0021-9193/93/020395-06$02.00/0 ... Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. ... nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is.
JOURNAL OF BACTERIOLOGY, Jan. 1993, p. 395-400
Vol. 175, No. 2
0021-9193/93/020395-06$02.00/0 Copyright C 1993, American Society for Microbiology
Oxidation of Biphenyl by a Multicomponent Enzyme System from Pseudomonas sp. Strain LB400 JOHN D. HADDOCK,* LOUISE M. NADIM,t AND DAVID T. GIBSON
Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242 Received 18 September 1992/Accepted 29 October 1992
Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl. Incorporation of both atoms of molecular oxygen into the substrate was shown with 1802. The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components. Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity. Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring.
rachlorobiphenyl (24). However, it is not known whether biphenyl 2,3-dioxygenase or a different 3,4-dioxygenase catalyzes the reaction. Therefore, we have initiated studies of the biphenyl dioxygenase of Pseudomonas sp. strain LB400 to determine the enzyme's substrate specificity and mode of attack on PCBs. In this report, we describe the resolution of biphenyl dioxygenase into three protein fractions that are required for the oxidation of biphenyl to cis-biphenyl dihydrodiol.
Aromatic hydrocarbons are growth substrates for aerobic bacteria that possess enzymes that add molecular oxygen to the ring. Dioxygenases initiate the degradation of benzene (17), toluene (16), naphthalene (21), and biphenyl (18) by catalyzing the oxidation of these substrates to the corresponding cis-dihydrodiols. The first three dioxygenases are multicomponent proteins that form short electron transport chains with flavins and iron-sulfur clusters as redox components (19). The initial component of the chain is a flavoprotein that oxidizes reduced pyridine nucleotides and passes the electrons to the terminal dioxygenase via a ferredoxin electron carrier. The ferredoxin and dioxygenase contain [2Fe-2S] redox centers involved in electron transport, and the latter protein contains the active site for the incorporation of 02 into the aromatic substrate. Biphenyl is an aromatic hydrocarbon that supports the growth of a large number of bacterial isolates (see, for example, references 8 and 26). The initial reaction in the degradation of biphenyl by a Beijerinckia sp. is catalyzed by biphenyl 2,3-dioxygenase. The reaction product has been identified as cis-(2R,3S)-dihydroxy-1-phenyl-cyclohexa-4,6diene (cis-biphenyl dihydrodiol) (18, 38). This reaction (Fig. 1) appears to be used by almost all bacteria that grow with biphenyl as the sole source of carbon and energy. Interest in these organisms stems from observations that after growth with biphenyl, many of them can oxidize several chlorinated biphenyl (CB) congeners that are present in commercially produced polychlorinated biphenyls (PCBs) (1, 8, 13). However, the degradation of PCBs in which chlorine occupies the 2,3- positions and the identification of metabolites formed from certain CB congeners have led to the suggestion that oxidation may occur at locations on the ring other than the 2,3- position (4, 6, 7, 14, 22, 23, 35). This has been confirmed forAlcaligenes eutrophus H850 and Pseudomonas sp. strain LB400. Biphenyl-grown cells from both organisms oxidized 2,5,2',5'-CB to cis-3,4-dihydroxy-3,4-dihydro-2,5,2',5'-tet-
MATERIALS AND METHODS Organisms and growth conditions. Pseudomonas sp. strain LB400 was isolated from a site contaminated with PCBs (9) and was provided by Herman Finkbeiner, General Electric Co., Schenectady, N.Y. The organism was maintained on a mineral salts medium (28) supplemented with 0.05% yeast extract and biphenyl vapors supplied from crystals placed in the lid of an inverted petri dish. The growth temperature was 30°C. Growth in liquid culture was carried out in the same medium, with 0.2% biphenyl added directly to the medium. The organism was mass cultured in a 100-liter fermentor with medium that was modified by increasing the concentrations of ammonium sulfate and biphenyl by a factor of four. Cells were harvested by centrifugation at the end of the log phase of growth. Excess biphenyl was removed from the culture prior to being harvested by filtration through glass wool. The cells were washed once in 20 liters of 20 mM NaK phosphate buffer (pH 7.0) and centrifuged. Cell paste was frozen in liquid nitrogen and stored at -70°C. Eschenchia coli JM109(pDTG614) contains the cloned todB gene which encodes the ferredoxin component of toluene dioxygenase (ferredoxinTOL) fromPseudomonasputida F1 (19a). Expression of the gene is under control of the isopropyl-p-Dthiogalactopyranoside (IPTG)-inducible tac promoter carried on the expression vector pKK223-3 (Pharmacia LKB Biotechnology, Piscataway, N.J.). The strain was grown in a 10-liter fermentor on medium that contained tryptone (20 g/liter), yeast extract (10 g/liter), NaCl (5 g/liter), and ampicillin (100 ,ug/liter). Expression of todB was induced by addition of IPTG (1 mM) when the optical density of the
Corresponding author. t Present address: Division of Biochemistry, The University of Texas at Austin, Austin, TX 78712. *
395
396
HADDOCK ET AL.
N'~
J. BAcT1ERIOL.
~~~0
X is.H
NAD(P)H
Biphenyl FIG. 1.
+
H+ NAD(P)+
OH
'OH
cis-Biphenyl dihydrodiol The reaction catalyzed by biphenyl clioxygenase.
culture at 600 nm had reached 3.0. Growth waIS continued for 3 h, after which the cells were harvested and stored at -700C. Preparation of cell extract. Forty grams wet weight) of frozen LB400 cells was thawed and resuspenided in 80 ml of breakage buffer composed of 50 mM 2-(N-nnorpholino)ethanesulfonic acid (MES) buffer, 5% (vol/vol) ethanol, and 5% (vol/vol) glycerol adjusted to pH 6.0 with qaOH. Phenylmethylsulfonyl fluoride and dithiothreitol wer*e added to final concentrations of 0.2 and 1 mM, respectivelyy. DNase I and RNase A (Sigma Chemical Co., St. Louis, Mlo.) were added to final concentrations of 10 pg/ml each. 'The cells were broken by passage through a chilled French ]pressure cell at 20,000 lb/in The crude extract was centrifuge,ed at 146,000 x g for 1 h at 4VC. The supernatant was pe]Ileted in liquid nitrogen and stored at -70'C. Cell extract fFrom 10 g of E. coli(pDTG614) cells was prepared in an anallogous manner. LB400 cells used for the O incorporation eiKperiment were washed with breakage buffer prior to lysis. Fractionation of cell extract. LB400 cell extrract (100 ml [2.2 g of protein]) was loaded onto a column (5.0 by 16 cm) of Q Sepharose Fast Flow anion-exchange resin (F'harmacia LKB Biotechnology) equilibrated with breakage biuffer. Unbound protein was washed from the column with C).4 liters of the same buffer at a flow rate of 8 mlmin. Bouind protein was eluted with a 1.5-liter linear gradient from 0 to 0.35 M KCI, and 12-ml fractions were collected. The col umn was operated with a Pharmacia fast protein liquid c]hromatography system at 50C. 180 incorporation. Following ultracentrif ugation of the crude LB400 cell extract, ammonium sulfatee was added to the supernatant to a final concentration of 70' Yo of saturation. The precipitated protein was recovered by centrifugation, dissolved in the buffer used to lyse the cells, and desalted on a gel filtration column (Presto Desalting Ccolumn; Pierce Chemical Co., Rockford, Ill.). Incubations vere performed in 28-ml flasks fitted with Teflon-lined septaLand screw-onhole caps (Supelco, Bellefonte, Pa.). The dlesalted extract (10 mg of protein) was incubated in 10 ml c)f 50 mM MES buffer (pH 6.0) that contained 400 p.M ferm ous ammonium sulfate, 20 mg of NADPH, and 200 p.M biphenyl. The headspace (18 ml) contained air or air plus 4iml of 98 atom% 1802 (Cambridge Isotope Laboratories, W4oburn, Mass.). The flasks were incubated at 300C with shakkng at 250 rpm for 30 min. The reactions were terminated by heating at 750C in a water bath for 10 min, followed by addiltion of 10 ml of ethyl acetate containing 1 mg of phenylboroni ic acid (PBA) to derivatize the cis-biphenyl dihydrodiol. The fIasks were then .
v
returned to the water bath for 15 min. The ethyl acetate was removed, and the aqueous phase was extracted with another 10 ml of ethyl acetate. The combined extracts were dried with sodium sulfate, concentrated to approximately 100 l, and analyzed by gas chromatography-mass spectrometry. Authentic cis-2,3-dihydroxy-2,3-dihydrobiphenyl was also derivatized with PBA to serve as a reference. Gas chromatography-mass spectrometry. A 1-jul volume of the concentrated ethyl acetate extract was injected onto a capillary column (0.2 mm by 25 m) with a 0.33-pm-thick film of cross-linked dimethylpolysiloxane stationary phase (Hewlett-Packard, Avondale, Pa.). The carrier gas was helium at a flow rate of 0.45 ml/min. Injections were split (100:1). The injection port temperature was 2200C, and the column temperature was increased from 150 to 2750C at a rate of 100C/min. Mass spectra were obtained with a Hewlett-Packard model 5970 Mass Selective Detector with an electron impact ion source. The ionizing voltage and source temperature were 70 eV and 2800C, respectively. A 5-Pl sample of the headspace atmosphere was similarly analyzed, except that the column temperature was kept at 1500C. Protein concentrations. Protein concentrations were estimated by the method of Bradford (10), with bovine serum albumin as the standard. Enzyme assays. The standard assay for biphenyl dioxygenase activity in cell extract was to measure the formation of radioactive biphenyl dihydrodiol at room temperature. Reaction mixtures (0.25 ml) contained 50 mM MES buffer (pH 6.0), 400 p.M NADPH, 400 pM ferrous ammonium sulfate, 160 P.M [U-14C]biphenyl (9.5 or 7.6 Ci/mol), and 5 PId of cell extract (0.1 to 0.15 mg of protein). The reaction was initiated by the addition of biphenyl, which was dissolved in 2 P. of dimethylformamide. After incubation at room temperature for an appropriate time period (typically 5 to 20 min), 25 PI of 37% formaldehyde was added to terminate the reaction. A 5-pl sample of each mixture was applied to the origin of a plastic-backed thin-layer silica gel plate (E. Merck, Darmstadt, Germany) and air dried. The plate was developed with 100% n-heptane to remove unreacted biphenyl from the origin. The origin, containing the reaction product, was then excised, and the radioactivity was measured by liquid scintillation counting. The concentrations of NADPH, biphenyl, and MES buffer were optimized to produce the maximum linear rate. This method was also used to assay the activity of partially purified components from chromatographic column fractions. Each assay mixture contained 25 P. of each of the peak fractions of two of the components and 25 P1 of the fraction containing the third component to be assayed. Thus, the peak fractions from components A and B, A and C, and B and C were combined to assay fractions containing components C, B, and A, respectively. In some assay mixtures, component B was replaced with cell extract from E. coli (pDTG614) containing ferredoxinTOL. One unit of biphenyl dioxygenase activity was defined as the amount of protein that oxidized 1 nmol of biphenyl per min. The reduction of cytochrome c was also used to assay components A and B. The reaction mixture (1.0 ml) contained 50 mM bis(2-hydroxyethyl)imino-tris(hydroxymethyl) methane (Bis-Tris) buffer (adjusted to pH 7.0 with HCl), 300 p.M NADPH, 87 pM horse heart cytochrome c (type III; Sigma), 29 pg of protein of partially purified component A, and 109 pg of protein of partially purified component B. The rate of the reaction was monitored spectrophotometrically at 550 nm, and a molar absorbance value of 21,000 M-1 cm-1 for reduced cytochrome c minus that for oxidized cyto-
BIPHENYL DIOXYGENASE
VOL. 175, 1993
397
TABLE 1. Requirements for biphenyl dioxygenase activity in cell extract Presence or absence of the following assay components':
Cell Biphenyl NADPH NADH extract
Fe2"
FAD FMN
Activityb
(nmol/min/mg of protein)
8.5c 7.7 7.7 10 9.5 0.34 0.39 Boiled a Final concentrations were as follows: cell extract, 0.64 mg/ml; [14C]biphenyl, 160 IM; NADPH and NADH, 400 pM; Fe(NH4)2SO4, 400 FM; FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), 1 PM. +, +
+
+
-
+
-
-
+ + + + +
+ + + + + +
+ + + +
+ -
+ + + + +
+ -
+ -
component present; -, component absent. I Averages of duplicate determinations with a 20-min incubation time. c Determined by the standard assay as described in Materials and Methods.
chrome c was used to calculate activity (34). One unit of activity was defined as the amount of protein that reduced 1 tmol of cytochrome c per min. Chemicals. 2,3-Dihydroxybiphenyl was obtained from R. Unterman, General Electric. cis-2,3-Dihydroxy-2,3-dihydrobiphenyl was obtained from the transformation of biphenyl by Beijerenckia sp. strain B8/36 (18). All other reagents were purchased commercially and were of the highest available purity. RESULTS Enzyme activity in cell extract. Biphenyl dioxygenase activity in crude cell extract was dependent on the presence of NADPH or NADH as the electron donor (Table 1). The rate with NADP+ was 17% of the rate found with NADPH. No other cofactors were required; however, the addition of ferrous iron and flavins stimulated activity. The rate of the reaction was linear for 20 min (data not shown). The optimum pH was 6.0 in 50 mM MES buffer. The activities at pH 5.5, 6.5, and 7.0 in 50 mM MES buffer were 77, 69, and 36%, respectively, of the activity at pH 6.0. After incubation for 24 h, the rate was 83% of the initial value for cell extract kept on ice and 73% for cell extract kept at 50C. The requirement for protein-protein interactions for catalysis was suggested
14
E 12 U,
c.10 CL10 8
._
--
6 c_ 6 5 C)
4 L
K
0.0
0.2
0.4
0.6 0.8 1.0 1.2 1.4 Protein, mg/ml FIG. 2. The effect of protein concentration on biphenyl dioxygenase activity in cell extract.
FIG. 3. Autoradiogram of the products formed from ["4C]biphenyl by biphenyl dioxygenase in cell extract. Lanes: 1 and 2, NADPH as electron donor; 3, NADH as electron donor; 4, without a reductant; 5, with boiled cell extract; 6, without cell extract. The incubation times were 10 min for the reaction depicted in lane 1 and 30 min for all others. The standards were as follows: A, biphenyl; B, 2,3-dihydroxybiphenyl; C, cis-2,3-dihydroxy-2,3-dihydrobiphenyl. The origin (lower arrow) and solvent front (upper arrow) are indicated. The developing solvent was chloroform-acetone (80:20).
by the nonlinear correlation between the reaction rate and the protein concentration in the assay (Fig. 2). Reaction products. Biphenyl dihydrodiol was the only product detected by autoradiography when NADPH was added as the electron donor for the reaction (Fig. 3). When NADH was substituted for NADPH, less of the dihydrodiol was detected and the reaction mixture turned yellow, indicating that the ring cleavage product was produced. Ring cleavage was also indicated by the accumulation of radioactive polar products that remained at the origin of the thinlayer plate. 180 incorporation. The mass spectrum of the PBA derivative of biphenyl dihydrodiol produced by cell extract under an atmosphere of air showed a base peak of 274 mlz (Fig. 4A), which is predicted from the addition of one phenylboronate moiety to biphenyl dihydrodiol. PBA was shown to react in a 1:1 ratio with cis-1,2-diols of cyclopentane and cyclohexane and in a 2:1 ratio for the corresponding trans isomers (32). The gas chromatography retention time and mass spectrum were identical to those for the PBA derivative of cis-biphenyl dihydrodiol produced by the Beijerenckia sp. strain B8/36 that was prepared to confirm the results. The PBA derivative of biphenyl dihydrodiol produced under a headspace that contained 1602, 160-180, and 1802 (55:1:44) (Fig. 4B) showed a mixture of ions at 274, 276, and 278 mlz (56:2:42), showing that both atoms of molecular oxygen are incorporated into the substrate by biphenyl dioxygenase. The ion intensity at 276 mlz was corrected for the contribution arising from 278 - 2 mlz before the ratio calculations were made. Resolution of biphenyl dioxygenase. Fractionation of cell extract on an ion-exchange column yielded three protein fractions that were required in combination for biphenyl dioxygenase activity (Fig. 5; Table 2). The required fractions containing protein that eluted at the lowest salt concentra-
398
HADDOCK ET AL.
J. BACTERIOL. TABLE 2. Requirement for three protein components for biphenyl dioxygenase activity
Act(dp
Component (25 ,jj)a
75j-
C
A + B ........
-2 25 ~
270
160
B + C .......
620
A + B + C .......
272
274
276
280
278
150
A+ C ........
6,600
A + EcC .........
120
B + Ec .......
160
C + Ec ....... A + C + Ec .......
a b
C
550
4,300
Refer to the legend to Fig. 5 for component descriptions. Determined by the standard assay with a 20.min incubation time. Ec, E. coli(pDTG614) cell extract (0.31 mg of protein).
.~100 C
0 751-
C
25270
272
276
274
280
278
MLZ FIG. 4. Mass spectra of the PBA derivative of biphenyl dihydrodiol produced by cell extract in the presence of air (A) and of air enriched with 1802 (B).
tion were yellow and were designated component A. Fractions required for activity that eluted next were reddish brown and were designated component C. A third group of necessary fractions eluted from the column near the end of the salt gradient. These fractions were brown and were designated component B. This component could be replaced
by cell extract from E. coli(pDTG614) which expresses the ferredoxinTOL component of toluene dioxygenase from P. putida F1 (Table 2). The maximum level of biphenyl dioxygenase activity was found when the peak fractions of all three components were combined. Less than 10% of the maximum rate was found when components B and C alone were combined. This activity likely resulted from partial overlap of component C with component A (Fig. 5). Cytochrome c reductase activity. The yellow appearance of fraction A and the stimulation of biphenyl dioxygenase activity by flavin nucleotides (Table 1) suggested that a flavoprotein was involved in catalysis. The ability of E. coli cell extract containing ferredoxinTOL to replace component B in the reaction (Table 2) suggested that a ferredoxin was also involved. In combination, the reductase and ferredoxin components of toluene dioxygenase from P. putida F1 catalyze the reduction of cytochrome c (37). Components A and B from strain LB400 had cytochrome c reduction rates of 0.17 and 0.022 pmolmin/mg of component protein, respectively, when assayed separately. When combined, the rate increased to 1.2 pmol/min/mg of protein. This result
2.0 1.8
1.6
-
0
