kinetic mechanism of the dechlorinating flavin-dependent ...10.1074/jbc.m116... · as the subunit...

Kinetic Mechanism of HadA monooxygenase

1

Supplemental Information

Kinetic Mechanism of the Dechlorinating Flavin-Dependent Monooxygenase HadA

Panu Pimviriyakul1, Kittisak Thotsaporn2, Jeerus Sucharitakul2 and Pimchai Chaiyen1*

1Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology,

Faculty of Science, Mahidol University, Bangkok, Thailand 10400. 2Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand 10300.

*Running title: Kinetic Mechanism of HadA monooxygenase

*Corresponding authors

E-mail: [email protected]. Phone: +66-2201 5596. Fax: +66-2354 7174.

Sequence similarity network (SSN) for HadA

Figure S1. Clusters of HadA homologs arranged based on sequence similarity. HadA (yellow dot) was arranged into clusters of enzymes that have sequence identities greater than >27.74% % for each cluster. These enzymes can be arranged into two major clusters in which HadA is associated with a larger cluster. Only 11 proteins (red dots) have been investigated at the protein level.


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Expression and Purification of HadA. Recombinant HadA was purified according to the protocol described in the Experimental

Procedures. The purity of HadA was analyzed by SDS-PAGE (12% SDS) which is stained with coomassie brilliant blue. The results showed that the purified HadA was more than 95% pure and has a subunit molecular mass ~59 kDa (Fig. S2).

Figure S2. SDS-PAGE (15%) analysis of purified HadA. Lane 1: AccuProtein Chroma molecular weight markers (Enzmart Biotech, Thailand), Lane 2: Purified HadA.


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Determination of the native molecular mass of HadA.

The native molecular mass of HadA was determined using a fast protein liquid chromatography (FPLC) instrument from ÄKTA (Amersham Biosciences). SephacrylTM S-300 high resolution (Amersham Pharmacia) size exclusion chromatographic medium was packed into a column as the stationary phase for protein separation. Protein samples were eluted in 50 mM sodium phosphate (NaPi) pH 7.0 containing 150 mM NaCl as the mobile phase with a flow rate of 0.5 ml/min. Protein concentration was determined by measuring the absorbance at 280 nm. A protein standard curve was constructed using standards with known native molecular weights consisting of blue dextran (2000 kDa, as void volume, V0), ferritin (440 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum alubumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa). Chromatograms of all standard proteins are shown in Fig. S3A. A plot of logMW versus Ve/V0 was linear (Fig. S3B) and the native molecular mass of HadA was determined as 194 kDa. As the subunit MW of HadA is 59 kDa, the quaternary structure of HadA can be calculated as 3.3 subunits/native protein. Because X-ray structures of HadA homologs TcpA and TftD (Webb BN et. al, J. Biol. Chem., 2014; Hayes et. al, Int. J. Mol. Sci., 2012) indicate that these enzymes are tetramers, it is likely that HadA is a tetrameric protein.

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1 Blue Dextran2 Ferritin3 β-Amylase4 Alcohol dehydrogenase5 Bovine serum albumin6 Carbonic anhydrase7 Cytochrome c

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Figure S3. Native molecular mass determination of HadA. (A) Chromatograms of protein standards from large to small sizes, blue dextran (2000 kDa), ferritin (440 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). (B) Elution volume of HadA (blue diamond) when compared to a standard curve of standard protein molecular weights (red circles).


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Flavin specificity of HadA. Binding of FADH- or FMNH- to HadA was investigated by stopped-flow spectrophotometry (Fig.

S4) to identify whether FADH- or FMNH- can be bound by HadA as described in the main text.

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Time (s) Figure S4. Kinetic traces resulting from the mixing of solutions of reduced flavin and air-saturated HadA. Solutions of (A) FADH- or (B) FMNH- (final concentration of 12.5 µM) were mixed against air-saturated HadA (final concentration 37.5 µM) in 20 mM HEPES pH 7.5 at 25oC in the stopped-flow spectrophotometer. The re-oxidation of (A) FADH- and (B) FMNH- in the absence (red line) and presence of HadA (blue lines) were monitored at 380 nm (solid line) and 450 or 446 nm (dashed line).


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HPLC chromatograms of various chlorophenols and products obtained from single turnover reactions.

Samples from single turnover reactions of HadA and various substrates were prepared and analyzed by HPLC/DAD or HPLC/DAD/MS using a Nova-Pak® (Water) C18 reverse phase column with a 4 µm particle size and 3.9 x 150 mm column size as the stationary phase. Chromatograms for substrates and products in Table 1 are shown in Figs. S5A-S5C.

Figure S5A. HPLC chromatogram profiles of single turnover reactions of HadA with 4-CP. HPLC/DAD analysis was performed using a mobile phase with a gradient of water/methanol containing 0.1% formic acid with a flow rate of 0.5 ml/min. The mobile phase gradient for analysis was carried out from 10-70% methanol. Chromatograms of 4-CP and product were monitored at retention times 26.8 and 4.4 min, respectively. The retention time of the standard HQ is the same as those for the product of 4-CP.

Figure S5B. HPLC chromatogram of single turnover reactions of HadA with 2-CP. Mobile phase was a gradient of H2O/methanol containing 0.1% formic acid with a flow rate of 0.5 ml/min. The gradient was carried out from 5-50% methanol. The hydroxylated product of 2-CP was monitored at 294 nm. Products have HPLC and mass spectroscopic profiles identical to standard compounds CHQ. These data indicate that HadA catalyzes para-hydroxylation of the ortho-substituted chlorophenol.


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Figure S5C. HPLC chromatograms of single turnover reactions of HadA with chlorophenols with multiple substituents. Mobile phase was a gradient of H2O/acetonitrile containing 0.1% formic acid with a flow rate 0.5 ml/min. The gradient was carried out from 2.5-90 % acetonitrile. Substrate and product were detected by absorbance at 300 nm and their retention times are as indicated. Products from (A) 2,4-DCP, (B) 2,4,5-TCP, and (C) 2,4,6-TCP reactions were identified as CHQ, 2,5-DCHQ, and 2,6-DCHQ, respectively based on retention times of the standard compounds.


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Identification of the order of hydroxylation in the reaction of HadA with 2,4,5-TCP.

To identify the second step of 2,4,5-TCP oxygenation, multiple turnover reactions of 2,5-DCHQ (first product) were performed. Reactions (5 mL) contained 2,5-DCHQ (100 µM), G-6-P (1 mM), G-6-PD (0.5 unit/ml), C1 (1 µM), NAD+ (5 µM), FAD (10 µM), HadA (10 µM), and ascorbic acid (1 mM) in 20 mM HEPES pH 7.5. G-6-PD was added to start the reaction. Samples from the reactions were taken and quenched at different time points during the period of 2-10 hr. Samples were prepared and analyzed by HPLC/DAD or HPLC/DAD/MSD (see Experimental Procedures). Chromatograms of the multiple turnover reactions of HadA and 2,5-DCHQ are shown in Fig. S6A. The retention time of the product was found to be 4.3 min with m/z of 160.9 (Fig. S6B). The product from the 2,5-DCHQ reaction is likely 5-chlorohydroxyquinol (5-CHQL), which has a molecular mass of 160.5. Overall, the results indicate that HadA first converted 2,4,5-TCP (m/z of 197.0) to 2,5-DCHQ (m/z of 177.0). 2,5-DCHQ was then further converted into 5-chlorohydroxyquinol (5-CHQL, m/z of 160.9) which is unstable (Fig. S6B). These results suggest that HadA can degrade CPs via sequential hydroxylation with dechlorination at position 4 and then at position 2 to generate trihydroxy aromatic compounds (Fig. 3, main text).


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Figure S6. Multiple turnover reaction of 2,5-DCHQ. (A) HPLC chromatograms of multiple turnover reactions of HadA and 2,5-DCHQ. Mobile phase was a gradient of H2O/acetonitrile containing 0.1% formic acid with a flow rate 0.5 ml/min. The gradient was carried out from 2.5-90% acetonitrile. 2,5-DCHQ and its product were detected at retention times of 15.7 min and 4.3 min, respectively. (B) Mass spectra of products from 2,4,5-TCP biodegradation. Products from the first and subsequent steps of 2,4,5-TCP conversion catalyzed by HadA were identified as 2,5-DCHQ and 5-CHQL, respectively.


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Time (s) Figure S7. Reactions of HadA in the presence and absence of ascorbic acid. A solution of HadA:FADH- was mixed with air-saturated 20 mM HEPES pH 7.5 in the presence of 1 mM ascorbic acid (solid line) or in the absence of ascorbic acid (dashed line) at 25oC in the stopped-flow spectrophotometer. Absorption changes at wavelength 380 and 450 nm were monitored and analyzed according the description in the main text.


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Figure S8. Binding of HadA and FADH- and the conformational changes between the two forms of the HadA:FADH- complex. Double-mixing stopped-flow experiments were carried out to demonstrate that longer incubation of HadA with FADH- resulted in greater conversion of the HadA:FADH- complex into the form that reacts slower with oxygen. Under the first mixing, HadA (75 µM) was incubated with FADH- (25 µM) at various age times before buffer containing O2 was added in the second mixing (0.13 mM O2). (A) Absorption changes at 450 nm were monitored to follow the binding of HadA to FADH- (resulting in enzyme-bound flavin oxidation that is slower than free FADH- oxidation) and (B) at 380 nm for monitoring the change from fast to slow reacting species (slow reacting species formed C4a-hydroperoxyFAD around 0.1-1 s). The inset in (A) is a plot between ΔA450 at 2 s versus the incubation time to calculate the HadA:FADH- binding rate under this condition. The inset in (B) is a plot between ΔA380 at 2 s versus the incubation time to calculate the rate of the enzyme conformational change.


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Figure S9. Kinetic traces of the reaction of HadA:FADH- with various concentrations of oxygenated 4-CP. Changes in absorption at 380 nm (solid line) and 450 nm (dashed line) of the reaction between HadA:FADH- (75 µM and 25 µM respectively) and air saturated buffer (0.13 mM) in the presence of various concentrations of 4-CP (0.1-10.0 mM) in 20 mM HEPES pH 7.5, 1 mM ascorbic acid were monitored.


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[4-CP] (mM) Figure S10. Rate of formation of the HadA:4-CP dead-end complex. Double-mixing stopped-flow experiments were carried out to measure the rate of formation of the HadA:4-CP complex. HadA (75 µM) was mixed with various concentrations of 4-CP and incubated at various age times in the first mixing. The resulting solution was mixed with anaerobic FADH- (25 µM) under the second mixing. (A) ΔA450nm at 2 s which indicated the amount of free FADH- that could not bind to HadA due to the binding of 4-CP to the enzyme when using 4-CP at 0.8 (red circle), 1.6 (blue square), 3.2 (green diamond), and 6.4 mM (black triangle) was plotted against the incubation time. (B) Observed rate constants were plotted against 4-CP concentrations. The plot shows a linear relationship, indicating that the binding of 4-CP to HadA is a single-step reaction, consistent with kon (slope) and koff (intercept) for the 4-CP binding to HadA of 25 ± 2 M-1 s-1 and 10 ± 2 × 10-4 s-1, respectively.


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Time (s) Figure S11. Comparison of C4a-hydroperoxy-FAD formation in a different mixing mode on stopped-flow spectrophotometer. Kinetic traces obtained from two stopped-flow mixings (HadA:FADH- binary complex with 4-CP/O2 (solid red line) versus HadA:FADH- in the presence of 4-CP with O2 (dash blue line) were compared to determine whether 4-CP can binding to the HadA:FADH- binary complex.

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Figure S12. No C4a-hydroxy-FAD formation is observed in the absence of substrate. Experiments similar to the red lines in Fig. 9 (main text) were carried out in the absence of 4-CP to determine whether the signal detected in Fig. 9 (main text) was indeed due to formation of C4a-hydroxy-FAD. The reaction of HadA:FADH- with air saturated buffer was detected using excitation wavelengths at 380 nm (solid line) and 450 nm (dashed line) with emission wavelength >495 nm. Only oxidized FAD was detected in these experiments.


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Figure S13. C4a-hydroperoxy-FAD and C4a-hydroxy-FAD formation in the presence of 4-chlorophenol at various pHs. A solution of HadA:FADH- (75 µM and 25 µM) was mixed with air-saturated 4-CP (0.8 mM with 0.13 mM oxygen) at various pHs from 6.7-10.0 (final pH after mixing). Absorbance 380 nm was detected for C4a-hydroperoxy-FAD formation at various pHs.


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Derivation of equations used for calculation a dissociation constant for HadA:4-CP complex (used for analysis of the data in inset of Fig. 8B)

Define E = HadA, A=4-CP, and B = FADH- From this model KD,A=

E [A][EA]

Eq. S1

KD,B=E [B][EB]

or EB = E [B]KD,B

Eq. S2

Total protein ET = E + EA +[EB] Eq. S3 Substitute [EB] from Eq. S2 in Eq. S3

ET = E + EA +E [B]KD,B

ET = EA + E 1+[B]KD,B

E = ET -[EA]KD,B

KD,B+[B] Eq. S4

Substitute [E] from Eq. S4 in Eq. S1

KD,A=ET -[EA]

KD,BKD,B+[B]

[A]

[EA]

KD,A EA 1+BKD,B

= ET A -[EA][A]

KD,A EA 1+BKD,B

+ EA A = ET A

[EA] KD,A 1+BKD,B

+ A = ET A

[EA][ET]

=[A]

KD,A 1+ BKD,B

+ A

This equation can be related to absorption signals and concentration of each species as

∆A450∆A450max

=[HadA:4-CP][HadAT]

=[4-CP]

KD,4-CP 1+ FADH-

KD,FADH-+ 4-CP

As we know that [FADH-] in this experiment is 25 µM and 𝐾=,>?=@A = 2.0 µM Then,

∆A450∆A450max

=[4-CP]

13.5KD,4-CP+ 4-CP𝐸𝑞. 𝑆5

kinetic mechanism of the dechlorinating flavin-dependent ...10.1074/jbc.m116... · as the subunit...

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