The planetary biology of cytochrome P450 aromatases

Abstract

Background: Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system.

Results: Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases-enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene.

Conclusions: This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems.

Figure 1

Reactions catalyzed by aromatases on multiple androgenic substrates.

Figure 2

Dating the pig duplication events. An evolutionary tree, following the topology of Figure 5, showing estimated TREx distances for individual branches calculated from reconstructed ancestral sequences. The scale corresponds to evolutionary time (in million years) estimated from the TREx's using a first order rate constant for transitions of 3 × 10^-9 changes per base per year.

Figure 3

The amino acid alignment of exon 4 of two aromatase isoforms from both peccary and babirusa sequences with exon 4 of pig aromatase isoforms ovarian, fetal, and placental. Asterisks represent conserved sites.

Figure 4

Cladogram of the order Artiodactyla showing the extant families and some selected extinct ones. Ruminantia includes the modern families Tragulidae, Giraffidae, Bovidae, Moschidae, and Cervidae, plus a number of extinct families. "Dichobunidae" is a paraphyletic assemblage of primitive taxa considered broadly ancestral to the later families. The interrelationships of the families reflect the "traditional" relationship based on morphology [85]. Different arrangements based on molecular information [86, 87] would alter the placement of the Camelidae and Hippopotamidae but would make no difference to the arguments presented here concerning the Suoidea. The interrelationships within the Suidae are based on information in several studies [32, 67, 88, 89]. Note that only a couple of extinct suid subfamilies are shown, and that only extant genera of Suinae are shown. Thick, medium-thick and thin lines represent family or above, subfamily and genera, respectively.

Figure 5

Phylogenetic tree for the 18 vertebrate aromatase genes. Numbers above the branches represent the K_A/K_s ratios, while numbers below indicate branches highlighted in Figure 6. Single and double asterisks represent bootstrap values of 95–99% and 100%, respectively. The following sequences were used: Tilapia nilotica (rainbow trout), gi:1613859, Oryzias latipes (medaka), gi:1786171, Danio rerio (zebrafish), gi:2306966, Carassius auratus (goldfish, ovary), gi:2662330, Ictalurus punctatus (catfish), gi:912802, Carassius auratus (goldfish, brain), gi:2662328, Sus scrofa (pig) placental, isoform 2, gi:1762232, Sus scrofa (pig) embryo, isoform 3, gi:1244543, Sus scrofa (pig) ovary, isoform 1, gi:1928957, Bos taurus (ox), gi:665546, Equus caballus (horse), gi:2921277, Mus musculus (mouse), gi:3046857, Rattus norvegicus (rat), gi:203804, Oryctolagus cuniculus (rabbit), gi:2493381, Homo sapiens (human), gi:28846, Gallus gallus (chicken), gi:211703, Poephila guttata (zebra finch, ovary), gi:926845, Ovis aries (sheep), gi:7673985.

Figure 6

The distribution of amino acid replacements on the tertiary structure of cytochrome P450 homolog. Amino acid replacements occurring along branches highlighted in Figure 5 are shown in red. The substrate binding pocket and nicotinamide co-factor are colored yellow and purple, respectively. The sites that bind the co-reductant are highlighted in green for reference.