Concerning P450 Evolution: Structural Analyses Support

Bacterial Origin of Sterol 14a-Demethylases

David C. Lamb,1 Tatiana Y. Hargrove,2 Bin Zhao,2 Zdzislaw Wawrzak,3 Jared V. Goldstone,4 WilliamDavid
Nes,5 Steven L. Kelly,1 Michael R. Waterman,2 John J. Stegeman,4 and Galina I. Lepesheva *,2,6
1

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Institute of Life Science, Swansea University Medical School, Swansea, United Kingdom
2
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN
3
Synchrotron Research Center, Life Science Collaborative Access Team, Northwestern University, Argonne, IL
4
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA
5
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX
6
Center for Structural Biology, Vanderbilt University, Nashville, TN
*Corresponding author: E-mail: galina.i.lepesheva@vanderbilt.edu.
Associate editor: Julian Echave

Abstract
Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the
bacterium Methylococcus capsulatus. Sterol 14a-demethylation is an essential step in this pathway and is catalyzed by
cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin
domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the
first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit
with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to
activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced
the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from
human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an
“orphan” P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked
differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our
results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that
early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis,
revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx
fusion proteins.
Key words: sterol biosynthesis, evolution, cytochrome P450, CYP51 redox partner, crystallography.

Article
Introduction regulatory molecules, such as hormones and brassinosteroids
(Lepesheva et al. 2018). Sterols are often considered as a de-
Cytochrome P450 enzymes (CYP; P450) are heme-containing fining signature (biomarkers) of eukaryotic organisms and are
monooxygenases that play essential roles in sterol/steroid rarely found in bacteria, which usually employ hopanoids to
biosynthesis and degradation, vitamin biosynthesis, detoxifi- facilitate cell membrane integrity (Jackson et al. 2002). Like
cation and activation of many drugs and environmental pol- sterols, hopanoids are cyclic isoprenoidal lipids, and are
lutants, enzymatic activation of carcinogens, and the cyclized by closely related cyclase enzymes (Abe 2007), but
biosynthesis of vast arrays of secondary metabolites their synthesis does not require atmospheric molecular oxy-
(Guengerich 2001). One such P450 is sterol 14a-demethylase gen. Thus, hopanoids have been used as molecular proxies for
(CYP51), an enzyme found in all kingdoms of life, catalyzing ancient microbial life predating the enrichment of oxygen in
the same three-step monooxygenation reaction that removes Earth’s atmosphere (Ourisson and Albrecht 1992; Saenz et al.
the 14a-methyl group from the nucleus of the sterol precur- 2015).
sor molecule (fig. 1). Ultimately, this reaction is required for In 1971, production of sterols in bacteria was first discov-
the biosynthesis of cholesterol in animals, ergosterol in fungi ered biochemically in the aerobic methanotroph
and protozoa, and stigmasterol in plants and algae. The ster- Methylococcus capsulatus Bath. A truncated postsqualene
ols function as integral components of eukaryotic cell mem- sterol pathway was predicted because only the modified lan-
branes and also serve as precursors for a multitude of different osterol molecules were detected and identified

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Mol. Biol. Evol. doi:10.1093/molbev/msaa260 Advance Access publication October 08, 2020 1
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FIG. 1. CYP51 reaction. The 14a-methyl group is first converted to the alcohol, then to the aldehyde intermediate, and at the third step is removed
as formic acid with the introduction of the double bond into the sterol core.

experimentally (fig. 2) (Bird et al. 1971). These observations has a CYP51 gene and it makes sterols. Furthermore,
were verified following the complete sequencing of the M. capsulatus is one of a handful of organisms discovered
M. capsulatus genome, in which only a small number of genes to-date carrying a gene encoding P450 heme domain fused
encoding sterol enzyme orthologs were seen and mapped to to a ferredoxin (fx) redox partner domain (Jackson et al. 2002;
the sterol biosynthetic pathway (Ward et al. 2004). Hannemann et al. 2007; McLean et al. 2007).
Subsequently, further examples of sterol-producing bacteria Herein, we report: 1) CYP51 genes are found in >1,000
were identified, including Methylosphaera hansonii (Schouten bacteria from nine different phyla establishing that horizontal
et al. 2000), Stigmatella aurantiaca, Nannocystis excedens gene transfer to bacteria is extremely unlikely and supporting
(Bode et al. 2003), Gemmata obscuriglobus (Pearson et al. a prokaryotic origin (and thus ancestral role) of CYP51 in
2003), Plesiocystis pacifica (Desmond and Gribaldo 2009), P450 evolution; 2) the existence of CYP51 ferredoxin fusion
Methylomicrobium alcaliphilum (Banta et al. 2015). With proteins (CYP51fx) in >50 bacterial organisms, suggesting
the rapid advances in genomic and bioinformatic analysis, that early cytochrome P450s may have initially been fusion
the numbers of bacteria known to possess genes for sterol proteins; and 3) the first crystal structure of a bacterial sterol
synthesis are continually increasing (Wei et al. 2016). biosynthetic enzyme. The P450 domain of M. capsulatus
Questions of the evolutionary origin of bacterial sterol bio- CYP51fx is similar in protein architecture to eukaryotic
synthetic machinery, as well as structural relationship of bac- CYP51s, and not to My. tuberculosis CYP51, indicating an
terial water-soluble CYP51 proteins to eukaryotic CYP51s evolutionary linkage between bacterial and eukaryotic sterol
(which are membrane-bound microsomal proteins) remain 14a-demethylases.
unanswered. Mycobacterium tuberculosis is the only bacterial
species with a P450 with CYP51 activity whose 3D crystal Results and Discussion
structure has been resolved (Podust et al. 2001). However, In this study, we first performed biochemical characterization
that structure differs profoundly from bone fide eukaryotic of M. capsulatus CYP51fx to confirm its enzymatic compe-
CYP51 crystal structures, both in the composition of the tency. Then we crystallized the fusion protein, determined the
substrate-binding cavity/active site volume and in the loca- crystal structure of its P450 domain, in the ligand-free and a
tion of the substrate access channel (Lepesheva et al. 2010). detergent-bound state, and compared these with the known
Moreover, the CYP51 gene was shown to be not essential in eukaryotic and My. tuberculosis CYP51 structures. Finally, we
My. tuberculosis physiology (McLean et al. 2006) nor essential used bioinformatic approaches to complement our
for cholesterol metabolism (van Wyk et al. 2019), whereas the structure-functional data and provide further support for a
CYP51 gene is essential in eukaryotes. Thus, two opposite and bacterial origin of CYP51.
contradicting hypotheses on CYP51 evolution exist and re-
main unresolved to-date: 1) CYP51 and sterol biosynthesis Biochemical Characterization of M. capsulatus
has a bacterial origin with soluble (nonmembrane-bound) CYP51fx
enzymes that are possible evolutionary ancestors for the Optical Properties
membrane-bound eukaryotic CYP51 P450 family (Yoshida The absolute absorbance spectrum of the purified protein
et al. 1997; Nelson 1999; Yoshida et al. 2000) or 2) bacterial was typical of ferric, low-spin P450 with the maxima of the
CYP51s are the result of horizontal gene transfer from some a-, b-, and c (Soret) bands at 568, 535, and 419 nm, respec-
early eukaryote (Debeljak et al. 2003; Rezen et al. 2004). tively, the spectrophotometric index A419/A278 ¼ 1.33, and
Although the origin and evolution of the cytochrome P450 the ratio DA393–470/DA419–470 ¼ 0.38 (fig. 3A). The heme
superfamily as a whole is unknown, current thinking suggests iron was readily reduced by sodium dithionite and the differ-
a role for CYP51 as the ancestral P450 progenitor with its ence spectrum of the ferrous CO-complex had an absorbance
ability to fix atmospheric oxygen resulting in increased P450 maximum at 448 nm (a hallmark of the cysteinate-
importance in generating metabolites for membrane integ- coordinated heme iron) with no quantifiable denatured cy-
rity. Subsequently, this was followed by gene duplication and tochrome P420 form (fig. 3B) (Omura and Sato 1964).
evolutionary diversification leading to broad P450 function-
ality in many roles (Sezutsu et al. 2013). Binding of Sterol Substrates
To address these fundamental questions regarding P450 Substrate titration experiments were first conducted with
evolution and sterol biosynthesis, the bacterium lanosterol and eburicol to clarify which of the two sterols
M. capsulatus is an excellent organism of choice because it has a higher affinity for M. capsulatus CYP51fx (and therefore
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FIG. 2. Sterol biosynthesis and final sterol products in Methylococcus capsulatus (A) versus eukaryotes (B). (A) Methylococcus capsulatus was shown
to encode a truncated postsqualene sterol pathway that synthesizes the modified lanosterol molecules 4,4-dimethylcholesta-8,24-dien-3-ol, 4,4-
dimethylcholesta-8-en-3-ol, 4-methylcholesta-8,24-dien-3-ol, and 4-methylcholesta-8-en-3-ol. Subsequent studies have demonstrated the pro-
duction of similar sterols in other aerobic methanotrophs of the Methylococcales order within the c-Proteobacteria. (B) Chemical structures of the
major eukaryotic sterol molecules: cholesterol in animals; stigmasterol in plants and algae; and ergosterol in fungi and protozoa.

should be selected for cocrystallization). Though with both sterols produced typical Type I-binding spectra,
M. capsulatus is known to synthesize lanosterol in vivo reflecting changes in the spin state of the heme Fe atom,
(Bird et al. 1971), the CYP51 family amino acid sequence with a blue shift in the Soret band maximum from 419 to
alignment suggested that the enzyme might prefer eburicol, 393 nm, and a DA393–470/DA418–470 ratio (2.12–2.14), indicat-
due to the isoleucine residue at position 81. The I81 in ing >95% heme iron transition from low- to high-spin form,
M. capsulatus CYP51fx corresponds to I105 in CYP51 from and the charge transfer band at 650 nm (fig. 4). The apparent
the protozoan pathogen Trypanosoma cruzi, (the only eu- spectral dissociation constants (Kd) derived from the titration
karyotic sterol 14a-demethylase having Ile in this position), plots were also similar, although binding of eburicol appeared
and we have previously found that T. cruzi CYP51 prefers to be about twice as tight (fig. 4, Inset, Kd¼48 6 4 vs.
eburicol over lanosterol (Lepesheva et al. 2006). Titration 94 6 11 nM for lanosterol).

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FIG. 3. The UV-visible absolute (A) and reduced carbon monoxide complex difference (B) absorbance spectra of purified Methylococcus capsulatus
CYP51fx. The P450 concentration was 3.8 mM, the optical path length was 1 cm. The specific heme content of the preparation was 15.6 nmol per
mg protein (98% of the ideal specific content [15.9 nmol per mg protein] calculated from the predicted molecular weight of the M. capsulatus
CYP51 sequence [63 kDa]).

FIG. 4. Spectral changes observed during titration of Methylococcus capsulatus CYP51fx with (A) lanosterol and (B) eburicol. Absolute (top) and
difference (bottom) spectra. The P450 concentration was 2 mM. Inset: The titration curves with hyperbolic fitting (quadratic Morrison equation).
The experiments were performed in duplicates, the results are presented as means 6 SD.

Enzymatic Activity demethylation of both C4-dimethylated sterols and C4-

Both lanosterol and eburicol were metabolized by monomethylated obtusifoliol at appreciable rates, perhaps
M. capsulatus CYP51fx to the 14a-demethylated products, contrary to suggestions based on the presence of Ile81 and
3b-hydroxy-4,4-dimethyl-cholesta-8,14,24-triene, and 3b-hy- the apparent binding affinities.
droxy-4,4-dimethyl-24-methylene-cholesta-8,14-diene, with Because lanosterol is the M. capsulatus CYP51fx substrate
the same efficiency and comparable rates, 1.8 and 1.6 min1, in vivo (Bird et al. 1971), this substrate was next used to verify
respectively. The turnover of obtusifoliol was 1.2 min1 (sup- the ability of the fused ferredoxin domain to transfer elec-
plementary table S1, Supplementary Material online). By trons to the P450 heme iron. There was no activity observed
comparison, the turnover of obtusifoliol by My. tuberculosis at 2 mM M. capsulatus CYP51fx (5 mM spinach ferredoxin re-
CYP51 (its preferred substrate) was 0.5 min1, when its activ- ductase) under these reaction conditions, but increasing the
ity was reconstituted under the same conditions (Lepesheva concentration to 10 mM (25 mM ferredoxin reductase)
et al. 2004), that is, using Escherichia coli flavodoxin/flavodoxin resulted in 25% of lanosterol conversion in a 2-h reaction
reductase as electron donor partners, as detailed in Materials (supplementary fig. S1, Supplementary Material online), sug-
and Methods. Thus, M. capsulatus CYP51fx catalyzes 14a- gesting a possibility of both inter- and intrareduction, as was
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FIG. 5. Crystal structure of ligand-free Methylococcus capsulatus CYP51 (PDB ID 6mi0). (A) A Coot snapshot of the 2F0–Fc electron density map
(contoured at 1.5 r), map radius 50 Å. The density for the near P450 chains from the neighboring unit cells is seen at the top, left and right, the
density for the fx domain is missing. (B) Ribbon representation of the P450 domain structure (blue, upper P450 face) and the fx domain model
(magenta). Orientation is about the same as in (A). The residues that are expected to be involved in the (productive) protein–protein interaction
are shown and labeled. (C) Superimposition of the M. capsulatus P450 domain with the Trypanosoma brucei (plum, PDB ID 3g1q, Ca RMSD, 1.6 Å),
human (tan, PDB ID 4uhi, Ca RMSD, 1.7 Å), and Candida albicans (pink, PDB ID 5tz1, Ca RMSD, 1.9 Å) CYP51 orthologs. (D) Superimposition with
CYP51 from Mycobacterium tuberculosis (red, PDB ID 1e9, Ca RMSD, 5.4 Å). The substrate entrances are marked with the arrow of the
corresponding color. (E) Heme support in M. capsulatus (blue), My. tuberculosis (red), and T. brucei (plum) structures. Y103, R361, and H420
(T. brucei numbering) are invariant across the whole CYP51 family, Y116 corresponds to F in bacterial and plant sequences, and R124 is protozoa-
specific, its role being played by a Lys (located one turn downstream of helix C) in animal and fungal CYP51. Distal P450 face. The residues that
correspond to T. brucei Y116 and R124 in the bacterial structures are shown in wire representation and labeled.

proposed for flavocytochrome P450 BM3 (CYP102) from the online) and contained one P450 molecule per asymmetric
soil bacterium Bacillus megaterium (a natural fusion of a P450 unit. However, no electron density for the ferredoxin domain
to a cytochrome P450 reductase) (Kitazume et al. 2007). was observed. Instead, the space above the P450 proximal
surface (the region where ferredoxin is expected to bind)
Structural Characterization of M. capsulatus CYP51fx looked like a “black hole” (fig. 5A and B). We believe this is
Crystal Structure of the P450 Domain with the Bound Water because, in the absence of the substrate, the P450/ferredoxin
Molecule complex, even if formed, is weak, likely representing multiple
We first crystallized M. capsulatus CYP51fx in the resting “encounter states” (also called transient complexes) where
(ferric) ligand-free form. The structure was solved to 2.7 Å ferredoxin can have many orientations that it adopts before
resolution (supplementary table S2, Supplementary Material forming a final, “productive” complex (Schilder and Ubbink
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Lamb et al. . doi:10.1093/molbev/msaa260 MBE
2013; Bowen et al. 2018) with the P450 domain. Crystal Structure of CYP51fx with the Bound Detergent
Physiologically, the electron transfer process between redox Anapoe-X-114
partner proteins and P450s takes place after a P450 enzyme The crystal structure was solved to 2.4 Å resolution and also
binds its substrate (Guengerich and Yoshimoto 2018). The contained one P450 molecule per asymmetric unit with no
large-scale conformational switch that accompanies binding electron density for the observed ferredoxin domain.
of the substrate in eukaryotic CYP51 structures (both proto- Although addition of substrate (either lanosterol or eburicol)

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zoan, Hargrove et al. 2018 and human, Hargrove et al. 2020) produced >95% high-spin P450 (fig. 3), and the sample
alters the topology of the P450 proximal surface that is known remained in the high-spin state at least until the crystal plates
to be involved in the interaction with the electron donor were set, upon crystallization the substrate was expelled from
protein partners. We conclude that the presence of substrate the CYP51 active site by the detergent: the spectra of the
in the M. capsulatus CYP51fx active site must be required for dissolved crystals revealed low-spin P450, with the Soret band
the formation of a “productive” P450/ferredoxin complex, maximum returned to 417 nm. In the detergent-bound struc-
one which might be tight enough to be detected crystallo- ture (fig. 6A and B), the heme iron is indeed hexa-
graphically. Indeed, the P450 domain in the 6MI0 structure coordinated: the detergent (23-(4-(2,4,4-trimethylpentan-2-
(fig. 5) has a typical substrate-free (i.e., water-/inhibitor-bound yl)phenoxy)-3,6,9,12,15,18,21-heptaoxatricosan-1-ol) lies
like) CYP51 conformation (Hargrove et al. 2018): the sub- above the water molecule, which serves as the sixth (distal)
strate channel is open, the proton delivery route is closed, ligand, like it does in structure 6MI0, though here it is shifted
and helix C is not crossing the heme plane. 0.7 Å toward the I-helix, and its distance to the iron is 2.4 Å
When overlaid with the other substrate-free CYP51 struc- (vs. 2.6 Å in 6mi0). The proximal methyl group of the deter-
tures, the P450 domain of M. capsulatus CYP51fx revealed the gent 2,4,4-trimethylpentan moiety is hanging 4.2 Å above the
highest similarity with protozoan T. brucei CYP51, Ca root- iron, the phenoxy ring plane is perpendicular to the heme
mean-square deviation (Ca RMSD) of 1.6 Å. The Ca RMSDs plane, with the oxygen atom directed toward the I helix. The
with human and fungal (Candida albicans) CYP51s are only largest portion of the 3,6,9,12,15,18,21-heptaoxatricosan-1-ol
arm is exposed to the bulk solvent, protruding above the
slightly larger, 1.7 and 1.9 Å, respectively (fig. 5C). The greatest
protein surface. This explains its higher B-factor (supplemen-
differences were observed with CYP51 from the nonsterol-
tary table S2, Supplementary Material online), though it
producing bacterium My. tuberculosis (Ca RMSD of 5.4 Å),
anchors to the outer wall of the F00 helix through a weak
both in the conformation of the secondary structural ele-
(3.7 Å) H-bond, linking oxygen atom 9 to the nitrogen of
ments that define the active site topology (fig. 5D), and in
the Gln182 side chain. Thus, contrary to all other known
the “eukaryotic” location of the substrate channel entrance,
CYP51-bound small molecules, the detergent must have en-
on the distal P450 face, bordered by the A0 and F00 helices and tered the active site not through the substrate access channel,
the tip of the b4 hairpin, the same as in eukaryotic CYP51 between helices A0 , F00 , and the tip of the b4 hairpin (although
(Lepesheva et al. 2010) and unlike My. tuberculosis CYP51, it probably expelled the substrate via this channel), but
which has the channel entrance on the upper P450 face, through the “water channel” that is sometimes seen between
bordered by helix B0 , B0 C loop, and helix C (Podust et al. 2001). helices I, F, and the tip of the b4 hairpin in the CYP51 struc-
The protein support for the heme propionates, however, tures (Lepesheva et al. 2010; Lepesheva and Waterman 2011)
in both bacterial CYP51 structures is the same and includes (fig. 6A and C). It appears that the detergent reorients the
the H-bonds from only three residues: Tyr (Y79 and Y76 imidazole ring of His260 (the CYP51 family signature histi-
[M. capsulatus and My. tuberculosis, respectively], B0 helix) dine) involved in the proton delivery network (Hargrove et al.
and Arg (R327 and R326, b strand 1–4) to the ring A propi- 2018; Hargrove et al. 2020), and its oxygen 18 forms a 3.0-Å H-
onate and His (H392, the heme bulge) to the ring D propio- bond with the imidazole N1 atom (fig. 6A), thus interfering
nate (fig. 5E). These three residues are invariant in all CYP51 with the “proper” closing of the conserved His260/Glu178 salt
sequences, whereas in protozoan, fungal, and animal CYP51s, bridge (3.4 Å bond length), the event expected in substrate-
two more amino acid side chains are involved in the interac- free/inhibitor-bound CYP51 molecules (fig. 6D). Overall, ex-
tion with the ring D propionate (Lepesheva et al. 2010; cept for the 2 Å rearrangements in helix F00 , the ligand-free
Hargrove et al. 2017). One of them is a Tyr on the B00 turn and detergent-bound structures are very similar (Ca RMSD of
(Y116 in fig. 5E), the residue conserved across all these three 0.37 Å), indicating that the closed (active) CYP51 conforma-
kingdoms, but always represented by Phe in plants and in tion that is observed while the substrate is productively
most bacteria (F92 in M. capsulatus and F89 in bound in the active site does not last when it is replaced
My. tuberculosis CYP51, fig. 5E), although some bacterial by the detergent.
CYP51s, carry a Tyr in this position (table 1 and fig. 8B), like
fungal, animal, and protozoan orthologs. The other residue is Structural Insights into the Broad Substrate Tolerance of
an Arg/Lys in helix C (R124 in fig. 5E). There is no conservation M. capsulatus CYP51fx
of the corresponding amino acid among plant and bacterial A structural explanation for the lack of obvious M. capsulatus
CYP51 sequences (N100 in M. capsulatus and K97 in CYP51fx substrate preferences toward eburicol over lano-
My. tuberculosis; fig. 5E, see also supplementary fig. S3A, sterol or obtusifoliol (supplementary table S1,
Supplementary Material online). Supplementary Material online) could be that Ile81 in
6
 Table 1. Taxonomy of Selecteda Bacteria Containing a CYP51 Gene, Sequence Identity, and “Phylum-Marker” Residues.
Bacteria CYP51

Group Phylum (Class) Order Family Examples Sequence Identity, % P450/Fx I81/F105b F92/Y116
c
Terrabacteria Actinobacteria Corynebacteriales Dietziaceae Dietzia timorensis 43 F F
Gordoniaceae Gordonia sp. HY186 43 F F
Skermania piniformis 46 F F
Nocardiaceae Nocardia paucivorans 47 F F
Williamsia limnetica 45 F F
Rhodococcus opacus 45 F F
Mycobacteriaceae Mycobacteroides abscessus 45 F F
Mycolicibacter algericus 47 F F
Mycobacterium tuberculosis 47 F F
Propionibacteriales Nocardioidaceae Marmoricola ginsengisoli 42 F F
Pimelobacter simplex 44 F F
Pseudomonas sp. SLBN-26 42 F F
Streptosporangiales Streptosporangiaceae Planobispora rosea 46 F F
Nonomuraea sp. ATCC 55076 46 F F
Thermomonosporaceae Actinocorallia populi 43 F F
Spirillospora albida 46 F F
Actinomadura kijaniata 46 F F
Concerning P450 Evolution . doi:10.1093/molbev/msaa260

Pseudonocardiales Pseudonocardiaceae Amycolatopsis halophila 46 F F

Amycolatopsis rhizosphaerae 47 F F
Amycolatopsis kentuckyensis 45 F F
Saccharopolyspora dendranthemae 48 F F
Acidimicrobiales Acidimicrobiaceae Acidimicrobiaceae bacterium 48 F F
Microthrixaceae Candidatus Microthrixd
parvicella Bio17-1 45/37 F F
parvicella RN1 44/37 F F
Streptomycetales Streptomycetaceae Streptomyces curacoi 38 F Y
Cyanobacteria Oscillatoriales Oscillatoriaceae Moorea sp. SIOASIH 40 F Y
Nostocales Calotrichaceae Calothrix sp. NIES-4071 38 F Y
Calothrix rhizosoleniae 43 F v
Oscillatoriales Gomontiellaceae Hormoscilla sp. GUM202 44 F Y
Chloroflexi Unclassified Unclassified Chloroflexi bacterium 36 F Y
(Dehalococcoidia) Unclassified Unclassified Dehalococcoidia bacterium 34 F Y
Firmicutes Bacillales Thermoactinomycetaceae Kroppenstedtia sanguinis 42 F F
— Proteobacteria Methylococcales Methylococcaceae Methylococcus capsulatusd 100/100 I F
(Gammaproteobacteria) Methylomagnum ishizawaid 69/47 L F
Methylocaldum marinumd 65/55 I F
Methylotetracoccus oryzaed 64/42 I F
Methylocaldum szegediensed 63/48 I F
Methylomicrobium alcaliphilumd 58/42 I F
Methylomicrobium kenyensed 58/42 I F
Methylobacter whittenburyid 57/40 I F
Methylobacter luteusd 56/40 I F
Methylomicrobium buryatensed 53/42 I F
(continued)
MBE

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Table 1. Continued
Bacteria CYP51

Group Phylum (Class) Order Family Examples Sequence Identity, % P450/Fx I81/F105b F92/Y116
d
Methylomonas lenta 52/38 I F
Methylobacter marinusd 53/38 I
Methylosarcina lacusd 52/40 I F
Methylovulum sp.d 51/40 I F
Methylicorpusculum oleiharenaed 55/33 I F
Nevskiales Sinobacteraceae Stenotrophob. rhamnosiphilumd 46/32 L F
Sinimarinibacterium sp. NLF-5-8d 49/37 L F
Algiphilaceae Algiphilus aromaticivoransd 45/35 L F
Oceanospirillales Alcanivoracaceae Ketobacter alkanivoransd 48/30 L F
Alcanivoracaceae TS13_700d 55/35 L F
Oceanospirillaceae Oceanospirillaceae bacterium 41 A F
Lamb et al. . doi:10.1093/molbev/msaa260

Unclassified Unclassified Gammaproteobacteria-HGWd 63/38 I F

Gammaproteobacteria WB5_2Bd 61/36 I F
Cellvibrionales Halieaceae Halioglobus japonicus 45 A F
Parahaliea mediterranea 42 F Y
Spongiibacteraceae Spongiibacter tropicusd 50/33 A F
Oceanicoccus sp. KOV_DT_Chl 44 A F
Unclassified Cellvibrionales bacteriumd 44/22 L F
Chromatiales Unclassified Chromatiales bacterium 39 F Y
Pseudomonadales Unclassified Pseudomonadales bacteriumd 47/36 L F
(Deltaproteobacteria) Myxococcales Nannocystaceae Nannocystis exedens 40 L Y
Plesiocystis pacifica 43 L Y
Sandaracinaceae Sandaracinus amylolyticusd 35/30 I Y
Sandaracinus sp. NAT8d 37/31 L F
Polyangiaceae Polyangium sp. SDU3-1 35 F Y
Unclassified Enhygromyxa salina 41 L Y
Minicystis rosead 42/37 I Y
Myxococcales bacteriumd 37/30 I Y
(Alphaproteobacteria) Rhodospirillales Acetobacteraceae Zavarzinia sp. HR-ASd 50/33 L F
Zavarzinia compransorisd 49/31 L F
Oleomonas sp. K1W22B-8d 46/30 L F
Sphingomonadales Sphingomonadaceae Novosphingobium tardaugens 43 L Y
PVCe Plantomyces Unclassified Planctomycetes bacterium 36 F Y
FCBf Gemmatimonadetes Gemmatimonadales; Unclassified Gemmatimonadales bacterium 36 F Y
— Spirochetes Spirochaetales Spirochaetaceae Spirochaeta sp.d 41/36 F F
— Nitrospirae Nitrospirales Nitrospiraceae Nitrospira sp. SB0677_bin_15 36 F Y
Unclassified Candidatus Rokubacteria 41 Y Y
a
Representative species from each family and genus are shown, including bacteria that do not make sterols, for example, Actinobacteria.
b
Methylococcus capsulatus/Trypanosoma brucei numbering.
c
Identity to M. capsulatus CYP51 (H260).
d
In these organisms, CYP51 is naturally fused to ferredoxin (CYP51fx).
e
Named after the phyla Planctomycetes, Verrucomicrobia, and Chlamydiae.
f
Named after the main member phyla Fibrobacteres, Chlorobi, and Bacteroidetes (NCBI taxonomy).
MBE

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Concerning P450 Evolution . doi:10.1093/molbev/msaa260 MBE

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FIG. 6. Crystal structure of the detergent-bound P450 domain of Methylococcus capsulatus CYP51fx (PDB ID 6mcw). (A) Interaction of Anapoe X
114 (cyan) with surrounding residues (within 4.5 Å, shown in plum sticks and labeled). Red dotted lines are H-bonds. Selected red sphere is a water
molecule. (B) The 2F0–Fc electron density map (1.2 r) for the detergent area. Orientation is similar to that in (A). (C) Surface representation. Heme
ring A propionate (magenta) is seen through the substrate access channel. (D) Overlaid detergent-bound and ligand-free (blue, PDB ID 6mi0)
structures, Ca RMSD of 0.37 Å. Distal P450 face in both cases.

M. capsulatus CYP51, probably due to some structural disor- type I response to cucurbitadienol was seen. Based on the
der observed in this region, is positioned more like L134 in Soret band maximum at 417 nm, this was probably the result
human CYP51 (fig. 7), which also has similar preference for of an unproductive binding mode via a water molecule;
lanosterol, eburicol, or obtusifoliol (Hargrove et al. 2016). This Kd ¼ 0.67 mM (supplementary fig. S2C, Supplementary
is unlike the I105 in T. cruzi CYP51, which prefers eburicol over Material online). As no spectral response to these sterols is
lanosterol and even more over obtusifoliol (Lepesheva et al. produced by eukaryotic orthologs (human and T. cruzi
2006), or the F105 in T. brucei CYP51, which has a strict CYP51; data not shown), these results support our structural
requirement for obtusifoliol (Lepesheva et al. 2004). To fur- findings. Consequently, we propose that ancestral bacterial
ther test M. capsulatus CYP51fx substrate tolerance, we spec- CYP51 may have had broad substrate specificity, and this may
trally titrated the enzyme with three naturally occurring be a reflection of various substrates made available to the
lanosterol isomers, cycloartenol (the initial sterol in plants enzyme from preceding steps. Bacteria encode for oxidosqua-
and some bacteria), parkeol (one of the sterol end-products lene cyclases (OSC) that act either as a lanosterol synthase or
in an abbreviated pathway in bacterium G. obscuriglobus a cycloartenol synthase whereas in eukaryotes generally only a
[Planctomyces]; Pearson et al. 2003), and cucurbitadienol (a single class of OSC is found in any specific phylum (cyclo-
phytosterol precursor found in some plant families; Shibuya artenol synthase in plants and algae vs. lanosterol synthase in
et al. 2004). The classical type I spectral response was observed animals, fungi, and protozoa). This may be of importance
for cycloartenol and parkeol, showing 88% and 73% low- to because the final products of many eukaryotic sterol biosyn-
high-spin transition of the heme iron with the calculated Kds thetic pathways are utilized by other biochemical pathways
of 0.46 and 0.26 mM, respectively (supplementary fig. S2A and to produce other important molecules, for example, hor-
B, Supplementary Material online). Additionally, a modified mones, brassinosteroids, signaling molecules, etc. Bacteria,
9
Lamb et al. . doi:10.1093/molbev/msaa260 MBE
Phylogenetic Analysis, Sequence Conservation, and
the Evolutionary Origins of CYP51
Strikingly, bioinformatic analysis of >247,000 bacterial
genomes (https://www.ncbi.nlm.nih.gov/genome/browse#!/
prokaryotes/) reveals that >1,000 bacteria possess a CYP51
gene even though the vast majority of them do not biosyn-

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thesize sterols. Specifically, these CYP51 genes are found both
in Gram-positive and in Gram-negative bacteria that include
the Terrabacteria group [phyla Actinobacteria, Firmicutes,
Chloroflexi, and Cyanobacteria], the PVC group [phylum
Planctomycetes], the FCB group [phyla
Gemmatimonadetes, Fibrobacteres, Chlorobi, and
Bacteroidetes], and ungrouped phyla Nitrospirae,
Spirochetes, and Proteobacteria (classes Gamma, Alpha,
and Delta) (table 1 and fig. 8A). Alignment of 150 CYP51
representatives from nine bacterial phyla (including
FIG. 7. Ile81 in Methylococcus capsulatus CYP51 (blue, PDB ID 6mi0). A CYP51fx fusion proteins) and their phylogenetic tree can be
flexible loop-like turn can be seen in the middle of the B0 helix. seen as supplementary figure S3A and B, Supplementary
Overlaid with Trypanosoma brucei (plum, PDB ID 3g1q), human Material online, respectively. These organisms are evolution-
(tan, PDB ID 4uhi), and T. cruzi (green, PDB ID 4ck8) CYP51 structures. arily very distant, and, accordingly, their CYP51 sequence
identities across bacterial phyla are low (30–45%). Even
among order Methylococcales (where M. capsulatus belongs)
the identity varies from 98% to 52%, the mean being 70%.
on the other hand, may have relaxed CYP51 substrate spe-
The average CYP51 sequence identity in order Myxococcales
cificity because the pathways are abbreviated and the sterol
is 45% (between 32% and 79%). Nevertheless, all these
end-product(s) is not part of downstream enzymatic sequences possess the unique CYP51 signature motifs
modifications. (fig. 8B). The B0 helix/B0 C loop conservation is essential for
Unfortunately, determining the complete structure of the interaction with the sterol substrates, and the I-helical –
M. capsulatus CYP51fx (which would be P450–ferredoxin HTt(s)– triad is required for the CYP51-specific proton deliv-
complex) by X-ray crystallography remains problematic due ery network (Lepesheva and Waterman 2011; Hargrove et al.
to the lack of electron density for tracing the ferredoxin do- 2020). Our finding of CYP51 sequences in four alpha-
main. The complex, at least in the absence of the substrate in proteobacteria, Zavarzinia sp. HR-AS (NCBI protein accession
the P450 active site, is either not strong enough or (more number WP_109905014.1), Zavarzinia compransoris
likely, in our opinion) the density for the small (8.4 kDa) fer- (WP_109920848.1), Oleomonas sp. K1W22B-
redoxin is not seen because it can form multiple intermediate 8 (WP_119777160.1), Novosphingobium tardaugens
(transient) interactions (Schilder and Ubbink 2013) with the (WP_021691104.1) (table 1), may indicate an alpha proteo-
P450 molecule (52 kDa), reaching the/a productive binding bacterial ancestor of mitochondria as a source of the first
mode only when the substrate is bound, and the electron eukaryotic P450 given that Lokiarcheaum, the closest known
transfer is expected. Indeed, to date only the X-ray structures living archaeon relative of eukaryotes, encodes no P450 genes
of two artificial (genetically engineered) P450–ferredoxin (Spang et al. 2015).
complexes have been resolved: the cross-linked Furthermore, >50 bacterial CYP51s (mostly
CYP101(P450cam)-putidoredoxin (Tripathi et al. 2013) and Proteobacteria [including three alpha-proteobacteria, family
the partial structure of CYP11A1(P450scc)-adrenodoxin, a syn- Acetobacteraceae] but also Actinobacteria and Spirochaeta)
thetic fusion (Strushkevich et al. 2011). In both of these cases, (noted in table 1), like M. capsulatus CYP51, are native fusion
the P450 domain was substrate-bound. The most recently proteins, where the P450 domain (N-terminus) is connected,
reported structure of a self-sufficient P450 monooxygenase via a 20–30 residues linker, to a ferredoxin domain (C-termi-
CYP116B46 (a P450-reductase-ferredoxin in one polypeptide nus). Methylococcus capsulatus-based alignment of ferredoxin
chain), although outlining the interaction between the FMN domains from bacterial CYP51fx proteins is shown as supple-
of the reductase domain and the Fe-S cluster of the ferredoxin mentary figure S4, Supplementary Material online. These fer-
domain, does not clearly reveal the productive interaction redoxins are 64–82 amino acid residues long, possessing three
between the ferredoxin and the P450 domains (Zhang et al. invariant cysteines that form the sulfur–iron cluster. The FeS
2020). Most likely this is because imidazole and not the sub- cluster is responsible for the functioning of ferredoxins as
strate is bound in the P450 active site. The continued work on electron transfer protein partners of most bacterial and all
cocrystallization of M. capsulatus CYP51fx with bound lano- eukaryotic mitochondrial P450s (accepting electrons from
sterol/eburicol is currently in progress, the spin state of the NADPH/NADH and transferring to the P450 heme iron).
P450 heme iron in the crystals being monitored prior to data The average sequence identity of the ferredoxin domains is
collection, as described in Materials and Methods. 45%, ranging between 22% and 98%. Existence of such a large
10
Concerning P450 Evolution . doi:10.1093/molbev/msaa260 MBE

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FIG. 8. CYP51 sequences from bacterial genomes. For simplicity, only one species per genus is shown. Trypanosoma brucei, T. cruzi, and human
orthologs are used as references. (A) Phylogenetic tree (rendered in TreeDyn 198.3). Colored branches represent different bacterial phyla:
Actinobacteria (blue), Cyanobacteria (cyan), Firmicutes (sky-blue), Chloroflexi (navy), Proteobacteria, a- (saddle brown), c- (dark red), d- (coral),
Planctomycetes (seagreen), Gemmatomonadales (limegreen), Nitrospirae (spring green), and Spirochetes (olive). Species that make sterols are
highlighted in bold. (B) Two CYP51 signature motives in the aligned bacterial sequences. Trypanosoma brucei numbering is presented on the top.
Residues crucial for the CYP51 function and absolutely conserved in all biological domains are in yellow. Alignment and phylogenetic tree of 150
bacterial CYP51 proteins are available as supplementary figure S3A and B, Supplementary Material online, respectively.

number of CYP51fx fusion proteins in different bacterial spe- CYP51 genes present in the genomes of sequenced and an-
cies is unprecedented to date, suggesting that it is unlikely to notated sterol and nonsterol-producing bacteria (fig. 9 and
be a coincidence but rather provided an evolutionary advan- supplementary fig. S6, Supplementary Material online) pro-
tage, for example, guaranteed electron transfer in conditions vides additional evidence that bacterial CYP51s are not a
when molecular oxygen (dioxygen) was scarce in the bur- result of horizontal gene transfer. On the contrary, bioinfor-
geoning Earth atmosphere, the event preceding the forma- matics analysis suggests a divergence hypothesis, that is, mod-
tion of sterol-enforced membranes and leading to the ern bacteria that do not make sterols but whose genomes still
appearance of eukaryotic forms of life (Nes and McKean possess a CYP51 gene (or rather did not lose the CYP51 gene)
1977). are derived from ancestors that used to make sterols but
A separate root for bacterial sequences seen in the phylo- evolved to stop producing them for some reasons, for exam-
genetic tree that includes CYP51 representatives from each ple, as symbionts, parasites, etc.
biological kingdom (supplementary fig. S5, Supplementary It is possible that within some species the resulting gene
Material online) is also consistent with common ancestry product evolved to new biological function(s). Such P450s
for eukaryotic CYP51s and CYP51s in modern bacteria. could be recruited to other lipid (nonsterol) biochemical
Different and variable gene synteny around CYP51fx and pathways involved in membrane biogenesis given that

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Lamb et al. . doi:10.1093/molbev/msaa260 MBE

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FIG. 9. Examples of bacterial CYP51 gene synteny. (A) CYP51fx and CYP51 genes found in sequenced and annotated genomes of sterol-producing
bacteria (M. capsulatus Bath, Methylococcus capsulatus Bath; P. pacifica, Plesiocystis pacifica; S. amylolyticus, Sandaracinus amylolyticus; E. salina,
Enhygromyxa salina; S. aurantica, Stigmatella aurantica; M. luteus, Methylobacter luteus; M. alcaliphilum, Methylomicrobium alcaliphilum; C. fuscus,
Cystobacter fuscus; C. coralloides, Corallococcus coralloides; N. excedens, Nannocystis excedens) and (B) synteny of CYP51 in Mycobacterium
tuberculosis, a representative bacterium that possess a CYP51 gene but does not make sterol. As predicted at the BioCyS Database (https://
biocyc.org/gene? orgid¼MTBH37RV&id¼G185E-4912#tab¼TU), genes RV0767c–RV0763c form a putative 6-gene operon and may act in a
pathway with CYP123.

many of the nonsterol-producing bacteria synthesize com- Supplementary Material online). This is probably due to the
plex lipids, for example, Mycobacteria sp. CYP51 is the third highly selective nature of the OSC catalytic reaction, being
postsqualene step of sterol biosynthesis, preceded by squa- one of the most complex biochemical reactions known (the
lene monooxygenase (SQM) and OSC reactions. In a similar formation of four rings along the long chain of the substrate
fashion, an analogous scenario seems likely for the gene [oxidosqualene], producing lanosterol). It is likely that genes
encoding SQM in bacteria. In sterol-producing bacteria, the encoding OSC were not retained following loss of sterol bio-
SQM, which utilizes atmospheric oxygen, is fixed in sterol synthesis because the complexity of its chemistry and lack of
biosynthesis. However, it is observed that SQM genes are ease of application to new substrates and functions.
also distributed and retained in nonsterol-producing bacteria, Conversely, SQM and CYP51, which fix atmospheric oxygen,
also suggesting that these enzymes have evolved to new could use their chemistry efficiently with other molecules in
function(s) as sterol biosynthesis was lost over time. In con- evolving to new function(s), an example being reported for a
trast, the OSC enzyme is seemingly only found in sterol- CYP51 evolved to new function in the biosynthesis of avena-
producing organisms (supplementary fig. S6B and C, cin in the roots of oat plants (Qi et al. 2006). We await crystal
12
Concerning P450 Evolution . doi:10.1093/molbev/msaa260 MBE
structural analysis of bacterial SQMs (from sterol and MUSCLE-Gblocks-PyML) (http://www.phylogeny.fr/index.
nonsterol-producing species) and subsequent comparison cgi) (Dereeper et al. 2010).
to the recent eukaryotic SQM structures (Padyana et al.
2019). Such comparisons may also reveal alterations in the Protein Expression and Purification
enzyme SQM structural architecture through evolving to new Methylococcus capsulatus CYP51fx (P450Fx native fusion) in
functionality, as we have described herein for CYP51. the pET17-b plasmid (Novagen) (Jackson et al. 2002) was

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expressed in E. coli. HMS-174 (DE3) (Novagen). The expres-
Conclusions sion level was 300–400 nmol l1. A single colony of bacteria
Our crystallographic data showed that the P450 domain of was used to inoculate 5 ml of LB media containing
the sterol-producing M. capsulatus CYP51fx enzyme displays 0.1 mg ml1 ampicillin. An overnight culture was incubated
a greater overall structural similarity to eukaryotic CYP51s at 37  C and 250 rpm for 16 h and then diluted into 500 ml of
(20–26% amino acid sequence identity) than to nonsterol- TB media supplemented with 100 mM potassium phosphate
producing My. tuberculosis CYP51, even though they share buffer (pH 7.4) containing 0.1 mg ml1 ampicillin and 125 ml
47% amino acid sequence identity, and yet the H-bond net- of trace elements salt solution. After incubation at 37  C and
work between the apoprotein and the heme propionates in 250 rpm for 5 h, the flasks were cooled to 22  C, the cultures
M. capsulatus CYP51fx is the same as in My. tuberculosis were supplemented with 1 mM d-aminolevulinic acid, 1 mM
CYP51 (and presumably in plant CYP51 orthologs [no struc- IPTG, and 1 mM FeCl3 (to ensure proper FeS cluster forma-
ture available yet]). This finding strongly reinforces the notion tion) and incubated for 20 h at 22  C and 180 rpm. The cells
that sterol 14a-demethylases, an essential enzyme of sterol were harvested by centrifugation at 1,850  g for 15 min, the
biosynthesis, most probably derived from a bacterial origin pellet was resuspended in 50 mM potassium phosphate
and may indeed have been the ancestral progenitor for the buffer (pH 7.4) containing 0.1 mM EDTA, 100 mM NaCl,
whole P450 superfamily (Yoshida et al. 2000). The 10% glycerol (v/v), and 0.1% Triton X-100 (v/v) (buffer A).
M. capsulatus CYP51fx enzyme may be evolutionarily closer Lysozyme (25 mg ml1) was added, and the suspension was
to a predecessor from some ancient sterol-making bacteria placed on ice for 15 min and then, after addition of 0.5 mM
that was cenancestor for: 1) all eukaryotic CYP51s (which PMSF, frozen at 80  C. All purification steps were done at
4  C, and all buffers contained 0.1 mM PMSF and 0.1 mM
further diverged to enable faster sterol flow), 2) modern bac-
dithiothreitol, which were added fresh daily. The pellet was
terial CYP51s, and 3) possibly for other bacterial P450s (which
homogenized in buffer A, the suspension was sonicated on ice
evolved to acquire new, mostly simplified functions, such as
(Sonic Dismembrator model 500, Fisher Scientific), and stirred
one-step vs. three-step monooxygenation reactions, abilities
with 0.2% Triton X-100 (v/v) at 4  C for 1 h. The solubilized
to accommodate and metabolize compounds of various che-
protein was separated from the insoluble material by centri-
motypes or to accept electrons from different protein
fugation at 82,000  g for 30 min. The supernatant was frozen
donors).
in liquid nitrogen and stored at 80  C. Methylococcus cap-
Finally, a wide distribution of CYP51 genes across bacterial
sulatus CYP51fx fusion was purified in two steps, including
phyla, a broad variability of bacterial CYP51 sequences, a sep-
affinity chromatography on Ni2þ-NTA agarose, and anion
arate cluster in the phylogenetic tree, and particularly the fact
exchange chromatography on Q-Sepharose. The thawed su-
that bacterial CYP51 carry all of the amino acid residues that
pernatant was diluted 2-fold with buffer A and applied to
serve as phyla-specific markers for eukaryotic CYP51 ((table 1
Ni2þ-NTA agarose equilibrated with the same buffer and the
and fig. 8), for example, in bacterial sequences there can be I (NTA-) bound protein was washed with equilibration buffer
(T. cruzi only), Y (plants/other protozoa), or L(animals/fungi) and then with 50 mM potassium phosphate buffer (pH 7.4)
and more rarely, M, V, or A at the position that aligns with I81 containing 500 mM NaCl, 10% glycerol (v/v), and 1 mM im-
in M. capsulatus CYP51, also, there can be either F or Y (in- idazole until the Triton X-100 was eliminated (as judged by
variant F in plants but always Y in animals/fungi/protozoa) A280 measurements).Then the protein was washed with
that aligns with F92 (see also fig. 5E)), implies that both plant 20 mM potassium phosphate buffer (pH 7.4) containing
and animal/fungi sterol biosynthesis pathways could be de- 200 mM NaCl, 10% glycerol (v/v), 5, 10, and then 15 mM
rived from pathways initially developed in ancient aerobic imidazole, and eluted with a linear gradient of imidazole
bacteria. (20–200 mM) in 20 mM potassium phosphate buffer (pH
Materials and Methods 7.4) containing 500 mM NaCl, 10% glycerol (v/v). The frac-
tions with a spectrophotometric index (A425/A280) 1 were
Bioinformatic Analysis pooled and concentrated using an Amicon Ultra 50K
The search for bacterial CYP51 sequences in the National (Millipore) to a volume of 2–4 ml. The protein was then di-
Center for Biotechnology Information sequence databases luted 25-fold with 20 mM potassium phosphate buffer (pH
was performed using NCBI protein–protein BLAST (TBlast 8.0) containing 10% glycerol (v/v), 0.1 mM EDTA (Q buffer),
suite) (Altschul et al. 1997) and M. capsulatus CYP51 as a and applied to a Q-Sepharose column equilibrated with Q
protein query. Multiple sequence alignment was carried out buffer containing 20 mM NaCl. The column was washed with
in Clustal Omega and analyzed in GeneDoc. The phylogenetic the equilibration buffer, and the protein was eluted with
tree was rendered in TreeDyn; branch support values (%) 20 mM potassium phosphate buffer (pH 7.6) containing
were generated in phylogeny analysis (one-click mode: 180 mM NaCl, 10% glycerol (v/v), and 0.1 mM EDTA, pooled
13
Lamb et al. . doi:10.1093/molbev/msaa260 MBE
and concentrated to 500 lM. The purity and molecular detector (INUS Systems). In order to verify the ability of the
weight of the protein (63 kDa, 561 amino acid residues in fusion M. capsulatus ferredoxin domain to transfer electrons
length, including 451 residues of the P450 domain, 22 residues to the P450 domain, we used spinach ferredoxin reductase
of the linker, 78 residues of the ferredoxin domain, and a 10 (Jackson et al. 2002). In this case, the CYP51fx concentration
His tag at the C-terminus; Jackson et al. 2002) was confirmed was increased to 10 mM, the concentration of ferredoxin re-
by SDS–PAGE. The correctness of the genes was confirmed by ductase was 25 mM. At these conditions, 25% of lanosterol

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DNA sequencing. Escherichia coli flavodoxin and flavodoxin conversion was observed in a 1-h reaction.
reductase were expressed and purified as described (Jenkins
and Waterman 1998). Spinach ferredoxin reductase was pur- Crystallization and Determination of the X-ray
chased from Sigma. Structures
Crystals were obtained at 24  C by vapor diffusion in hanging
Spectroscopic Characterization drops containing equal volumes of the protein and well so-
UV-visible spectra were recorded at ambient temperature in a lution. The crystals of ligand-free M. capsulatus CYP51fx were
dual-beam Shimadzu UV-240IPC spectrophotometer. P450 grown from a 250-mM protein sample in 10 mM potassium
concentrations were determined from the Soret band absor- phosphate buffer (pH 7.4) containing 100 mM NaCl, 5.6 mM
bance in the absolute spectrum, using an absolute molar tris(2-carboxyethyl)phosphine (TCEP), 5% glycerol (v/v) and
extinction coefficient (e417) of 117 mM1 cm1 for the low- 2% ANAPOE X-114 (a-[(1,1,3,3-tetramethylbutyl)phenyl]-w-
spin oxidized form of the protein or a difference molar ex- hydroxy-poly(oxy-1,2-ethanediyl)) (Anatrace) mixed with
tinction coefficient (De446–490) of 91 mM 1 cm1 for the 0.2 M lithium acetate (pH 7.5) and 18% PEG 3,350 (w/v).
reduced carbon monoxide complex in the difference spectra. Methylococcus capsulatus CYP51fx was crystallized in the
The spin states of P450 samples were estimated from the presence of the detergent since it could not be crystallized
absolute spectra as the ratio DA393–470/DA418–470 in its absence. The crystals of M. capsulatus CYP51fx in com-
(Lepesheva et al. 1999). plex with the detergent were obtained in an attempt to
cocrystallize substrate-bound protein, because we expected
Substrate Binding Assay that binding of the substrate should induce the large-scale
Binding of lanosterol, eburicol, cycloartenol, parkeol, and conformational rearrangement in the CYP51 molecule, which
cucurbitadienol to 2 lM M. capsulatus CYP51fx was moni- should strengthen P450–ferredoxin interaction (Hargrove
tored in a 50-mM phosphate buffer, pH 7.4, containing et al. 2018; Hargrove et al. 2020). The M. capsulatus
100 mM NaCl and 0.1 mM EDTA. The sterols were dissolved CYP51fx sample (5 lM in 20 mM potassium phosphate
in 45% (w/v) 2-hydroxypropyl-b-cyclodextrin (HPCD) to buffer [pH 7.4] containing 200 mM NaCl, 5.6 mM TCEP,
0.5 mM concentration and added in 1 ml aliquots to 2 ml and 10% glycerol [v/v]) was gradually saturated with eburicol
protein samples (concentration range 0.25–3 mM). Equal (using a 0.5-mM stock solution in 45% [w/v] HPCD, because
amounts of 45% HPCD solution were added to the reference of the limited solubility of this highly hydrophobic sterol, Log
cuvette to correct for the solvent-induced spectral perturba- P 8.6; Hargrove et al. 2018), incubated for 20 min at room
tions. The Kd values were calculated by fitting the data for the temperature, concentrated using an Amicon Ultra 50K
substrate-induced absorbance changes in the difference (Millipore) to 500 mM, diluted 2-fold with 5 mM phosphate
spectra D(AmaxAmin) versus substrate concentration to buffer (pH 7.4), and mixed with ANAPOE X-114 (final con-
the quadratic Morrison equation (DA=(DAmax/ centration 2%). The high-spin (substrate-bound) state of the
2E)((L þ EþKd)((L þ EþKd)24LE)0.5)) using GraphPad P450 heme in the mixture was spectrally confirmed. The well
Prism 6 (GraphPad, La Jolla, CA). solution consisted of 0.2 M lithium acetate (pH 7.5) and 15%
PEG 6,000 (w/v). After harvesting, crystals were cryoprotected
Reconstitution of Enzymatic Activity with 25% (v/v) glycerol and frozen in liquid nitrogen. The X-
The sterol 14a-demethylase activity of M. capsulatus CYP51fx ray diffraction data were collected at the Advanced Photon
was reconstituted with radiolabeled (3-3H) lanosterol, ebur- Source, Argonne National Laboratory, 21-ID-F (0.97872 nm),
icol, and obtusifoliol, specific activity 4,000 dpm nmol1, as and 21-ID-D (1.07807 nm) beamlines, respectively. The dif-
described previously for CYP51 from My. tuberculosis fraction images were processed with HKL-2000, and crystal
(Lepesheva et al. 2001), except that the sterols were added structures solved by molecular replacement with Phaser MR
from a 0.5-mM solution in 45% (w/v) HPCD. Escherichia coli (CCP4 Program Suite; Potterton et al. 2003) using T. brucei
flavodoxin and flavodoxin reductase (molar excess over P450 CYP51 (PDB ID 3g1q) as a search model. The structure was
18 and 2, respectively), served as electron donor partners. The built with Coot (Emsley et al. 2010) and refined with Refmac5
enzyme/substrate molar ratio was 1/25 (2/50 mM). The final (CCP4 Suite). Details of the data collection and refinement
reaction volume was 500 ml and contained 20 mM MOPS (pH statistics are listed in supplementary table S2, Supplementary
7.4), 50 mM KCl, 5 mM MgCl2, 10% (v/v) glycerol, Material online. After determining the structure and observ-
0.4 mg ml1 isocitrate dehydrogenase, and 25 mM sodium ing ANAPOE X-114 instead of the substrate in the P450 active
isocitrate. The reaction was initiated by addition of 5 mM site, the crystals were reproduced at the same conditions,
NADPH and stopped by extraction of the sterols with ethyl harvested, dissolved in 10 mM potassium phosphate buffer
acetate. The extracted sterols were analyzed by a reversed- (pH 7.4) containing 100 mM NaCl, 5.6 mM TCEP, and 5%
phase HPLC system (Waters) equipped with a b-RAM glycerol (v/v), and the absorbance spectra (2 ml samples)
14
Concerning P450 Evolution . doi:10.1093/molbev/msaa260 MBE
were taken using a Nanovue 4282 V1.7.3 spectrophotometer Emsley P, Lohkamp B, Scott WG, Cowtan K. 2010. Features and devel-
as described previously (Hargrove et al. 2018). The Soret max- opment of Coot. Acta Crystallogr D Biol Crystallogr. 66(4):486–501.
Guengerich FP. 2001. Common and uncommon cytochrome P450 reac-
imum of the enzyme was 417 nm, exactly corresponding to tions related to metabolism and chemical toxicity. Chem Res Toxicol.
the low-spin water coordinated P450 (see fig. 6A) and indi- 14(6):611–650.
cating that the detergent replaced the substrate upon crys- Guengerich FP, Yoshimoto FK. 2018. Formation and cleavage of C-C
tallization. Structure superimposition and RMSD calculation bonds by enzymatic oxidation-reduction reactions. Chem Rev.

Downloaded from https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msaa260/5919591 by MBLWHOI Library user on 16 December 2020

was performed in lsqcab (CCP4 Suite). Molecular graphics 118(14):6573–6655.
Hannemann F, Bichet A, Ewen KM, Bernhardt R. 2007. Cytochrome P450
were rendered using Chimera. The model of M. capsulatus systems—biological variations of electron transport chains. Biochim
ferredoxin was built in Modeler (CCP4 Suite) based on the Biophys Acta. 1770(3):330–344.
structure of adrenodoxin (PDB ID 1AYF) with 32 truncated Hargrove TY, Friggeri L, Wawrzak Z, Qi A, Hoekstra WJ, Schotzinger RJ,
amino acid residues at the N-terminus. York JD, Guengerich FP, Lepesheva GI. 2017. Structural analyses of
Candida albicans sterol 14a-demethylase complexed with azole
Supplementary Material drugs address the molecular basis of azole-mediated inhibition of
fungal sterol biosynthesis. J Biol Chem. 292(16):6728–6743.
Supplementary data are available at Molecular Biology and Hargrove TY, Friggeri L, Wawrzak Z, Sivakumaran S, Yazlovitskaya EM,
Evolution online. Hiebert SW, Guengerich FP, Waterman MR, Lepesheva GI. 2016.
Human sterol 14a-demethylase as a target for anticancer chemo-
Acknowledgments therapy: towards structure-aided drug design. J Lipid Res.
57(8):1552–1563.
The study was supported by National Institutes of Health Hargrove TY, Wawrzak Z, Fisher PM, Child SA, Nes WD, Guengerich FP,
(Grant No. R01 GM067871 to G.I.L.) and by a UK-USA Waterman MR, Lepesheva GI. 2018. Binding of a physiological sub-
Fulbright Scholarship and the Royal Society (to D.C.L.). strate causes large-scale conformational reorganization in cyto-
chrome P450 51. J Biol Chem. 293(50):19344–19353.
Data Availability Hargrove TY, Wawrzak Z, Guengerich FP, Lepesheva GI. 2020. A require-
ment for an active proton delivery network supports a Compound I
The atomic coordinates and structure factors (accession mediated C-C bond cleavage in CYP51 catalysis. J Biol
codes 6MI0 and 6MCW) have been deposited in the Chem. 295(29):9998–10007.
Protein Data Bank (http://wwpdb.org/), and all other data Jackson CJ, Lamb DC, Marczylo TH, Warrilow AG, Manning NJ, Lowe DJ,
underlying this article and in its online Supplementary Kelly DE, Kelly SL. 2002. A novel sterol 14a-demethylase/ferredoxin
fusion protein (MCCYP51FX) from Methylococcus capsulatus repre-
Matarial . sents a new class of the cytochrome P450 superfamily. J Biol Chem.
277(49):46959–46965.
References Jenkins CM, Waterman MR. 1998. NADPH-flavodoxin reductase and
flavodoxin from Escherichia coli: characteristics as a soluble micro-
Abe I. 2007. Enzymatic synthesis of cyclic triterpenes. Nat Prod Rep. somal P450 reductase. Biochemistry 37(17):6106–6113.
24(6):1311–1331. Kitazume T, Haines DC, Estabrook RW, Chen B, Peterson JA. 2007.
Altschul SF, Madden TL, Sch€affer AA, Zhang J, Zhang Z, Miller W, Obligatory intermolecular electron-transfer from FAD to FMN in
Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation dimeric P450BM-3. Biochemistry 46(42):11892–11901.
of protein database search programs. Nucleic Acids Res. Lepesheva GI, Friggeri L, Waterman MR. 2018. CYP51 as drug targets for
25(17):3389–3402. fungi and protozoan parasites: past, present and future. Parasitology
Banta AB, Wei JH, Welander PV. 2015. A distinct pathway for tetrahy- 145(14):1820–1836.
manol synthesis in bacteria. Proc Natl Acad Sci U S A. Lepesheva GI, Nes WD, Zhou W, Hill GC, Waterman MR. 2004. CYP51
112(44):13478–13483. from Trypanosoma brucei is obtusifoliol-specific. Biochemistry
Bird CW, Lynch JM, Pirt FJ, Reid WW, Brooks CJW, Middleditch BS. 1971. 43(33):10789–10799.
Steroids and squalene in Methylococcus capsulatus grown on meth- Lepesheva GI, Park HW, Hargrove TY, Vanhollebeke B, Wawrzak Z, Harp
ane. Nature 230(5294):473–474. JM, Sundaramoorthy M, Nes WD, Pays E, Chaudhuri M, et al. 2010.
Bode HB, Zeggel B, Silakowski B, Wenzel SC, Reichenbach H, Müller R. Crystal structures of Trypanosoma brucei sterol 14a-demethylase
2003. Steroid biosynthesis in prokaryotes: identification of myxobac- and implications for selective treatment of human infections. J
terial steroids and cloning of the first bacterial 2,3(S)-oxidosqualene Biol Chem. 285(3):1773–1780.
cyclase from the myxobacterium Stigmatella aurantiaca. Mol Lepesheva GI, Podust LM, Bellamine A, Waterman MR. 2001. Folding
Microbiol. 47(2):471–481. requirements are different between sterol 14a-demethylase (CYP51)
Bowen AM, Johnson EOD, Mercuri F, Hoskins NJ, Qiao R, McCullagh JSO, from Mycobacterium tuberculosis and human or fungal orthologs. J
Lovett JE, Bell SG, Zhou W, Timmel CR, et al. 2018. A structural Biol Chem. 276(30):28413–28420.
model of a P450-ferredoxin complex from orientation-selective dou- Lepesheva GI, Strushkevich NV, Usanov SA. 1999. Conformational dy-
ble electron-electron resonance spectroscopy. J Am Chem Soc. namics and molecular interaction reactions of recombinant cyto-
140(7):2514–2527. chrome P450scc (CYP11A1) detected by fluorescence energy
Debeljak N, Fink M, Rozman D. 2003. Many facets of mammalian lan- transfer. Biochim Biophys Acta. 1434(1):31–43.
osterol 14a-demethylase from the evolutionarily conserved cyto- Lepesheva GI, Waterman MR. 2011. Structural basis for conservation in
chrome P450 family CYP51. Arch Biochem Biophys. 409(1):159–171. the CYP51 family. Biochim Biophys Acta. 1814(1):88–93.
Dereeper A, Audic S, Claverie J-M, Blanc G. 2010. BLAST-EXPLORER Lepesheva GI, Zaitseva NG, Nes WD, Zhou W, Arase M, Liu J, Hill GC,
helps you building datasets for phylogenetic analysis. BMC Evol Waterman MR. 2006. CYP51 from Trypanosoma cruzi: a phyla-
Biol. 10(1):8–8. specific residue in the B0 helix defines substrate preferences of sterol
Desmond E, Gribaldo S. 2009. Phylogenomics of sterol synthesis: insights 14a-demethylase. J Biol Chem. 281(6):3577–3585.
into the origin, evolution, and diversity of a key eukaryotic feature. McLean KJ, Dunford AJ, Sabri M, Neeli R, Girvan HM, Balding PR, Leys D,
Genome Biol Evol. 1:364–381. Seward HE, Marshall KR, Munro AW. 2006. CYP121, CYP51 and

15
Lamb et al. . doi:10.1093/molbev/msaa260 MBE
associated redox systems in Mycobacterium tuberculosis: towards Schouten S, Bowman JP, Rijpstra WI, Sinninghe Damste JS. 2000. Sterols
deconvoluting enzymology of P450 systems in a human pathogen. in a psychrophilic methanotroph, Methylosphaera hansonii. FEMS
Biochem Soc Trans. 34(6):1178–1182. Microbiol Lett. 186(2):193–195.
McLean KJ, Girvan HM, Munro AW. 2007. Cytochrome P450/redox Sezutsu H, Le Goff G, Feyereisen R. 2013. Origins of P450 diversity. Philos
partner fusion enzymes: biotechnological and toxicological pros- Trans R Soc B. 368(1612):20120428.
pects. Expert Opin Drug Metab Toxicol. 3(6):847–863. Shibuya M, Adachi S, Ebizuka Y. 2004. Cucurbitadienol synthase, the first
Nelson DR. 1999. Cytochrome P450 and the individuality of species. Arch committed enzyme for cucurbitacin biosynthesis, is a distinct en-

Downloaded from https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msaa260/5919591 by MBLWHOI Library user on 16 December 2020

Biochem Biophys. 369(1):1–10. zyme from cycloartenol synthase for phytosterol biosynthesis.
Nes WR, McKean MR. 1977. Biochemistry of steroids and other isopen- Tetrahedron 60(33):6995–7003.
tenoids. Baltimore: University Park Press. Spang A, Saw JH, Jørgensen SL, Zaremba-Niedzwiedzka K, Martijn J, Lind
Omura T, Sato R. 1964. The carbon monoxide-binding pigment of liver AE, van Eijk R, Schleper C, Guy L, Ettema T. 2015. Complex archaea
microsomes. J Biol Chem. 239:2379–2385. that bridge the gap between prokaryotes and eukaryotes. Nature
Ourisson G, Albrecht P. 1992. Hopanoids. 1. Geohopanoids: the most 521(7551):173–179.
abundant natural products on Earth? Acc Chem Res. 25(9):398–402. Strushkevich N, MacKenzie F, Cherkesova T, Grabovec I, Usanov S, Park
Padyana AK, Gross S, Jin L, Cianchetta G, Narayanaswamy R, Wang F, H-W. 2011. Structural basis for pregnenolone biosynthesis by the
Wang R, Fang C, Lv X, Biller SA, et al. 2019. Structure and inhibition mitochondrial monooxygenase system. Proc Natl Acad Sci U S A.
mechanism of the catalytic domain of human squalene epoxidase. 108(25):10139–10143.
Nat Commun. 10(1):97. Tripathi S, Li H, Poulos TL. 2013. Structural basis for effector control and
Pearson A, Budin M, Brocks JJ. 2003. Phylogenetic and biochemical ev- redox partner recognition in cytochrome P450. Science
idence for sterol synthesis in the bacterium Gemmata obscuriglobus. 340(6137):1227–1230.
Proc Natl Acad Sci U S A. 100(26):15352–15357. van Wyk R, van Wyk M, Mashele SS, Nelson DR, Syed K. 2019.
Podust LM, Poulos TL, Waterman MR. 2001. Crystal structure of cyto- Comprehensive comparative analysis of cholesterol catabolic
chrome P450 14a-sterol demethylase (CYP51) from Mycobacterium genes/proteins in Mycobacterial species. Int J Mol Sci. 20(5):1032.
tuberculosis in complex with azole inhibitors. Proc Natl Acad Sci U S Ward N, Larsen Ø, Sakwa J, Bruseth L, Khouri H, Durkin AS, Dimitrov G,
A. 98(6):3068–3073. Jiang L, Scanlan D, Kang KH, et al. 2004. Genomic insights into
Potterton E, Briggs P, Turkenburg M, Dodson E. 2003. A graphical user methanotrophy: the complete genome sequence of Methylococcus
interface to the CCP4 program suite. Acta Crystallogr D Biol capsulatus (Bath). PLoS Biol. 2(10):e303.
Crystallogr. 59(7):1131–1137. Wei JH, Yin X, Welander PV. 2016. Sterol synthesis in diverse bacteria.
Rezen T, Debeljak N, Kordis D, Rozman D. 2004. New aspects on lano- Front Microbiol. 7:990.
sterol 14a-demethylase and cytochrome P450 evolution: lanosterol/ Yoshida Y, Aoyama Y, Noshiro M, Gotoh O. 2000. Sterol 14-demethylase
cycloartenol diversification and lateral transfer. J Mol Evol. P450 (CYP51) provides a breakthrough for the discussion on the
59(1):51–58. evolution of cytochrome P450 gene superfamily. Biochem Biophys
Qi X, Bakht S, Qin B, Leggett M, Hemmings A, Mellon F, Eagles J, Werck- Res Commun. 273(3):799–804.
Reichhart D, Schaller H, Lesot A, et al. 2006. A different function for a Yoshida Y, Noshiro M, Aoyama Y, Kawamoto T, Horiuchi T, Gotoh O.
member of an ancient and highly conserved cytochrome P450 fam- 1997. Structural and evolutionary studies on sterol 14-demethylase
ily: from essential sterols to plant defense. Version 2. Proc Natl Acad P450 (CYP51), the most conserved P450 monooxygenase: II.
Sci U S A. 103(49):18848–18853. Evolutionary analysis of protein and gene structures. J Biochem.
Saenz JP, Grosser D, Bradley AS, Lagny TJ, Lavrynenko O, Broda M, 122(6):1122–1128.
Simons K. 2015. Hopanoids as functional analogues of cholesterol Zhang L, Xie Z, Liu Z, Zhou S, Ma L, Liu W, Huang JW, Ko TP, Li X, Hu Y,
in bacterial membranes. Proc Natl Acad Sci U S A. et al. 2020. Structural insight into the electron transfer pathway of a
112(38):11971–11976. self-sufficient P450 monooxygenase. Nat Commun. 11(1):2676.
Schilder J, Ubbink M. 2013. Formation of transient protein complexes.
Curr Opin Struct Biol. 23(6):911–918.

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