Oxygenation of Monoenoic Fatty Acids by CYP175A1, an Orphan

Aug 31, 2012 - Oxygenation of Monoenoic Fatty Acids by CYP175A1, an Orphan ... been investigated to assess the enzymatic properties of this orphan ...
0 downloads 0 Views 1MB Size
Subscriber access provided by University Libraries, University of Memphis

Article

Oxygenation of monoenoic fatty acids by CYP175A1, an orphan cytochrome P450 from Thermus thermophilus HB27 Sandeep Goyal, Shibdas Banerjee, and Shyamalava Mazumdar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/bi300514j • Publication Date (Web): 31 Aug 2012 Downloaded from http://pubs.acs.org on September 6, 2012

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Oxygenation of monoenoic fatty acids by CYP175A1, an orphan cytochrome P450 from Thermus thermophilus HB27†

Sandeep Goyal1, Shibdas Banerjee1 and Shyamalava Mazumdar* Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.

Running title: Oxygenation of monoenoic fatty acids by CYP175A1

Keywords: P450, CYP175A1, Monoenoic fatty acids, Epoxidation, Allyl-hydroxylation.

*Correspondence: Prof. S Mazumdar, Phone no.: +91 22 2278 2363, FAX: +91 22 2280 4610, E-mail: [email protected], Web: http://www.tifr.res.in/~shyamal/ 1

These two authors contributed equally to this work.



This work was supported by Tata Institute of Fundamental Research.

1 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 45

Abstract The

catalytic

activity

of

CYP175A1

towards

monooxygenation

of

saturated

and

monounsaturated fatty acids of various chain lengths (C16-C24) has been investigated to assess the enzymatic properties of this orphan thermostable cytochrome P450 enzyme. The results showed that the enzyme could catalyze the reaction of monounsaturated fatty acids but not of saturated fatty acids. The product analyses using ESI-MS and GC-MS revealed an important regioselectivity in the CYP175A1 catalyzed monooxygenation of the monoenoic fatty acids depending on the ethylenic double bond (C=C) configuration. When the double bond was in cisconfiguration, an epoxy fatty acid was found to be the major product and two allyl-hydroxy fatty acids were found to be the minor products. But, when the double bond was in trans-configuration the product distribution was reversed. The oxygenation efficiency was found to be the highest for Palmitoleic acid (chain length C16) but there was no direct correlation of the activity with the chain length or the position of unsaturation of the fatty acid. Molecular docking calculations showed that the ‘U’-type conformations of the monoenoic fatty acids are particularly responsible for their binding in the enzyme pocket and that is also consistent with the observed regioselectivity in the oxygenation reaction. The present results provide evidences that CYP175A1 can catalyze regioselective oxygenation reaction of several monoenoic fatty acids though it cannot catalyze the oxygenation of the corresponding saturated analogues. These studies may provide critical information on the nature of the enzyme pocket and of the possible the natural substrate of this orphan enzyme.

2 ACS Paragon Plus Environment

Page 3 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Abbreviations Heme: Iron protoporphyrin IX regardless of oxidation and ligation state; WT: Wild type; Tm: Melting temperature; PDB: Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, http://www.rcsb.org/pdb/; PMSF: Phenyl methyl sulphonyl fluoride; IPTG: Isopropyl-β-D-1-thiogalactopyranoside;

TBHP:

tert-Butyl

hydroperoxide;

m-CPBA:

m-

chloroperbenzoic acid; EDTA: Ethylenediaminetetraacetic acid dipotassium salt; H2O2: Hydrogen peroxide; NADH: β-Nicotinamide adenine dinucleotide, reduced dipotassium salt; ESI-MS: Electrospray ionization mass spectrometry; GC-MS: Gas chromatography mass spectrometry; CID: Collision induced dissociation; GOLD: Genetic optimization for ligand docking.

3 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cytochrome P450 forms a superfamily of hemoproteins1 that catalyze the monooxygenation of various organic compounds2 including aromatic and aliphatic hydroxylation, N-oxidation and also epoxidation of C=C double bonds. These enzymes often catalyze regio- and stereospecific oxygenation of nonactivated hydrocarbons under mild conditions, which is a challenging task for chemical catalysis in organic synthesis.3 Thus, cytochrome P450s have great potential as biocatalysts to perform numerous industrially important reactions such as synthesis of drugs and other fine chemicals.3 However, low tolerance to various solvents and high temperature have generally limited the usefulness of cytochrome P450 for industrial application. Interestingly, two thermophilic cytochrome P450s e.g. CYP119 and CYP175A1 have been isolated, purified and characterized 4-7 in recent years. Both of them possess extreme stability (Tm = 91oC for CYP1198 and Tm = 88oC for CYP175A15) and might be used to overcome the above limitations. The natural substrates and the biological functions of both of these enzymes (CYP119 & CYP175A1) are unknown9 and therefore they are considered as the so-called orphan P450.10 However, there have been recent advances in using an engineered CYP119 to catalyze the fatty acid hydroxylation11 and mutant Q67G/Y68I of CYP175A1 to catalyze testosterone hydroxylation.12 Native CYP175A1 has been used to hydroxylate β-carotene at 3- and 3'positions.13 CYP175A1 shows the highest sequence identity of 26% with cytochrome P450BM-3 (CYP102A1)5 from Bacillus megaterium among all the structurally characterized bacterial cytochrome P450 enzymes. A close inspection of the substrate binding pocket from the crystal structure of CYP175A1 and CYP102A1 shows that the pockets/active sites of these two enzymes are similar and mostly surrounded by hydrophobic residues. Hence, both of them should prefer 4 ACS Paragon Plus Environment

Page 4 of 45

Page 5 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

hydrophobic substrates to show catalytic activity. Long chain saturated fatty acids (Lauric acid, myristic acid, palmitic acid and stearic acid) were found to be the natural substrate of P450BM3.14 However, no substrate other than β-carotene13 has been reported for the native CYP175A1 so far. It is known that Thermus thermophilus HB27 produces lipolytic enzymes15, 16, and it has also its own lipolytic activity.17 Therefore, we systematically screened several fatty acids (saturated and monounsaturated) for their potential as substrates for CYP175A1. Here we report that CYP175A1 is able to catalyze the monooxygenation reaction of several monoenoic fatty acids though it cannot catalyze the oxygenation of the corresponding saturated analogues. Along with the substrate specificity, we also investigated the regioselectivity of the monooxygenation reactions of those monoenoic fatty acids both experimentally and theoretically.

Experimental Materials The various components of bacteriological culture media were purchased from Himedia, India. Ampicillin sodium salt, chloramphenicol, DNaseI (from bovine pancrease), PMSF, IPTG, sodium cholate, anhydrous sodium sulphate, potassium bicarbonate, hexamethyldisilazane, trimethylchlorosilane, pyridine, 70% aqueous solution of TBHP, (Trimethylsilyl) diazomethane, NADH, and several fatty acids (saturated and monounsaturated) were purchased from Sigma Aldrich. The lysozyme (from hen egg white) and m-CPBA was purchased from Fluka. The salt ammonium sulphate, dipotassium hydrogen ortho phosphate and potassium dihydrogen orthophosphate, tris-HCl (2-Amino-2-hydroxymethyl-propane-1, 3-diol hydrochloric acid) and tris-base were purchased from USB Chemicals. The column material Phenyl Sepharose was

5 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

purchased from GE Healthcare Bio-Sciences AB, Uppasala. Hydroxyapetite material was purchased from Biorad Laboratories Inc, Hercules, CA. HPLC and/or GC grade methanol, chloroform, diethyl ether, dichloromethane, toluene, cyclohexane and other required solvents were purchased from Merck Chemicals. EDTA and was purchased from s. d. Fine Chemicals, India. Hydrogen peroxide was purchased from Qualigens. All other buffer components were purchased from Qualigen/s. d. fine chemicals. Millipore water was used for buffer preparation and for all other experimental work wherever required. The concentration of H2O2 was measured spectrophotometrically using the molar extinction coefficient (ε) 39.4 M-1cm-1 at 240 nm.18 m-CPBA was re-crystallized in a 1:3 diethyl ether/petroleum ether solution using the method described by Davies et al.19

Protein Expression and Purification The pKK-223 plasmid, encoding the gene for WT CYP175A1, was a kind gift from Prof. V. B. Urlacher (University of Stuttgart, Stuttgart, Germany). The wild type CYP175A1 was expressed and purified by earlier reported method.5, 20, 21 In brief, CYP175A1 was grown by taking a single colony of the BL21-DE3 codon plus RP cells, harboring the plasmid encoding the gene for WT protein and inoculating it into the 2×YT media containing ampicillin (100 µg/ml) and chloramphenicol (50 µg/ml). Expression of protein was induced by addition of IPTG (1mM final concentration), and the culture was grown at 30oC for 48 hr. After the cell lysis, the protein was precipitated by (NH4)2SO4 (35 to 50 % saturation), re-dissolved in buffer and purified by a hydrophobic column (Phenyl Sepharose), and fractions having Rz (Reinheitszahl, A417/A280) ≥ 1.4 were collected. The protein was further purified on a hydroxyapatite column and fractions having Rz ≥ 1.5 were collected.20 The purified fractions were concentrated using centriprep

6 ACS Paragon Plus Environment

Page 6 of 45

Page 7 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

concentrators (10 kD cut-off membrane, Millipore), dialyzed in 50 mM KPi, pH 7.5 buffer and stored at -25oC in 50 % glycerol. The concentration of CYP175A1 was estimated spectrophotometrically from the absorbance at 418 nm (ε417 = 104 mM

−1

cm−1).5 The purity of the

protein was checked by SDS PAGE, and the ESI-mass spectrum of the purified protein (See Figure S1 in the Supporting Information) confirms the molecular weight of the protein ~44.87 kD. The plasmids encoding wild type putidaredoxin (PdX) and putidaredoxin reductase (PdR) were kindly supplied by Prof. S. G. Sligar (University of Illinois, USA). The wild type putidaredoxin (PdX) and putidaredoxin reductase (PdR) were expressed and purified using a reported procedure22 and the concentration of PdX was determined from the absorbance at 455 nm (ε455 = 5.9 mM−1cm−1)23 and that of PdR was estimated from the absorbance at 454 nm (ε454 = 10.9 mM−1 cm−1).24 Enzymatic Oxygenation of Fatty Acids Fatty acid oxygenation by CYP175A1 was carried out by incubating 500 µL reaction mixture containing 20 µM CYP175A1, 600 µM fatty acid and 20 mM H2O2 in 50 mM KPi buffer (pH= 7.5) at 45oC unless otherwise mentioned. The controls for the reactions were carried out that contained all the reactants except CYP175A1 or except H2O2 in the assay mixture. Each reaction was allowed to proceed for 60 min unless otherwise stated. The reaction mixture was then extracted with chloroform and the chloroform layer was collected, dried over anhydrous sodium sulfate, filtered through 0.2 µm syringe filter and then concentrated for mass spectrometric analysis. The kinetic parameters for the enzymatic reaction were determined by initial rate method at 45oC by mixing 20 mM of H2O2 to a solution containing different concentrations of the 7 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

substrate fatty acid (varied between 100 - 500 µM), 20 µM CYP175A1 in 50 mM buffer (pH 7.5). The reaction was stopped by acetic acid (1%) after 1 min, and the unreacted fatty acid along with the products of the enzymatic reaction were extracted in CHCl3 and subjected to ESI-mass spectrometric identification. The concentrations of the reactants and products were determined from the ratio of peak currents, as well as by comparing with the ion currents from standard solutions of the reactants and the products. The initial rates of formation of monooxygenated fatty acid obtained from the mass spectrum were estimated at different concentrations of the substrate and analysed by Lineweaver-Burk method to evaluate the kinetic parameters Km and kcat of the reaction. Synthesis of Standard Samples of the Oxygenation Products Epoxidation of monoenoic fatty acids The epoxidation of the ethylenic double bond present in the monoenoic fatty acid was performed following a slight modification of the literature procedure.25 5 mg monoenoic fatty acid (16C9, 18C9, 18T9, 18C6, and 18C11, see Table 1) was reacted with 4 mg m-CPBA in 500 µL chloroform at room temperature overnight. The epoxy fatty acid derivative was solvent extracted with diethyl ether after addition of 1 ml 5% aqueous potassium bicarbonate solution to the reaction mixture. These epoxy derivatives of the monoenoic fatty acids were used as standards in the tandem mass spectrometry for identification of the enzymatic reaction products of these substrates. Allylic hydroxylation of monoenoic fatty acids Allyl-hydroxy fatty acids were synthesized following a slight modification of the literature procedure.26 100 µL 70% aqueous solution of tert-butyl hydroperoxide (TBHP) mixed with ~1.5

8 ACS Paragon Plus Environment

Page 8 of 45

Page 9 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

mg selenium dioxide in 200 µL dichloromethane (DCM) was stirred for 30 min This reaction mixture was added to 200 µL of DCM solution of the monoenoic fatty acid (~25 mM, 16C9, 18C9, 18T9, 18C6, and 18C11, see Table 1) and the solution was kept at shaker for 24 hr. The solution was subsequently mixed with 1 ml water and solvent extracted with DCM/brine solution. The unreacted fatty acids and the reaction products (allyl hydroxy- derivatives) were separated in the DCM phase and identified by ESI-MS, which showed formation of allylic mono-hydroxylation products from each of the monoenoic fatty acids (16C9, 18C9, 18T9, 18C6, and 18C11, see Table 1). There were two isomers of the allylic hydroxy- derivatives identified by tandem mass spectrometry, and these allyl hydroxy derivatives were used as standards for identification of the enzymatic reaction products of the monoenoic fatty acids in the present experiment. Esterification of the fatty acids and their derivatives for GC-MS analysis Esterification of the fatty acids was carried out following a slight modification of the literature procedure.27 ~1 mg fatty acid/epoxy fatty acid/hydroxy (allylic) fatty acid was dissolved in 150 µL toluene and 100 µL methanol followed by the addition of 2 M trimethylsilyl diazomethane (~5 µL in ether). The reaction mixture was kept in a shaker for 30 min at room temperature and subsequently concentrated to give the corresponding methyl ester of the fatty acid/epoxy fatty acid/hydroxyl fatty acid, that was used for GC-MS analysis. Trimethylsilylation of the hydroxyl functional group28, 29 for GC-MS analysis The ester of the allyl hydroxy product of the monoenoic fatty acid (as described above) was further converted to trimethylsilyl derivative of the hydroxy group for GC-MS analyses. The hydroxy fatty acid ester was reacted with 15 µL hexamethyldisilazane and 5 µL

9 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 45

trimethylchlorosilane in presence of 50 µL pyridine, and subsequently the trimethylsilyl ether derivative was extracted in cyclohexane and used for GC-MS analysis. UV-Visible Absorption Study The UV-Visible absorption spectra were recorded from 220-700 nm (as per needed) in quartz cuvette having path length of 1 cm on a double beam Perkin Elmer (Lambda 750-UV/Vis/NIR) spectrophotometer. The wavelength accuracy for the spectrophotometer was ± 0.15 nm and it was equipped with a Peltier cell temperature controller with an accuracy of ± 0.3-0.5 oC. Spectral titration of binding of the substrates: The dissociation constants for various fatty acids were determined by titrating the substrate free enzyme (~5 µM) with fatty acids at room temperature. A decrease in absorbance at Soret (417 nm) and increase in absorbance at 390 nm with the increasing concentration of fatty acids are indicative of binding of the substrate to CYP175A1 and of the conversion of heme iron from low-spin to high-spin state. Data were analysed by reported method 30 to obtain the dissociation constant (Kd) values from the intercept and the slope of the plot of 1/ΔA417 against 1/[Substrate] according to the following equation: 1/ΔA417 = (Kd/(PT × Δε417)) × (1/[Substrate]) + (1/(PT × Δε417)) where, ΔA417 is change in absorbance at 417nm. Kd (Slope/Intercept) = dissociation constant of enzyme-substrate complex. PT is the total concentration of the holo enzyme. Δε417 is difference in extinction coefficient of substrate-free and substrate-bound form at 417 nm. [Substrate] is total concentration of substrate. Electrospray Ionization Mass Spectrometry (ESI-MS) 10 ACS Paragon Plus Environment

Page 11 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ESI-MS and collision induced dissociation (ESI-MS/CID) studies were carried out using a Thermo Finnigan LCQ Deca Electrospray quadrupole ion trap mass spectrometer.31, 32 All the experiments were done under identical conditions unless otherwise stated. The flow rate of the analyte solution (in methanol) was maintained at 5 µL/min, and the solution was directly injected through the ion source kept in negative ion mode. Nitrogen was used as sheath and auxiliary gas. The ion source conditions were: sheath gas flow rate 25 (arbitrary unit), with no auxiliary gas flow. Capillary temperature (for desolvation) was maintained at approximately 200 oC and capillary voltage was kept at -15 V. The ion-spray voltage and tube lens offset were maintained at -5 kV and -30 V respectively. CID product ion spectra were acquired using an isolation width of 2 m/z under the normalized collision energy ranged from 40-50% with wide band activation turned on. The activation Q (related to the parameter q z in the Mathieu equation for the precursor ions)33 of 0.250 with an activation time 30 ms was used for CID-MS/MS (tandem mass spectrometry) studies. Helium was used as buffer as well as collision gas in the collision cell (ion trap). Data acquisition was performed for 1 min using XCalibur software (Thermo Fisher Scientific). Gas chromatography-mass spectrometry (GC-MS) The fatty acids and their oxidized products were derivatized (esterified and/or silylated) to make them amenable to GC-MS analysis. The GC-MS analysis was performed using a gas chromatograph (Trace GC ultra, Thermo Finnigan Electron Corporation) coupled with an ion trap mass spectrometer (Polaris Q, Thermo Finnigan).34 Agilent J & W GC capillary column (30 m length, 0.32 mm i.d., 0.50 µm film thickness) was utilized. Helium was used as a carrier gas (99.99% purity) maintained at a constant flow rate of 1.6 ml/min. The GC column temperature program was as follows: initial temperature 150°C for 2 min, from 150°C to 300°C at a rate of 11 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 45

10°C/min, finally kept at 300°C for 2 min. Analyte (in chloroform) injection (1 μl) was performed in a split less mode and the temperature at the injector was 280°C. The MS conditions were as follows: electron ionization (EI) mode at ionization energy of 70 eV, emission current of 250 μA, ion source and transfer line temperature at 230°C and 280°C, respectively. Standard samples were used to identify the retention times and the products were further verified by matching the fragmentation pattern with a database in the software provided with the instrument. Molecular docking calculations Docking of various fatty acids with CYP175A1 (PDB ID: 1N97) was performed using GOLD software (version 5.1).35, 36 GOLD is an automated ligand docking program used to calculate the possible conformations of the substrate that binds to the active site of the protein. It uses a genetic algorithm (GA) to check the ligand conformational flexibility and partial flexibility of the protein.37 It satisfies the fundamental requirement that the ligand must displace loosely bound water on binding.37 The genetic algorithm parameters used are: population size 100, number of islands 5, niche size 2, selection pressure 1.1, migration frequency 10, number of operations 100000, mutation frequency 95, and crossover frequency 95. The default speed selection was used to avoid a potential reduction in docking accuracy. One hundred genetic algorithm (GA) runs were performed without early termination with diverse solution generation. The substrate conformer with the highest binding score was taken into account for prediction of possible mode of binding of the substrate to the enzyme.

12 ACS Paragon Plus Environment

Page 13 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Results and Discussion Binding of monoenoic fatty acids with CYP175A1 The crystal structure analysis suggested that the active site (enzyme pocket) of CYP175A1 consists predominantly of hydrophobic residues analogous to that in case of cytochrome P450BM-3, which has high structural similarity with CYP175A1 . Since fatty acids are known to be the substrates of P450 BM-314, we screened the binding of several saturated and unsaturated (monoenoic) fatty acids in the enzyme pocket of CYP175A1 spectrophotometrically. However, among all the fatty acids tested only some unsaturated monoenoic fatty acids induced changes in the absorption spectra in the Soret region. Titration of CYP175A1 with different monoenoic fatty acids showed an increase in absorbance at 390 nm and decrease in absorbance at 417 nm (see Figure S2 in the Supporting Information). A typical isosbestic point at 405 nm was observed in the difference spectra of substrate-bound versus substrate free CYP175A1 (see Figure S2 in the Supporting Information). This result suggests a typical type I substrate binding.2 The calculated Kd values are reported in the Table 1. The observation of large dissociation constant (Kd) values (Table 1) suggests that the substrate may be weakly bound or binding of the substrate does not efficiently displace the axial water molecule from the heme center of the enzyme. Oxygenation of monoenoic fatty acids by CYP175A1 The catalytic activity of CYP175A1 was assayed using H2O2 as the source of oxidizing equivalents.2, 20, 38-44 The high-valent redox intermediate (cpdI) of the cytochrome P450 could be generated

by

the

peroxide

shunt

pathway

using

H2O2

or

analogues

(m-CPBA,

cumenehydroperoxide, peroxyacid etc.) to catalyze the oxygenation reaction of the substrate.2, 20

13 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 45

A reaction mixture containing CYP175A1, fatty acids and H2O2 (see experimental section for details) at 45oC showed the oxygenation of the monoenoic fatty acids. The product and the unreacted substrate extracted in organic phase were analyzed in ESIMS by direct injection in the negative ion mode (see Figure 1). In the negative ion mode of ESI, ionization of the fatty acid or oxygenated fatty acid occurs via deprotonation of the carboxylic acid functional groups.33, 45 The ionization efficiency45 of both substrate (fatty acid) and product (oxygenated fatty acid) were found to remains essentially the same. Control experiments with known concentrations of the fatty acids and their oxygenated derivatives showed almost the same peak current per M of the sample (e.g., a control mixture of 500 M of 16C9 and 500 M of its standard oxygenated derivative showed an average ion count of 4.92105 at m/z=253.4 for the fatty acid, and an average ion count of 4.3105 at m/z=269.4 for the oxygenated derivative, under identical instrument conditions).

Thus, the ratio of the concentration of the unreacted

substrate to the concentration of product in the enzymatic reaction could be calculated from the ratio of the peak current (ion signal) of the substrate (unreacted fatty acid) to that of the product (oxygenated fatty acid) recorded in ESI-MS (Figure 1). It is evident from Figure 1 that the monooxygenation has taken place (an increase by 16 Da in the molecular weight of the products relative to that of the corresponding substrates) in all the substrates under investigation (see Table 1). Moreover, no ion signals corresponding to addition of two or more oxygen atoms to the fatty acid were detected in the present studies, indicating that only single monooxygenation of the fatty acids is catalyzed by CYP175A1. The percentage yields for monooxygenation of each fatty acid are shown in the Figure 1. Figure 1 shows that the catalytic activity of CYP175A1 is maximum for 16C9 (64%) & 18C6 (60%), and minimum for 20C11 (12%), 22C13 (12%) and 24C15 (7%). No significant product formation

14 ACS Paragon Plus Environment

Page 15 of 45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

was observed in the control experiments containing the fatty acid with H2O2 but without CYP175A1, or containing the fatty acid with the enzyme but without H2O2 in the reaction mixtures (see Figure S3 in the Supporting Information for a typical substrate 16C9). Thus the oxygenation of the above fatty acids is essentially carried out by the enzymatic action of CYP175A1. These results thus indicate that the substrate binding cavity of the enzyme might be such that it can accommodate the monoenoic fatty acid 16C9 more favorably compared to the larger fatty acids. We also studied the fatty acid oxygenation in the reductive pathway using putidaredoxin (PdX)-putidaredoxin reductase (PdR) as the redox partners for the class-I type of CYP enzyme system and NADH as the electron donor. PdX-PdR redox partners were earlier13 shown to drive the reductive pathway for hydroxylation of β-carotene by CYP175A1. The reductive pathway also resulted in the oxygenation of the monoenoic fatty acids. However, the reaction efficiency (e.g., 21% for 16C9) was lower compared to that in the peroxide shunt pathway (64% for 16C9), indicating poor redox coupling of PdX-PdR system with CYP175A1 (see Figure S4 in the Supporting Information). The effects of the experimental conditions on the enzymatic efficiency were assessed by varying the time course, temperature and H2O2 concentration in the reaction mixture without changing the initial concentrations of the enzyme and of the substrate. The percentage yield of the product increased with incubation time and reached a plateau at ~30 min (Figure 2a). Variation of the reaction temperature from 30 oC to 70oC also showed distinct temperature dependence of the percentage yield of the product formation and the maximal activity temperature for the monoenoic fatty acid oxygenation was found to be 45 oC (Figure 2b).

15 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 45

Figure 3 shows that with the increase in the concentration of H2O2 from 1 mM to 20 mM in the reaction mixture (containing 16C9), the amount of product (after 1h reaction) increases and reaches a maximum at 20 mM H2O2 concentration. When the peroxide concentration was 200 µM and the reaction time was 24 h, the total yield of the oxygenated product for 16C9 was found to be 30% (data not shown). This yield is almost half of that obtained in the presence of 100 times more concentration of H2O2 (20 mM H2O2 ) in 1 h reaction time (64% yield for 16C9). It is however important to note that presence of H2O2 causes degradation of the enzyme, which becomes predominant at higher concentrations of the peroxide. Increase of H2O2 concentration above 20 mM causes decrease in the amount of the product formation (Figure 3) possibly due to the H2O2-dependent bleaching of the heme chromophore and degradation of the enzyme.38, 40 We also assessed the heme degradation of CYP175A1 in presence of the substrate (16C9) at different concentrations of H2O2 by monitoring the Soret absorption at 417 nm with time (see Figure S5 in the Supporting Information). The results indicated that even with 20 mM H2O2, there were ~38% degradation of the enzyme in 1 hr, though the overall yield of the enzymatic product at this concentration of peroxide were the highest (64%, Figure 1). Moreover, the degradation of the enzyme by peroxide was not linear with time, and there was only