Concerning P450 Evolution: Structural Analyses Support Bacterial Origin of Sterol 14α-Demethylases

Abstract

Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14α-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.

Keywords: CYP51 redox partner; crystallography; cytochrome P450; evolution; sterol biosynthesis.

Fig. 1.

CYP51 reaction. The 14α-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.

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 production of similar sterols in other aerobic methanotrophs of the Methylococcales order within the γ-Proteobacteria. (B) Chemical structures of the major eukaryotic sterol molecules: cholesterol in animals; stigmasterol in plants and algae; and ergosterol in fungi and protozoa.

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 µM, 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 µM. Inset: The titration curves with hyperbolic fitting (quadratic Morrison equation). The experiments were performed in duplicates, the results are presented as means ± SD.

Fig. 5.

Crystal structure of ligand-free Methylococcus capsulatus CYP51 (PDB ID 6mi0). (A) A Coot snapshot of the 2F₀–F_c electron density map (contoured at 1.5 σ), 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, Cα RMSD, 1.6 Å), human (tan, PDB ID 4uhi, Cα RMSD, 1.7 Å), and Candida albicans (pink, PDB ID 5tz1, Cα RMSD, 1.9 Å) CYP51 orthologs. (D) Superimposition with CYP51 from Mycobacterium tuberculosis (red, PDB ID 1e9×, Cα 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.

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–F_c electron density map (1.2 σ) 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, Cα RMSD of 0.37 Å. Distal P450 face in both cases.

Fig. 7.

Ile81 in Methylococcus capsulatus CYP51 (blue, PDB ID 6mi0). A flexible loop-like turn can be seen in the middle of the B′ helix. Overlaid with Trypanosoma brucei (plum, PDB ID 3g1q), human (tan, PDB ID 4uhi), and T. cruzi (green, PDB ID 4ck8) CYP51 structures.

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, α- (saddle brown), γ- (dark red), δ- (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.

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? orgid=MTBH37RV&id=G185E-4912#tab=TU), genes RV0767c–RV0763c form a putative 6-gene operon and may act in a pathway with CYP123.