Novel insights into oxidation of fatty acids and fatty alcohols by cytochrome P450 monooxygenase CYP4B1

Article abstract of DOI:10.1016/j.abb.2019.108216

CYP4B1 is an enigmatic mammalian cytochrome P450 monooxygenase acting at the interface between xenobiotic and endobiotic metabolism. A prominent CYP4B1 substrate is the furan pro-toxin 4-ipomeanol (IPO). Our recent investigation on metabolism of IPO related compounds that maintain the furan functionality of IPO while replacing its alcohol group with alkyl chains of varying structure and length revealed that, in addition to cytotoxic reactive metabolite formation (resulting from furan activation) non-cytotoxic ω-hydroxylation at the alkyl chain can also occur. We hypothesized that substrate reorientations may happen in the active site of CYP4B1. These findings prompted us to re-investigate oxidation of unsaturated fatty acids and fatty alcohols with C9–C16 carbon chain length by CYP4B1. Strikingly, we found that besides the previously reported ω- and ω-1-hydroxylations, CYP4B1 is also capable of α-, β-, γ-, and δ-fatty acid hydroxylation. In contrast, fatty alcohols of the same chain length are exclusively hydroxylated at ω, ω-1, and ω-2 positions. Docking results for the corresponding CYP4B1-substrate complexes revealed that fatty acids can adopt U-shaped bonding conformations, such that carbon atoms in both arms may approach the heme-iron. Quantum chemical estimates of activation energies of the hydrogen radical abstraction by the reactive compound 1 as well as electron densities of the substrate orbitals led to the conclusion that fatty acid and fatty alcohol oxidations by CYP4B1 are kinetically controlled reactions.

Full text of DOI:10.1016/j.abb.2019.108216

Journal Pre-proof
Novel insights into oxidation of fatty acids and fatty alcohols by cytochrome P450
monooxygenase CYP4B1
Florian A. Thesseling, Michael C. Hutter, Constanze Wiek, John P. Kowalski, Allan E.
Rettie, Marco Girhard
PII:
S0003-9861(19)30840-9
https://doi.org/10.1016/j.abb.2019.108216
YABBI 108216
DOI:
Reference:
To appear in: Archives of Biochemistry and Biophysics
Received Date: 23 October 2019
Revised Date: 26 November 2019
Accepted Date: 29 November 2019
Please cite this article as: F.A. Thesseling, M.C. Hutter, C. Wiek, J.P. Kowalski, A.E. Rettie,
M. Girhard, Novel insights into oxidation of fatty acids and fatty alcohols by cytochrome P450
monooxygenase CYP4B1, Archives of Biochemistry and Biophysics (2020), doi: https://doi.org/10.1016/
j.abb.2019.108216.
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Author statement
Florian A. Thesseling: Investigation, Data curation, Visualization, Writing – original draft.
Michael C. Hutter: Methodology, Software, Formal analysis. Constanze Wiek:
Conceptualization, Funding acquisition. John P. Kowalski & Allan E. Rettie: Writing –
review & editing, Resources, Funding acquisition. Marco Girhard: Conceptualization;
Funding acquisition; Data curation; Validation; Visualization; Supervision; Writing – review &
editing.

1
2
Novel insights into oxidation of fatty acids and fatty alcohols by
cytochrome P450 monooxygenase CYP4B1
3
4
Florian A. Thesselinga,1, Michael C. Hutterb, Constanze Wiekc, John P. Kowalskid,
Allan E. Rettied, Marco Girharda,*
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a Institute of Biochemistry, Heinrich-Heine University, 40225 Düsseldorf, Germany
florian.thesseling@hhu.de, marco.girhard@hhu.de
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9
b Center for Bioinformatics, Saarland University, 66123 Saarbrücken, Germany
10
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14
15
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18
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michael.hutter@bioinformatik.uni-saarland.de
c
Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich-Heine University,
40225 Düsseldorf, Germany
constanze.wiek@med.uni-duesseldorf.de
d
Department of Medicinal Chemistry, School of Pharmacy, University of Washington,
Seattle, WA 98195, United States of America
kowalj@uw.edu, rettie@uw.edu
* Corresponding author
Marco Girhard
Phone: +49 211 81-13866
Email: marco.girhard@hhu.de
25 Highlights
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28
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30
• CYP4B1 fatty acid hydroxylation patterns are more diverse than previously reported
• The presence of cytochrome b5 reduces uncoupling and increases CYP4B1 activity
• Computational docking allows interpretation of the observed hydroxylation patterns
•
Activation energy calculations imply that the reactions are kinetically controlled
1 Present address: VIB Center for Microbiology, Laboratory for Systems Biology, KU Leuven, 3001
Heverlee-Leuven, Belgium
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31 Abstract
32 CYP4B1 is an enigmatic mammalian cytochrome P450 monooxygenase acting at the
33 interface between xenobiotic and endobiotic metabolism. A prominent CYP4B1
34 substrate is the furan pro-toxin 4-ipomeanol (IPO). Our recent investigation on
35 metabolism of IPO related compounds that maintain the furan functionality of IPO
36 while replacing its alcohol group with alkyl chains of varying structure and length
37 revealed that, in addition to cytotoxic reactive metabolite formation (resulting from
38 furan activation) non-cytotoxic ω-hydroxylation at the alkyl chain can also occur. We
39 hypothesized that substrate reorientations may happen in the active site of CYP4B1.
40 These findings prompted us to re-investigate oxidation of unsaturated fatty acids and
41 fatty alcohols with C9-C16 carbon chain length by CYP4B1. Strikingly, we found that
42 besides the previously reported ω- and ω-1-hydroxylations, CYP4B1 is also capable
43 of α-, β-, γ-, and δ-fatty acid hydroxylation. In contrast, fatty alcohols of the same
44 chain length are exclusively hydroxylated at ω, ω-1, and ω-2 positions. Docking
45 results for the corresponding CYP4B1-substrate complexes revealed that fatty acids
46 can adopt U-shaped bonding conformations, such that carbon atoms in both arms
47 may approach the heme-iron. Quantum chemical estimates of activation energies of
48 the hydrogen radical abstraction by the reactive compound 1 as well as electron
49 densities of the substrate orbitals led to the conclusion that fatty acid and fatty
50 alcohol oxidations by CYP4B1 are kinetically controlled reactions.
51
52
53 Keywords
54 CYP4B1; cytochrome P450 monooxygenase; fatty acid; fatty alcohol; biocatalysis
55
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56 Introduction
57 Cytochrome P450 monooxygenases (P450 or CYP) are heme-thiolate containing
58 enzymes present in all domains of life encompassing ~ 350,000 sequences in
59 databases [1]. In the human genome, 57 different P450 genes encode related CYPs
60 that can be clustered in 18 families and 45 subfamilies based on homology in
61 sequence. CYP4B1 belongs to the cytochrome P450 family 4 that also includes the
62 CYP4A, 4F, 4V, 4X, and 4Z subfamilies [2]. Typically, the majority of enzymes of the
63 CYP4 family are involved in fatty acid and eicosanoid oxidation [3]. The predominant
64 reaction is ω-hydroxylation, which serves to remove excess free fatty acids to
65 prevent lipotoxicity, catabolize leukotrienes and prostanoids, and produces the
66 bioactive metabolite, 20-HETE, from arachidonic acid [4].
67 CYP4B1 is unique, because it appears to act at the interface between xenobiotic and
68 endobiotic metabolism [5-7]. In mammals, CYP4B1 is capable of ω- and ω-1-
69 hydroxylation of unsaturated fatty acids, but in contrast to other CYP4 family
70 members, this enzyme is also responsible for the metabolism of a range of
71 chemically unrelated pro-toxic xenobiotics, including 4-ipomeanol (IPO) 13 [8] and
72 perilla ketone (PK) 14 [9] (Figure 1). Strikingly, in humans this situation is very
73 different, because no catalytic activity has been reported so far for native human
74 CYP4B1. This intriguing observation can be explained by the fact that in the human
75 enzyme an otherwise evolutionarily highly conserved proline residue of the meander
76 region that should be located at position 427 of the so-called “ERR triad” and that is
77 important for heme binding, has been replaced by a serine amino acid rendering it
78 apparently inactive [10]. It has previously been reported that the exchange of serine
79 427 to proline can partially restore the catalytic activity of human CYP4B1 supporting
80 fatty acid hydroxylation (compounds 1, 2, 4; preferentially at the ω-position) [10], as
81 well as bioactivation of IPO 13 and PK 14 [11]. Moreover, by systematic screening
82 against the native rabbit CYP4B1, Wiek et al. [12] identified and introduced an
83 additional twelve amino acids into the human S427P variant resulting in an optimized
84 variant that was as stable and active in metabolizing IPO 13 as the native rabbit
85 CYP4B1.
86 As mentioned above, CYP4 enzymes preferentially oxygenate primary (energetically
87 less favored) C-H bonds over adjacent (energetically favored) secondary C-H bonds;
88 e.g. the rabbit CYP4B1 hydroxylates lauric acid (C12) 4 at ω:ω-1 positions at a ratio
89 of ~ 3:2 [13]. Recently the structure of native rabbit CYP4B1 in complex with octane
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90 (pdb 5T6Q) [14] was solved helping to identify structural features that explain this
91 preference. In short, the overall topographical features of CYP4B1 mirror those of
92 other P450s, but the crystal structure revealed that octane approaches the heme iron
93 'end on' meaning that the linear hydrocarbon is constrained to an extended
94 conformation within a slot-like active site cavity. This cavity narrows nearer the heme
95 so that the ω-1-hydrogens are more shielded from the active oxygen species than
96 the ω-carbons, thus promoting the observed regioselectivity. An unusual feature that
97 has evolved to contribute to this regioselectivity is an ester bond between the heme
98 5-methyl and a glutamic acid residue (E310 in rabbit CYP4B1 [15, 16], present also
99 in some members of the CYP4 family [17]). The preference for octane ω-
100 hydroxylation is retained in a mutated E310A variant albeit with a 4-fold loss in ω:ω-1
101 regioselectivity while maintaining a similar overall rate of metabolite formation [14]. In
102 contrast, C12 4 is metabolized by E310A at ~ 60% of the rate of the native enzyme
103 displaying a clear change in regioselectivity to predominantly ω-1 hydroxylation [16].
104 The recently solved structure of the CYP4B1 E310A variant in complex with octane
105 (pdb 6C93) disclosed that noncovalent interactions maintain heme deformation in the
106 absence of the ester linkage and that reduced rigidity probably underlies increased
107 E310A catalyzed ω-1-hydroxylation [18].
108 In a previous study reporting on metabolism of PK 14 by CYP4B1, we noticed that
109 besides cytotoxic reactive metabolite formation (resulting from furan activation) three
110 non-cytotoxic hydroxylation products were also formed via hydroxylations on the alkyl
111 chain [11]. In addition, in a very recent study, we examined a series of synthetically
112 derived IPO 13 related N-alkyl-3-furancarboxamides maintaining the furan
113 functionality while replacing the alcohol group with alkyl chains of varying structure
114 [Kowalski et al., manuscript submitted]. In this case the amount of alkyl hydroxylation
115 increased with increasing chain length, bolstering the hypothesis that substrate
116 reorientations can occur in the active site of CYP4B1.
117 These findings prompted us in the present study to re-investigate oxidation of
118 unsaturated fatty acids 1–8 and fatty alcohols 9–12 by rabbit and human S427P
119 CYP4B1. Strikingly, we found that besides previously reported ω- and ω-1-
120 hydroxylation, CYP4B1 is also capable of α-, β-, γ-, and δ-fatty acid hydroxylation of
121 mid chain length fatty acids. In contrast, fatty alcohols of the same chain length are
122 exclusively hydroxylated at ω, ω-1, and ω-2 positions. Docking studies of the
123 corresponding CYP4B1-substrate complexes and quantum chemical calculations on
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124 the substrates lead to the conclusion that fatty acid and fatty alcohol oxidations by
125 CYP4B1 are kinetically controlled reactions.
126
127
128 Materials and Methods
129 Chemicals and enzymes
130 The compounds investigated in this study are summarized in Figure 1. Unsaturated
131 fatty acids of C9 to C16 carbon chain length 1–8 and the fatty alcohols decanol (C10-
132 ol) 9, dodecanol (C12-ol) 10, tetradecanol (C14-ol) 11, and hexadecanol (C16-ol) 12
133 were purchased from Sigma-Aldrich (Germany).
134 Catalase from bovine liver and recombinant bovine superoxide dismutase were
135 obtained from Sigma-Aldrich.
136 Glucose dehydrogenase (GDH) from Bacillus megaterium (gdhIV; GenBank D10626)
137 was expressed via pET-22b(+) in E. coli BL21(DE3) as described previously [19].
138 Rat cytochrome P450 reductase (CPR) and human cytochrome b5 were expressed
139 and purified as described previously [20].
140
141 CYP4B1 plasmid construction, enzyme expression and purification
142 Construction of the plasmids r-P422∆1_pET (expressing the rabbit cyp4b1 cDNA,
143 GenBank NM_001082103 isoform 1, now termed r-∆1) and h-P427∆3 (expressing
144 the human cyp4b1 cDNA, GenBank NM_000779 transcript variant 2) mutant S427P
145 (now termed h-∆3P) has been reported previously [11]. The N- and C-terminal amino
146 acid modifications introduced for expression in E. coli are summarized in the
147 Supporting Information (Supplementary Table S1).
148 r-∆1 was expressed from pET-22b(+) (Novagen, Germany), while pCWOri [21] was
149 used to express h-∆3P. Expression conditions in E. coli OverExpress C43(DE3) ([F-
-
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150 ompT hsdSB (rB mB ) gal dcm (DE3)], Lucigen Corporation, USA) and purification by
151 affinity chromatography utilizing a C-terminal His6-tag have also been reported
152 previously [11].
153 CYP4B1 concentrations of non-purified enzymes were determined by the CO-
154 difference spectral assay with ε450-490 = 91 mM-1 cm-1 [22], while CO-difference
155 spectra of purified CYP4B1 preparations were measured according to an optimized
156 method by Zheng et al. [16].
157
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158 Influence of cytochrome b5 on lauric acid conversion by CYP4B1
159 Coupling efficiency and NADPH consumption
160 The coupling efficiency and NADPH consumption in the absence and presence of
161 cytochrome b5 were determined as follows: Reactions were carried out in 50 mM
162 potassium phosphate buffer, pH 7.5 in a total reaction volume of 100 µL. The
163 reaction mixtures contained 0.25 µM CYP4B1, 0.5 µM CPR, 100 U mL-1 superoxide
164 dismutase, 1,000 U mL-1 catalase, 25 µg mL-1 1,2-didodecanoyl-sn-glycero-3-
165 phosphocholine (DLPC). Optionally 0.25 µM cytochrome b5 were added. C12 4 (from
166 a 10 mM stock solution dissolved in DMSO) and NADPH were added in final
167 concentrations of 200 µM and the reaction mixtures were incubated at 30°C. The
168 consumption of NADPH was followed by measuring the absorbance decrease at
169 340 nm. NADPH oxidation rates were calculated with ε340 = 6.22 mM-1 cm-1.
170 After complete consumption of NADPH, the samples were prepared for GC/MS
171 analysis as described below. Since equimolar amounts of substrate and NADPH
172 were applied, the coupling efficiency directly corresponds to the amount of C12 4
173 converted by CYP4B1.
174
175
Influence of cytochrome b5 and cofactor regeneration on lauric acid conversion
176 Reaction mixtures were prepared as described above. Optionally, 25 U mL-1 GDH
177 and 20 mM glucose were added for cofactor recycling. Reactions with GDH
178 contained 200 µM NADPH, whereas 1 mM NADPH was added if GDH was not
179 present to ensure sufficient amounts of cofactor. Reaction mixtures were incubated
180 at 30°C for 30 or 120 min. Thereafter, samples were prepared for GC/MS analysis as
181 described below.
182
183 Fatty acid and fatty alcohol conversion by CYP4B1
184 Conversions of fatty acids 1–8 and fatty alcohols 9–12 were carried out in 50 mM
185 potassium phosphate buffer, pH 7.5 and a total reaction volume of 100 µL. Reaction
186 mixtures contained 0.25 µM CYP4B1, 0.5 µM CPR, 0.25 µM cytochrome b5,
187 100 U mL-1 superoxide dismutase, 1,000 U mL-1 catalase, 25 U mL-1 GDH, 20 mM
188 glucose, 25 µg mL-1 DLPC, 200 µM substrate (from a 10 mM stock solution dissolved
189 in DMSO) and 200 µM NADPH. Samples were incubated at 30°C for 90 min; this
190 reaction time was chosen as an almost complete substrate conversion (as achieved
191 for C12 4 after 120 min) was not desirable, so as to allow comparison of the
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192 conversion values for the individual substrates and also between the two CYP4B1
193 isoforms.
194
195 Sample preparation and gas chromatography / mass spectrometry (GC/MS)
196 analysis
197 After the desired reaction time, 100 µM of an internal standard (Supplementary
198 Table S2) and 2 µL of 37% HCl were added to each sample. Samples were extracted
199 with 500 µL diethyl ether, the ether was dried with water free magnesium sulfate and
200 evaporated completely. The residues were dissolved in 30 µL N,O-
201 bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane and
202 incubated at 80°C for 30 min.
203 0.5 µL of a sample was injected to a GC/MS instrument (GC/MS-QP2010 plus,
204 Shimadzu, Germany) equipped with an FS-Supreme-5 column (30 m x 0.25 mm x
205 0.25 µm, Chromatographie Service GmbH, Germany). Helium was used as carrier
206 gas with a linear velocity of 30 cm min-1. The column oven temperature profiles for
207 compounds 1–12 are provided in the Supplementary Table S2.
208 Identification of products from fatty acid 1–8 conversions was done as described
209 previously (Supplementary Figure S1) [19]. Hydroxylation positions of fatty alcohols
210 9–12 conversion products were identified by their characteristic mass fragmentation
211 patterns (Supplementary Figure S2).
212
213 Computational methods
214 The crystal structure of rabbit CYP4B1 (pdb 5T6Q) [14] was used for computational
215 analysis of r-∆1 (98.6% protein sequence identity; see Supplementary Table S1), and
216 served as template to generate a homology model of the h-∆3P variant (86.4%
217 protein sequence identity; see Supplementary Material for pdb-file). The homology
218 model of h-∆3P was built using SWISS-MODEL [23, 24], whereas for r-∆1 the
219 coordinates of the corresponding crystal structure were used. Further preparation for
220 docking was carried out by AutoDock Tools (Windows version 1.5.6r3) [25],
221 assigning AMBER charges to the amino acids of the protein, whereas Gasteiger-
222 Marsili charges were computed for the heme cofactor. The partial charge on the iron
223 atom was set to 0.400e and ‒0.348e to each of its ligating nitrogen atoms, as in
224 earlier docking studies [19]. Protonation states of the histidine residues were
225 assigned manually by visual inspection in order to optimize their local hydrogen-
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226 bonding network. The rectangular grid box (80 x 48 x 56 points) was dimensioned
227 large enough to comprise a linear alkane chain of length C16 in the binding pocket
228 above the heme cofactor. The grid spacing was kept at the default value of 0.375 Å.
229 Ligand molecules were prepared with HYPERCHEM on the basis of the octane
230 molecule that was present is the crystal structure and energetically optimized using
231 the MM+ force field parameters as implemented in HYPERCHEM version 6.02
232 (Hypercube Inc., Gainsville, FL, 1999). Atom types and Gasteiger-Marsili charges for
233 use in AutoDock version 4.2 [26, 27] were computed with in-house PERL scripts.
234 During docking all rotatable bonds of the ligands were treated as flexible, whereas
235 the protein part was kept rigid. A total of 250 runs of the Lamarckian Genetic
236 Algorithm for each ligand were carried out, otherwise default parameters of AutoDock
237 were applied.
238 Activation energies for the P450-mediated hydrogen radical abstraction were
239 computed according to the approach of Mayeno et al. [28] at AM1 level of theory,
240 which was also used for obtaining the coefficients of the substrate orbitals.
241
242
243 Results
244 Influence of cytochrome b5 on CYP4B1 activity
245 Cytochrome b5 is a microsomal heme protein that has an electron transfer role in
246 fatty acid desaturation and some other enzymatic reactions [29, 30]. The effects of
247 cytochrome b5 on P450 catalysis are diverse; in a number of studies either no effect,
248 stimulation, or inhibition with the addition of cytochrome b5 have been reported, often
249 in a P450- and substrate-dependent manner that is not well understood (reviewed in
250 [31]).
251 In order to investigate the influence of cytochrome b5 on CYP4B1 activity, reactions
252 containing C12 4 and r-∆1 with and without cytochrome b5 were performed. C12 4
253 was chosen, because this compound has traditionally been used as a model
254 substrate to probe for ω-hydroxylase activity toward physiologically relevant
255 substrates [5]. In addition, reactions with and without NADPH regeneration by GDH
256 were investigated (Figure 2A). The reaction containing just r-∆1 and CPR, but no
257 cytochrome b5 or GDH, reached 24% substrate conversion after 30 min and almost
258 twice as much (47%) after 120 min. The addition of cytochrome b5 to the reaction led
259 to an increase of C12 4 conversion to 45% and 84% after 30 and 120 min,
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260 respectively. A similar effect was observed after the addition of GDH without
261 cytochrome b5; here conversions of 59% and 95% were reached after 30 and
262 120 min, respectively. By combination of cytochrome b5 and GDH conversion further
263 increased to 69 % after 30 min and 97 % after 120 min.
264 To further investigate the effect of cytochrome b5 on C12 4 conversion, the NADPH
265 coupling efficiencies and conversion rates were determined under the chosen
266 reaction conditions. In general, the NADPH coupling efficiency of a P450 enzyme is
267 dependent on the substrate: If substrate binding is weak the enzyme-substrate-
268 complex can fall apart and intramolecular uncoupling reactions within the catalytic
269 cycle of the P450 can occur. In addition, the coupling efficiency depends on the
270 interaction between P450 and CPR, inefficient interaction can lead to intermolecular
271 uncoupling during electron transport between both enzymes. In both cases NADPH
272 is consumed, but instead of oxidizing the substrate, reactive oxygen species like
273 H2O2 are formed [32, 33].
274 The efficiency of NADPH consumption and substrate conversion can be tested by
275 adding both compounds in equimolar concentrations to the reaction. After complete
276 NADPH oxidation the percentage of converted substrate is equal to the coupling for
277 the P450 with this specific substrate under the given reaction conditions. In the case
278 of C12 4, addition of cytochrome b5 boosted the coupling efficiency from 26% to 54%
279 (Figure 2B). In contrast, monitoring of the NADPH consumption revealed that the
280 addition of cytochrome b5 led to a reduction from 8.4 pmol s-1 to 6.6 pmol s-1
281 (Figure 2C).
282 While analyzing the product distributions of the C12 4 conversions by r-∆1 in the
283 absence/presence of cytochrome b5 and/or GDH, we noticed to our surprise that in
284 addition to previously reported ω- and ω-1-hydroxylation metabolites [13], additional
285 NADPH-dependent chromatography peaks occurred in comparison to the no-P450
286 negative control (Figure 3A). These new products could clearly be attributed to
287 hydroxylations at the ω-10 (α), ω-9 (β), ω-8 (γ), and ω-7 (δ) carbon atoms of C12 4
288 (Supplementary Figure S1). In-depth analysis of the GC/MS data revealed that these
289 metabolites were present in all reactions at every time points and that their relative
290 distribution among total reaction products remained almost unaltered over the
291 reaction time of 120 min (Supplementary Table S3). Moreover, it was noted that the
292 addition of cytochrome b5 and GDH while enhancing reaction rates, had only
293 marginal effects on the product distributions (Supplementary Table S3).
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294
295
296
297
298 Conversion of fatty acids by CYP4B1
299 Since, to the best of our knowledge, hydroxylations at positions other than ω/ω-1 had
300 not been reported for any CYP4B1 isoform so far, this prompted us to evaluate
301 conversion of fatty acids 1–8 with C9 to C16 carbon chain lengths by both, r-∆1 and
302 h-∆3P, in reaction systems containing cytochrome b5 and GDH.
303 Indeed, hydroxylations at positions other than ω and ω-1 were found for compounds
304 C10 - C14 2–6 and for both r-∆1 and h-∆3P (Table 1). The conversion of C12 4
305 resulted in the most diverse product pattern amongst the tested substrates; seven
306 hydroxylation products could be detected via GC/MS. Most abundant — not only for
307 C12 4 but for most fatty acids 1–7 — was the ω-hydroxylation product. Unexpectedly,
308 r-∆1 generated large amounts (9 - 35%) of previously unknown ω-6 to ω-10 hydroxy
309 metabolites from compounds C10 - C13 2–5. Interestingly, no ω-3 (except of trace
310 amounts in case of C14 6), nor ω-4, nor ω-5 hydroxylations were observed for any
311 compound. In addition, hydroxylation did not occur at positions ≥ ω-11.
312 Notably, as a general trend, it was observed that independent of the carbon chain
313 length and CYP4B1 isoform, the positions α and γ were preferred over β and δ
314 positions, e.g. the α:β:γ:δ-distribution for C11 3 is 12:1:35:1 in the case of r-∆1 and
315 4:0:5:0 from h-∆3P.
316 Additionally, small amounts of two C12 4 hydroxy fatty acid derivatives could be
317 identified by their characteristic m/z ratios during MS analysis, namely γ-
318 dodecalactone 15 and δ-dodecalactone 16; these products result from non-
319 enzymatic intramolecular esterification of γ- and δ-hydroxy C12 (Supplementary
320 Figure S1). Noteworthy, γ- and δ-lactones have a variety of commercial uses as
321 flavorings and fragrances, e.g. δ-decalactone is a common fragrance compound with
322 a creamy peach note [34].
323 Detailed information about fatty acid conversion is given in Figure 4A, substrate
324 oxidation rates (SOXR) and product patterns observed for both CYP4B1 isoforms are
325 summarized in Table 1. Briefly, a comparison of the conversions by r-∆1 and h-∆3P
326 reveals an increased acceptance of medium- over shorter- and longer-chained fatty
327 acids. For r-∆1 an increase in substrate conversion from C9 1 (14%) to C12 4 (79%)
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328 was observed, whereas conversions for C13 5 and C14 6 dropped to 13% and 11%,
329 respectively. Notably, although not tremendously pronounced, the acceptance of h-
330 ∆3P of fatty acids with chain length higher than twelve carbon atoms seems to be
331 slightly better than r-∆1. Here, SOXR for C13 5 and C14 6 of 1.7 and
-1
332 1.2 nmol min-1 nmolP450 were reached, respectively, as compared to 1.2 and 1.0 by
333 r-∆1. Furthermore, fatty acids with chain length ≤ 12 carbon atoms were generally
334 less well accepted as substrates by h-∆3P compared to r-∆1. Conversion of C15 7
335 was low, whereas C16 8 was not converted by either enzyme.
336
337 Conversion of fatty alcohols by CYP4B1
338 Regarding the product distribution, in contrast to fatty acids of the same chain length
339 GC/MS analysis revealed only two major (namely ω and ω-1) and one minor (namely
340 ω-2) hydroxylation products in case of all tested fatty alcohols. By way of example,
341 the gas chromatogram of C12-ol 10 (Figure 3B), as well as m/z fragmentation
342 patterns of detected products are shown (Supplementary Figure S2). Detailed
343 information on SOXR and product distributions are summarized in Table 1.
344 Analogous to the fatty acids, the highest conversion was observed for C12-ol 10 in
345 the case of r-∆1 (62%), while h-∆3P converted C12-ol 10 and C10-ol 9 to nearly the
346 same extent (~40%) (Figure 4B).
347 Interestingly, the SOXR of h-∆3P for C10-ol 9 is 7-fold higher than that of C10 (3.5
348 vs. 0.5), whereas the SOXR of r-∆1 for C10-ol 9 (as well as C12-ol 10) are lower than
349 those of C10 2 and C12 4. (Table 1). Both, r-∆1 and h-∆3P were not capable to turn
350 over C16-ol 12.
351 As a general trend it can be stated that independent of the carbon chain length, the
352 ω-1 and ω-2 positions are more preferred for hydroxylation by r-∆1, whereas h-∆3P
353 is more selective for ω-hydroxylation.
354
355 Computational analyses of fatty acid and fatty alcohol conversions
356 As described in the Material and methods section, the crystal structure of the rabbit
357 CYP4B1 (pdb 5T6Q) [14] was used for computational analyses of r-∆1, and served
358 as template to generate a homology model of the h-∆3P variant. The most
359 pronounced difference between the crystal structure and the homology model is that
360 the unresolved residues, aspartic acid 196 to asparagine 200 and isoleucine 272 to
361 arginine 276 in the crystal structure, were reconstructed. Due to the high sequence
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362 similarity between rabbit and human enzyme, no notable deviations of the backbone
363 atoms were observed. Likewise, all side chain rotamers in the binding site were
364 conserved. Since the crystal structure shows a bound n-octanol molecule in the
365 active site, we expect no substantial change upon docking of similar ligands, e.g.
366 fatty alcohols and fatty acids that also possess linear carbon chains. The reported
367 QMEAN4 score [23] of SWISS-Model was -1.27, which is very similar to that of the
368 original crystal structure (−1.17) with a resolution of 2.7 Å. The MolProbity score [35]
369 after adding hydrogens, but without possible flips of corresponding side chains, was
370 1.60, residing in the 93rd percentile of structures with similar resolution.
371 Docking studies suggested that fatty acids can bind in two different bonding
372 conformations, such that carbon atoms in both arms may approach the heme-Fe
373 (Figures 5A & 5B). With increasing carbon chain length, the hydrophobic
374 hydrocarbon chain is increasingly twisted and bent inside the binding pocket.
375 Likewise, the loss of entropy upon binding longer chains also increases as more
376 rotatable bonds are frozen. Eventually, the binding pocket cannot accommodate
377 chain lengths that exceed C15 7 anymore. As a consequence of the chain bending
378 near the heme the hydroxylation pattern is shifted to the ω/ω-1 positions (Figure 5A).
379 Hydrogen-bond formation with the COOH-group of threonine 314, which is very close
380 to the heme and therefore strongly conserved, was observed for part of the fatty
381 acids (Figure 5B).
382 To reveal further factors that influence the hydroxylation of fatty acids, the electron
383 densities of the Highest Occupied Molecular Orbitals (HOMOs) of fatty acids C9 –
384
C14 1-6 and the activation energies for the associated hydrogen abstraction at
385 various carbon atoms of C12 4 were computed. The coefficients of the HOMOs
386 indicate that sufficient electron density is present far into the chains of fatty acid
387 molecules meaning that, in principle, all these positions would be available for
388 hydroxylation (Figure 6A). In contrast, whereas there is a high barrier for hydrogen
389 abstraction from the ω as well as ω-10 (α) carbon atom, the least energy is required
390 at position ω-1, which then slowly increases towards the carboxy group (Figure 6B).
391 Since all of the energies are within 3 kcal mol-1 no thermodynamic preference of a
392 particular position is apparent. Instead docking reveals that a U-shaped folding of the
393 carbon chain allows the front positions (α, β, γ, δ) to get close to the heme-Fe
394 (Figure 5A), suggesting a kinetic control of the hydroxylation.
- 12 -

395 The fatty alcohols appear to be less reactive, although alternative binding
396 conformations were also found. According to analysis of the orbitals of the fatty
397 alcohols, the following picture emerges: The presence of substantial electron density
398 throughout the chain is even more pronounced in comparison to the fatty acids, so
399 again all these positions could come into play for hydroxylation (Figure 6C).
400 Moreover, hydrogen abstraction of C12-ol 10 (Figure 6D) was computed to require
401 less activation energy for most of the positions (especially α and β) in comparison to
402 C12 4. However, docking reveals that in contrast to the corresponding fatty acids for
403 which two major binding modes were found, the fatty alcohols preferably bind with
404 the end of the chain facing the heme-Fe, thereby forming a hydrogen-bond to either
405 the side chain of glutamine 218 (Figure 5C) or to the carbonyl oyxgen atom of
406 glutamine 377. Consequently, since only ω to ω-2 positions are actually
407 hydroxylated, this indicates that these positions are the closest to the heme-Fe, again
408 suggesting kinetic control.
409
410 Computational analysis of 4-ipomeanol and perilla ketone conversions
411 We also performed docking for the two previously studied pro-toxins IPO 13 and PK
412 14. For IPO 13 the energetically most favorable docking position in both enzymes is
413 the one with the furan functionality in close proximity to the heme-Fe leading to furan
414 activation via epoxidation (Supplementary Figure S3A). In contrast, for PK 14, two
415 different conformations in r-∆1 as well as in h-∆3P were found for which the predicted
416 docking scores are practically equal in energy: The first leads to furan activation by
417 epoxidation (Supplementary Figure S3B), while the second allows hydroxylations at
418 the carbon atoms of the isopropyl group (Supplementary Figure S3C). The respective
419 pdb-files of the docked ligands in the corresponding reference frame are available as
420 Supplementary Material. In summary, these in silico data correspond very well with
421 the experimentally obtained results of our previous study (Supplementary Figure S4)
422 [11].
423
424
425 Discussion
426 Fatty acids are endogenous substrates of many P450s [36, 37]). Fatty acid
427 hydroxylation is of particular interest from a mechanistic point of view, since
428 members falling within certain P450 subfamilies exhibit preference for
- 13 -

429 thermodynamically disfavored oxygen insertion at positions remote from activating
430 functional groups resulting in the synthesis of products of high biological activity [38].
431 Hydroxylations may occur close to the carboxyl group giving rise to α- or β-
432 hydroxylated fatty acids (e.g. bacterial CYP152 peroxygenases [39]), in-chain (e.g.
433 plant CYP703 [40] or bacterial CYP116 [34]), at the subterminal ω-1 to ω-3 positions
434 (e.g. bacterial self-sufficient CYP102 [41]), or at the terminal ω-position (e.g. bacterial
435 CYP153 [42], yeast CYP52 [43], or plant CYP86 [44]). Similar to bacteria and plants,
436 mammalian species comprise a set of P450s covering fatty acid hydroxylation at
437 various positions: CYP1 and CYP2 concentrate on subterminal, bis-allylic and olefinic
438 oxidations, whereas the CYP4 family members generally favor ω- over ω-1-
439 hydroxylations [38].
440 Rabbit CYP4B1 was previously shown to metabolize C12 4 as well as C7 - C10 fatty
441 acids with emphasis on terminal ω-hydroxylation; the positional selectivity increased
442 with decreasing chain length [5]. Within our study this observation was confirmed;
443 whereas for C9 - C12 1–4 the ω:ω-1 ratio was ~ 3:2, in case of C14 6 and C15 7 the
444 ω:ω-1 the ratio decreased to 1:1. In addition, under the given reaction conditions we
445 frequently observed the formation of α-, β-, γ-, and δ-hydroxy fatty acids for carbon
446 chain length C10 – C13 2–5 with clear preference for α- and γ- over β- and δ-
447 positions. It has previously been reported that induction of CYP4B1 under hypoxic
448 conditions was correlated with ω-9-oxidations of arachidonic acid, albeit this type of
449 reaction appeared at odds with the prototypic oxygen insertion at the ω-position [45],
450 and hydroxylations at α- to δ-positions for unsaturated fatty acids have to the best of
451 our knowledge not been reported for CYP4B1 so far.
452 We aimed to explain formation of the new metabolites by in silico analysis based on
453 the recently solved crystal structure of rabbit CYP4B1 (pdb 5T6Q) [14] and the
454 homology model of h-3∆P. In silico fatty acid docking revealed that there were no
455 fundamental differences between the observed conformations of rabbit CYP4B1 (r-
456 ∆1) and the “activated” human CYP4B1 S427P variant (h-∆3P). Nevertheless, our
457 experimental data revealed that h-∆3P in comparison to r-∆1 showed generally a
458 stronger selectivity for ω- over ω-1 hydroxylation, accompanied by generally lower
459 formation of α- to δ-hydroxy fatty acids, albeit the preference for α- and γ- over β- and
460 δ-positions was retained. We hypothesize that these differences in product
461 distribution are attributed to varying residence times of the substrates in the active
462 sites of the P450s; the longer the substrate stays within the active site, the more
- 14 -

463 conformations can possibly be obtained leading to hydroxylations at all positions that
464 are able to get into close proximity to the heme-Fe and consequently to more diverse
465 hydroxylation patterns.
466 Consistent with our observation are the previously calculated KD values determined
467 from Type I spin state shifts of r-∆1 [11]. The KD values for C10 - C12 2–4 that were
468 hydroxylated with the lowest selectivity are in range of 30 - 33 µM, whereas those of
469 longer (C14 – C16) and shorter (C8 -C9) fatty acids are in range of 7 – 25 µM. The
470 tighter binding of short and long fatty acids may lead to less flexible arrangements
471 within the active site and consequently to higher ω/ω-1-selectivity.
472 Calculations of the localization of the HOMOs in the orbital analysis of fatty alcohols,
473 as well as fatty acids C9 - C14 1–6 [19] revealed that the electron densities are
474 concentrated towards the end of the chains with the exception of the ω-position.
475 Together with the calculated hydrogen abstraction energies these data suggest that
476 fatty acid and fatty alcohol hydroxylations by CYP4B1 are kinetically rather than
477 thermodynamically controlled reactions. This is also reflected by the preferred
478 docking conformations, which differ between fatty acids and fatty alcohols.
479 Similar observations of shifted regioselectivities have been made previously: Jóźwig
480 et al. [46] reported that for CYP267B1 from Sorangium cellulosum the regioselectivity
481 of hydroxylation was shifted from positions located closer to the methyl terminal
482 carbon (ω-1, ω-2, ω-3) towards the in-chain positions (ω-3 and ω-4) with increasing
483 chain length of the fatty acid. The authors also found evidence for C14 6 to be bound
484 U-shaped and in two different orientations [46]. Khatri et al. [19] investigated fatty
485 acid hydroxylation by CYP267A1 from the same organism and noticed that the wild-
486 type enzyme contains a natural heme-signature variant (a conserved phenylalanine
487 at position 366 exchanged by a leucine residue) that led to a shift in regioselectivity
488 from subterminal (ω-1 to ω-3) positions to in-chain (ω-4 to ω-9) positions. The “back-
489 mutation” L366F completely revoked this behavior resulting in exclusive subterminal
490 hydroxylation [19]. Fiorentini et al. [47] recently screened CYP153 family enzymes
491 that also exhibit very high ω-selectivity. The authors reported that they detected trace
492 amounts of α-, β-, and γ-hydroxy fatty acid products from conversion of C12 4 by a
493 CYP153 from Pseudomonas sp. and also explained this by the presence of a leucine
494 residue instead of a bulkier phenylalanine residue (conserved in CYP153) that
495 provides a higher flexibility to the substrate posing within the active site to allow
496 inverted orientations of fatty acid substrates [47].
- 15 -

497 The crystal structure of CYP4B1 solved by Hsu et al. [14] suggests a very narrow
498 substrate binding site directing the ω-position of octane close to the heme iron.
499 Nevertheless, the substrate binding site is at least big enough to accept the furan
500 compounds IPO 13 and PK 14 [11] as well as the even bulkier 2-aminoanthracene
501 [48] as substrates.
502 External factors such as the addition of cytochrome b5 can influence fatty acid
503 hydroxylation activity as well: Loughran et al. [49] reported that the turnover of C12 4
504 by CYP4A7 increased from 28 to 130 min-1 through the addition of cytochrome b5;
505 based on their data the authors suggested that cytochrome b5 in this particular case
506 did not serve as an electron donor for CYP4A7, but rather took a conformational role
507 in such that it docks at the proximal surface of the heme pocket to open the substrate
508 channel for greater access to substrates of varying flexibility, bulk, and chain length
509 [49].
510 Regarding the role of cytochrome b5 in CYP4B1 fatty acid hydroxylation, we
511 observed that under the given experimental conditions, addition of cytochrome b5 led
512 to an increase of C12 4 conversion from 24 to 45% within 30 min. The coupling
513 efficiency was improved from 26 to 54%, which may be caused by the ability of
514 cytochrome b5 to act as an electron buffer between P450 and CPR. This might also
515 be the reason for the observed decrease of the NADPH consumption rate from 8.4 to
516 6.6 pmol s-1 (corresponding to 10.1 and 7.9 nmol min-1 nmolCPR-1, respectively). Since
517 the product distribution was not affected by the addition of cytochrome b5 and
518 assuming that the intramolecular uncoupling reactions within the catalytic cycle of
519 CYP4B1 remain unaltered, our results imply that cytochrome b5 directly influences
520 electron transfer. However, it must be noted that the roles of cytochrome b5 in P450
521 catalysis are generally not only substrate dependent, but can also vary considerably
522 with regard to the chosen experimental conditions [31]. It also remains to be
523 elucidated whether the first, the second, or both electrons are transferred via
524 cytochrome b5 to CYP4B1, e.g. by stopped-flow spectroscopy of CPR flavin
525 reduction and P450 heme reduction.
526 Finally, for IPO 13 and PK 14, we previously observed major differences in the ligand
527 binding and metabolic conversion [11]. These two substrates differ only in the
528 hydrophobicity of the terminal portion of their alkyl chains, where the hydroxyl group
529 of IPO 13 is replaced by a methyl group in PK 14 (Figure 1). This results in very
530 substantially increased metabolic conversion of PK 14 by r-∆1 with hydroxylations at
- 16 -

531 the alkyl chain in three different positions making up the majority of oxidative
532 metabolites of PK 14, but not of IPO 13 (Supplementary Figure S4). Our hypothesis
533 that PK 14, in contrast to IPO 13, can enter the active site of CYP4B1 in two different
534 binding orientations (one resulting in alkyl chain hydroxylation, the other in furan ring
535 activation through epoxidation) is now confirmed by the in silico analysis carried out
536 within this study.
537
538
- 17 -

539 Funding information
540 This work was initially supported by the Strategic Research Fund of the Heinrich-
541 Heine University Düsseldorf, Germany [to C.W. and M.G.], the National Institutes of
542 Health [grant number R01 GM49054 to A.E.R.], and subsequently by the UW School
543 of Pharmacy Brady Fund for Natural Products Research [to A.E.R.] as well as the
544 National Institutes of Health [training grant number T32 GM07750 to J.P.K.].
545
546
547
548 Figures
549
550
551
552
553
Figure 1. Compound library investigated within this study.
- 18 -

554
555
Figure 2. Lauric acid 4 conversion by rabbit CYP4B1 (r-∆1). All reactions contained
100 U mL-1 superoxide dismutase and 1,000 U mL-1 catalase. All reactions (except No. 1)
also contained 0.25 µM r-∆1 and 0.5 µM CPR. Where it applies (+), 0.25 µM cytochrome b5
and/or 25 U mL-1 GDH and 20 mM glucose were added. Data represent the
average ± standard deviation of three experiments. (A) Influence of cytochrome b5 and
cofactor regeneration (GDH) on r-∆1 activity. NADPH concentration was 200 µM in reactions
containing GDH and 1 mM in reactions without GDH. The negative control (reaction 1) did
not contain r-∆1 and CPR. (B) Determination of the NADPH coupling efficiencies and (C)
NADPH consumption rates in the presence (+) and absence (–) of cytochrome b5. Reactions
6 and 7 contained equimolar amounts of C12 4 and NADPH (200 µM each).
556
557
558
559
560
561
562
563
564
565
- 19 -

566
567
Figure 3. GC/MS chromatograms of (A) lauric acid 4 and (B) dodecanol 11 conversions by
rabbit and human CYB4B1. Black dashed-dotted line: negative control without CYP4B1 and
CPR (NC). Green solid line: r-∆1. Blue dashed line: h-∆3P. Mass spectra of the individual
reaction products (ω, ω-1, ω-2, α, β, γ, δ) and lactones (15 and 16) are shown in the
Supplementary Material (Supplementary Figures S1 and S2). IS: internal standard, S:
substrate (C12 4 or C12-ol 11), γ-dodecalactone 15, δ-dodecalactone 16.
568
569
570
571
572
573
- 20 -

574
575
Figure 4. Conversion of (A) fatty acids 1-8 and (B) fatty alcohols 9-12 by r-∆1 and h-∆3P.
576
577
The product distributions of these reactions are summarized in Table 1.
- 21 -

578
- 22 -

579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
Figure 5. (A) Docking of lauric acid 4, into the crystal structure of rabbit CYP4B1
demonstrates a U-shaped folding of the carbon chain inside the binding pocket. Here, the ω-
3 position is closest to the Heme-Fe (3.04 Å) denoted as green line. The Heme moiety is
covalently linked via the OE2 atom of glutamine 310 and the sulfur atom of cysteine 448
furthermore ligates the iron atom. The carboxylate group of C12 4 forms a hydrogen-bond
with the backbone oxygen atom of glutamine 377. Also shown are the side chains of the
residues forming close hydrophobic contacts; parts of the helical secondary structure were
omitted for clarity. (B) A docked conformation of C12 4 showing an alternative binding pose
in which the front positions (α, β, γ, δ) get close to the Heme-Fe, whereby the α-position is
within 3.31 Å (green line). The carboxylate group of C12 4 forms a hydrogen-bond with the
side chain of threonine 314 denoted as light blue line. (C) The obtained docking conformation
of 1-dodecanol 10 shows that the terminal positions are preferably hydroxylated. Among
those the ω-1 position is closest to the Heme-Fe (2.94 Å), indicated as green line. The
hydroxyl group of C12-ol 10 forms a hydrogen-bond with the side chain of glutamine 218
denoted as light blue line. Color code for all Figures: grey = carbon; red = oxygen;
blue = nitrogen; yellow = sulfur; coral pink = iron; cyan = CYP4B1 amino acid backbone. The
respective pdb-files of the docked ligands in the corresponding reference frame are available
as Supplementary Material.
- 23 -

598
599
Figure 6. (A) Localization of the Highest Occupied Molecular Orbitals (HOMOs) in the
completely linear chains of fatty acids C9 – C14 1-6. (B) Computed activation energies for
hydrogen abstraction at carbon atoms of lauric acid 4. (C) Localization of the HOMOs in the
completely linear chains of fatty alcohols C10-ol – C16-ol 9-12. (D) Computed activation
energies for hydrogen abstraction at carbon atoms of 1-dodecanol 10.
600
601
602
603
604
- 24 -

Tables
Table 1. Substrate oxidation rates (SOXR) and product distributions of the fatty acids 1–7 and fatty alcohols 9–11 conversion by r-∆1 and h-∆3P.[a]
Product distribution [%] [b]
SOXR [nmol
Compound
C9 (1)
CYP4B1
-1
min-1 nmolP450
]
ω
ω-1
41
21
24
30
20
25
28
14
45
41
43
51
50
43
49
25
42
21
53
42
ω-2
–
ω-3
–
ω-6
-
ω-7
- *
– *
3
ω-8
x
ω-9
x
ω-10
r-∆1
h-∆3P
r-∆1
1.3
0.2
3.2
0.5
5.0
0.9
6.7
3.9
1.2
1.7
1.0
1.2
0.7
0.6
3.4
3.5
5.5
3.6
1.3
1.5
59
79
22
56
27
66
40
80
34
53
42
41
50
51
48
75
56
78
35
54
x
x
–
–
–
x
x
1
–
27
3
23 *
1 *
1
x
x
C10 (2)
h-∆3P
r-∆1
9
–
< 1
35
5
x
x
2
–
< 1
–
12 *
4 *
1
x
C11 (3)
h-∆3P
r-∆1
–
–
–
x
2
–
–
5
18
2
4 *
1 *
1
C12 (4)
h-∆3P
r-∆1
< 1
2
–
–
1
<< 1
9
–
–
5
5
C13 (5)
h-∆3P
r-∆1
–
–
–
–
–
6
<< 1
4
4
1
–
–
–
6
C14 (6)
h-∆3P
r-∆1
7
–
–
–
–
1
–
–
–
–
–
–
–
–
C15 (7)
h-∆3P
r-∆1
–
–
–
–
–
–
–
3
–
–
–
–
x
x
C10-ol (9)
C12-ol (10)
C14-ol (11)
h-∆3P
r-∆1
–
–
–
–
–
x
x
2
–
–
–
–
–
–
h-∆3P
r-∆1
1
–
–
–
–
–
–
12
4
–
–
–
–
–
–
h-∆3P
–
–
–
–
–
–
[a]
[b]
Data represent mean values of three experiments; standard deviations were within ≤ 14%.
ω-4, ω-5 and ≥ ω-11 were never detected; where the sum of
products for an individual compound is < 100%, low amounts of the corresponding lactones were observed (see Figure 3A). x: Formation of this product is not
possible for this compound. –: Product was not detected. * Indicates the α-carbon atom of the respective compound.
- 25 -

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