequence analysis

The complete 927 bp EcoRI–HindIII fragment containing

the bphC gene was amplified from strain LA-4 by PCR,

which contained an initiation codon ATG at nucleotides 7–9

and a stop codon at nucleotides 922–927. The deduced

amino acid sequence shared the highest similarity with

BphCs of B. xenovorans LB400 and Pseudomonas pseudoal-

caligenes KF707 (both only 74% identity) (Furukawa et al.,

1987; Hofer et al., 1993). The phylogenetic relationship of

related enzymes is shown in Fig. 1a. The phylogenetic

analysis of the type I extradiol dioxygenases indicated that

they were all derived from the same ancestral origin. The

branch with BphC_LA-4 was obviously distinct from other

known BphCs.

Comparing the amino acid sequences of BphC_LA-4,

BphC_KKS102 and BphC_LB400 (Fig. 1b), it was suggested

that BphC_LA-4 was divided into two domains, N-terminal

(residues 1–134) and C-terminal (residues 135–304) (Eltis &

Bolin, 1996; Senda et al., 1996). According to previous

reports, the BphC_LA-4 was a two-domain type I extradiol

dioxygenase in which the C-terminal domain binds iron (II)

and was catalytically active (Lee et al., 2003; Vaillancourt

et al., 2006). Eltis & Bolin (1996) found a fingerprint region

and nine strictly conserved residues by aligning 23 extradiol

dioxygenases. Therefore, the fingerprint region spanned

residues 238–259 in the BphC_LA-4 sequence with the

pattern (GRHSNDHMVSFYAVTPSGFDVE). Three of the

strictly conserved residues, His-145, His-209 and Glu-259 in

BphC_LA-4, were the metal ligands. Three additional active

site residues, His-194, His-240 and Tyr-249, were also

strictly conserved in BphC_LA-4, which played potential

catalytic roles. The other conserved residues, Gly-28, Leu-

164 and Pro-253, played structural or folding roles in

BphC_LA-4.

Structural prediction and modeling

The 3D structure of BphC_LB400 was used as a template to

generate the BphC_LA-4 model. As shown in Fig. 2a, the

modeled structure of BphC_LA-4 suggested that the N- and

C-terminal domains were structurally similar, and each

domain of BphC_LA-4 contained two repetitions of the

babbb motif. Comparing the 3D structures between the

constructed model and the template model, the relative

mean square deviation of C-a on superposition was 3.0 A.

This suggested that the constructed model of BphC_LA-4

was reliable with a perfect quality.

In BphC_KKS102, three less well-conserved residues, Ile-

174, Phe-201 and Thr-280, interacted with the benzene ring

moiety of 2,3-dihydroxybiphenyl, which determined varied

substrate specificities among the related enzymes (Senda

et al., 1996). Comparing the amino acid sequences and 3D

structures of BphC_LA-4 and BphC_KKS102 (PDB code

1kw9), only Thr-280 was replaced by Ile-279 in BphC_LA-4,

as shown in Figs 1b and 2b.

To investigate the specificity of BphC_LA-4, the enzy-

me–substrate complexes were constructed (Fig. 3). As in


233Enzyme–substrate complexes and characterization of BphC_LA-4

Vaillancourt’s report (Vaillancourt et al., 2005), the active

site of BphC was poorly complementary to catechol because

the volume in the active site occupied by the second ring of

biphenyl was excess ‘free’ volume when catechol binds.

Therefore, the specificity of BphC for bicyclic substrate was

higher than that for monocyclic substrate. According to the

enzyme–substrate complexes (Fig. 3a–e), the volume of the

second ring of biphenyl was completely free in the cate-

chol:BphC complex, but the volume was partially occupied

by the methyl group of 3-methylcatechol. The chlorine

substitution or methyl group at position C4 of catechol

could partially occupy the free volume, but the residue Ile-

279 was close to these substitutions. This indicated that the

substitution at position C4 was sterically hindered because

of lack of space to accommodate the chlorine atom or

methyl group. The specificity of BphC_LA-4 for 4-methyl-

catechol should be lower than that for 4-chlorocatechol

because the methyl group occupied a larger space than the

chlorine atom. The prediction of specificity for BphC_LA-4

was in the following order: 2,3-dihydroxybiphenyl43-methylcatechol4 catechol4 4-chlorocatechol4 4-methyl-

catechol.

Purification and characterization of BphC_LA-4

The His-Tag BphC in E. coli BL21 (pET-bphC) was purified

at room temperature using an AKTA Explorer. The protein

was estimated to be 4 95% pure by SDS-PAGE. The

subunit molecular mass is shown in Fig. 4. The result was

Fig. 2. 3D structure of BphC_LA-4 constructed

with SWISS-MODEL (a) and a superposition of

BphC_LA-4 active site structure with that of

BphC_KKS102. (b) BphC_LA-4 and

BphC_KKS102 are shown in green and purple,

respectively.

(b)(a)

BphC LB400

BphC KF707

BphC Cam-1

BphC OU83

BphC KF715

BphC B-356

BphC SMN4

BphC KKS102

BphC TK102

BphC LA-4

BphC RHA1

TodE DOT-T1

Edo2 RW1

BphC R04

BphC B1

NahC pPG7

XylE pWW0

100

86100

55

99

66

95

99

92

100

889782

0.00.20.40.60.8

Fig. 1. Sequence analysis of BphC_LA-4. (a) Phylogenetic relationships of type I extradiol dioxygenases. Sequences used were as follows: Pseudomonas

sp. KKS102 (M26433); Dyella sp. LA-4 (EU258607); Rhodococcus sp. R04 (DQ403247); Sphingobium sp. B1 (EF151283); Pseudomonas sp. OU83

(X91876); Rhodococcus sp. RHA1 (D32142); Comamonas sp. TK102 (AB086835); Comamonas sp. B-356 (U91936); Pseudomonas sp. Cam-1

(AY027651); Burkholderia sp. LB400 (X66122); Pseudomonas sp. KF715 (M33813); Pseudomonas sp. KF707 (M83673); Delftia sp. SMN4 (AY028943);

Pseudomonas sp. pWW0 (M64747); TodE Pseudomonas sp. DOT-T1 (Y18245); Sphingomonas sp. RW1 (AJ223219). (b) Comparison of amino acid

sequences of BphC_LA-4 with BphC_KKS102 and BphC_LB400. �Residues binding the Fe ion. #Residues that play potential catalytic roles. $Residues

that play structural or folding roles. &Residues that interact with the benzene ring moiety of 2,3-dihydroxybiphenyl.


234 A. Li et al.

identified with the prediction of molecular mass (36.8 kDa)

using the PROTPARAM tool.

The BphC_LA-4 activities for 2,3-dihydroxybiphenyl and

other dihydroxylated substrates were investigated. The spe-

cific activity was 118.3 U mg�1 for 2,3-dihydroxybiphenyl,

which was the best substrate for BphC_LA-4. The optimal

pH for BphC_LA-4 was 8.0, and the enzyme showed its

maximal activity at 40 1C (Fig. 5).

To determine the substrate specificity, the enzyme was

tested for its ability to oxidize 2,3-dihydroxybiphenyl, cate-

chol, 4-chlorocatechol, 3-methylcatechol and 4-methylcate-

chol. The apparent kinetic constants of BphC_LA-4 are

listed in Table 1. The Km for 2,3-dihydroxybiphenyl was the

lowest (18.9mM), and the Km for 4-methylcatechol the

biggest (359.7 mM). The biggest Kcat was for 2,3-dihydrox-

ybiphenyl, and the largest reduction of Kcat was for 4-

methylcatechol. According to the specificity constant (Kcat/

Km) (Table 1), BphC_LA-4 was able to cleave dihydroxylated

substrates in the following order of specificity: 2,3-

dihydroxybiphenyl4 3-methylcatechol4 catechol4 4-

chlorocatechol4 4-methylcatechol. The results agreed with

the prediction of specificity of BphC_LA-4 using enzyme–

substrate complexes.

Effects of metal ions on the enzyme activities

The effects of metal ions on BphC_LA-4 activity are shown

in Fig. 6a. The results indicated that Ca21 and Mg21 were

able to enhance the BphC activities, and Ba21 had no effect

on the activity of BphC_LA-4. However, other metal ions,

especially Cu21, were able to inhibit the activities signifi-

cantly.

Effects of chemical compounds on the enzymeactivities

BphC_LA-4 could be inhibited by various compounds, and

the results are shown in Fig. 6b–d. The enzyme activity of

BphC_LA-4 was completely inhibited when there was

4 25 mM L-ascorbic acid. KI 600 mM reduced the specific

activity of the enzyme by 35%. Meanwhile, it was observed

that 1.25 mM SDS inhibited nearly 60% of BphC_LA-4

activity. The oxidant H2O2 significantly inhibited the en-

zyme activity, and the activity was completely inhibited by

1 mM H2O2 (data not shown). In agreement with previous

reports, it was typical for all iron (II)-dependent extradiol

Fig. 3. Stereo pictures of enzyme–substrate complexes of BphC_LA-4.

(a) 2,3-Dihydroxybiphenyl:BphC complex; (b) catechol:BphC complex; (c)

4-chlorocatechol:BphC complex; (d) 3-methylcatechol:BphC complex;

(e) 4-methylcatechol:BphC complex.

44.3 kDa

29.0 kDa

36 kDa

M 1 2

Fig. 4. SDS-PAGE of purified BphC. Lane M: protein molecular weight

marker (Broad); lane 1: crude cell extracts; lane 2: purified His-Tag BphC.



dioxygenases that the enzyme activities were completely

inhibited in the presence of over 1 mM H2O2, while manga-

nese-dependent extradiol dioxygenases showed relatively

weak inactivation (Que et al., 1981; Boldt et al., 1995; Hatta

et al., 2003). This demonstrated that BphC_LA-4 is an iron

(II)-dependent extradiol dioxygenase.

Discussion

A bphC gene was amplified by PCR from PCB-degrading

bacteria D. ginsengisoli LA-4. The deduced amino acid

sequence of BphC_LA-4 showed <74% identity with other

known BphC. In the classification of extradiol dioxygenases

based on the phylogenetic relationship, the enzyme be-

longed to a superfamily with the sequences within the same

subfamily exhibiting over 54% identity (Eltis & Bolin, 1996).

Clearly, BphC_LA-4 belonged to the subfamily of BphCs

obtained from gram-negative bacteria such as BphC_LB400

and BphC_KF707, which were type I iron (II)-dependent

extradiol dioxygenases.

The metal ligands and catalytic residues were strictly

conserved in BphC_LA-4. The other residues playing struc-

tural or folding roles in BphC_LA-4 were also strictly

conserved (Eltis & Bolin, 1996). Among these residues, His-

194 took part in a weak hydrogen bond with the hydroxyl

group of the substrate (Uragami et al., 2001). The proto-

nated His-194 could stabilize a negative charge on the O2

molecule located in the hydrophobic O2-binding cavity and

seemed to function as a proton donor (Sato et al., 2002).

The residues interacting with the benzene ring moiety of

2,3-dihydroxybiphenyl, such as Ile-174, Phe-201 and Thr-

280 in BphC_KKS102, determined the substrate specificities

(Senda et al., 1996). However, these residues were less well

conserved among the related enzymes. Comparison of

BphC_LA-4 and BphC_LB400 showed that Ile-174 and Ile-

279 in BphC_LA-4 were replaced by Met and Pro in

BphC_LB400, respectively. But Phe-201 in BphC_LA-4 and

BphC_LB400 were conserved. The Vmax values of

BphC_LB400 for the dihydroxylated substrates were in the

following order: 2,3-dihydroxybiphenyl4 catechol4 3-

methylcatechol; the specific activity for 4-methylcatechol

was too low to be accurately determined (Eltis et al., 1993).

However, the Kcat value of BphC_LA-4 for 3-methylcatechol

was higher than that for catechol, and the kinetic parameters

for 4-methylcatechol could be determined. It was obvious

that the residues Ile-174, Phe-201 and Ile-279 determined

the specificity of BphC_LA-4.

According to previous reports, the structurally character-

ized proteins are limited compared with the number of

known protein sequences (Arnold et al., 2006). Crystal

structures of BphC_LB400 and BphC_KKS102 have been

reported for the substrate-free enzyme as well as for the

enzyme–substrate complexes (Han et al., 1995; Senda et al.,

1996; Vaillancourt et al., 1998; Dai et al., 2002; Sato et al.,

2002; Vaillancourt et al., 2002a b). However, the enzyme–

substrate complexes were mainly focused on only three

dihydroxylated substrates, such as 2,3-dihydroxybiphenyl,

catechol, and 3-methylcatechol. To our knowledge there

have been no reports demonstrating the specificity of BphC

for 4-chlorocatechol or 4-methylcatechol using enzyme–

substrate complexes. Therefore, we constructed a 3D struc-

ture of BphC and enzyme–substrate complexes. Comparing

the 3D structures of BphC_LA-4, BphC_LB400 and

BphC_KKS102, the b-strand between two a-helices was

absent in the second babbb motif of BphC_LA-4 in the N-

terminal domain (Han et al., 1995; Senda et al., 1996).

According to the enzyme–substrate complexes, the volume

in the active site occupied by the second ring of biphenyl

played an important role in the determination of the

specificity of BphC for bicyclic and monocyclic catecholic

substrates (Vaillancourt et al., 2005). As shown in Fig. 3, the

7.0 7.5 8.0 8.5 9.00

102030405060708090

100R

elat

ive

activ

ity (

%)

pH20 30 40 50 60 70 80

60

70

80

90

100

110

120

130

140

Rel

ativ

e ac

tivity

(%

)

Temperature (°C)

(a) (b)

Fig. 5. Effects of pH and temperature on

enzyme activity. (a) pH; (b) temperature.

Table 1. Apparent kinetic parameters of BphC_LA-4

Substrate Km (mM) Kcat (S�1)

Kcat/Km

(10�2 S�1 mM�1)

2,3-Dihydroxybiphenyl 18.9 84.1 445.0

Catechol 281.0 6.7 2.4

4-Chlorocatechol 49.6 1.0 2.0

3-Methylcatechol 68.8 16.0 23.3

4-Methylcatechol 359.7 0.7 0.2


236 A. Li et al.

volume was free in the enzyme–substrate complexes of

monocyclic catecholic substrates. It was suggested that the

specificity of BphC_LA-4 for bicyclic substrate was higher

than that for monocyclic substrate. Because the methyl group

of 3-methylcatechol partially occupied this free volume, the

specificity for 3-methylcatechol should be higher than that

for catechol. As per Senda’s report, the amino acid residues in

BphC_KKS102, Phe-186 and Thr-280 interacted with posi-

tion C6 of the catechol ring of biphenyl, which corresponded

with position C4 of monocyclic catecholic substrates. The

amino acid residue Phe-186 interacted with the catechol ring

and contributed to the O2-binding pocket, which was well

conserved in known BphCs (Senda et al., 1996; Sato et al.,

2002). This suggested that Phe-186 was important for the

enzymatic activity, but could not determine substrate speci-

ficities. However, Thr-280 was located at the entrance and

interacted with the benzene ring moiety of 2,3-dihydroxybi-

phenyl (Senda et al., 1996), indicating that Thr-280 was very

important for the specificity of BphC_KKS102. In BphC_LA-

4, Thr-280 was replaced with an Ile residue (Fig. 2b). In the

4-chlorocatechol:BphC complex and 4-methylcatechol:BphC

complex, the steric hindrance by Ile-279 played an important

role in the specificity of BphC_LA-4. Furthermore, the

specificity for 4-methylcatechol should be lower than that

for 4-chlorocatechol, because the methyl group was larger

than the chlorine atom. Therefore, we hypothesized that the

specificity of BphC_LA-4 was in the following order:

2,3-dihydroxybiphenyl4 3-methylcatechol4 catechol4 4-

chlorocatechol4 4-methylcatechol.

The purified BphC activities in 2,3-dihydroxybiphenyl

and other dihydroxylated substrates were determined. The

results showed that BphC_LA-4 exhibited higher cleavage

activity for 2,3-dihydroxybiphenyl and 3-methylcatechol.

However, catechol, 4-methylcatechol and 4-chlorocatechol

could be transformed with relatively low activities. The

specific activity for 2,3-dihydroxybiphenyl of BphC_LA-4

was higher than that of BphC_KF707 (87.2 U mg�1), but

lower than that of BphC_LB400 (191 U mg�1) (Furukawa &

Arimura 1987; Eltis et al., 1993). The optimal temperature

for BphC_LA-4 was 40 1C (Fig. 5), which was higher than

for BphC_LB400 and BphC2_P6 (Asturias et al., 1994;

Seeger et al., 1995). To our knowledge, the optimal tempera-

ture for BphC_LA-4 was the highest, with the exception of

BphC_JF8, which was a thermostable manganese-dependent

2,3-dihydroxybiphenyl 1,2-dioxygenase (Hatta et al., 2003).

Because the Km for 2,3-dihydroxybiphenyl was the lowest

among these dihydroxylated substrates, the BphC_LA-4

showed the highest affinity for 2,3-dihydroxybiphenyl, im-

plying a better fit with the bigger substrate. According to the

specificity constant (Kcat/Km) (Table 1), BphC_LA-4 cleaved

dihydroxylated substrates in the same order with the pre-

dicted specificity by enzyme–substrate complexes. The

(d)(c)

(b)(a)

0 5 10 15 20 25 30

0

20

40

60

80

100

Rel

ativ

e ac

tivity

(%

)

L-Ascorbic acid concentration (mM)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

40

50

60

70

80

90

100

Rel

ativ

e ac

tivity

(%

)

SDS concentration (mM)0 100 200 300 400 500 600

0

20

40

60

80

100

Rel

ativ

e ac

tivity

(%

)

KI concentration (mM)

Fig. 6. Effects of metal ions and inhibitors on

enzyme activity. (a) Effects of metal ions; (b)

effects of L-ascorbic acid; (c) effects of KI; (d)

effects of SDS.



specificity of BphC_LA-4 for dihydroxylated substrates was

different with BphC_JF8 and BphC_LB400 (Eltis et al., 1993;

Hatta et al., 2003). Moreover, BphC_LA-4 preferentially

cleaved these substituted catechols in the order 3-sub-

stituted4 4-substituted and 4-Cl4 4-methyl, which sug-

gested that catalysis was affected by steric determinants.

In summary, the bphC gene of strain LA-4 was amplified

and expressed successfully. BphC_LA-4 was an iron (II)-

dependent type I extradiol dioxygenase. This is an effective

way to predict or explain the specificity of BphC using the

enzyme–substrate complexes constructed by the method of

bioinformatics.

Acknowledgement

This work was supported by the National Natural Science

Foundation of China (No. 50608011).

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Documents

Enzyme–substrate interaction and characterization of a 2,3-dihydroxybiphenyl 1,2-dioxygenase from Dyella ginsengisoli LA-4