Steroids, triterpenoids and molecular oxygen

Abstract

There is a close connection between modern-day biosynthesis of particular triterpenoid biomarkers and presence of molecular oxygen in the environment. Thus, the detection of steroid and triterpenoid hydrocarbons far back in Earth history has been used to infer the antiquity of oxygenic photosynthesis. This prompts the question: were these compounds produced similarly in the past? In this paper, we address this question with a review of the current state of knowledge surrounding the oxygen requirement for steroid biosynthesis and phylogenetic patterns in the distribution of steroid and triterpenoid biosynthetic pathways. The hopanoid and steroid biosynthetic pathways are very highly conserved within the bacterial and eukaryotic domains, respectively. Bacteriohopanepolyols are produced by a wide range of bacteria, and are methylated in significant abundance at the C2 position by oxygen-producing cyanobacteria. On the other hand, sterol biosynthesis is sparsely distributed in distantly related bacterial taxa and the pathways do not produce the wide range of products that characterize eukaryotes. In particular, evidence for sterol biosynthesis by cyanobacteria appears flawed. Our experiments show that cyanobacterial cultures are easily contaminated by sterol-producing rust fungi, which can be eliminated by treatment with cycloheximide affording sterol-free samples. Sterols are ubiquitous features of eukaryotic membranes, and it appears likely that the initial steps in sterol biosynthesis were present in their modern form in the last common ancestor of eukaryotes. Eleven molecules of O2 are required by four enzymes to produce one molecule of cholesterol. Thermodynamic arguments, optimization of function and parsimony all indicate that an ancestral anaerobic pathway is highly unlikely. The known geological record of molecular fossils, especially steranes and triterpanes, is notable for the limited number of structural motifs that have been observed. With a few exceptions, the carbon skeletons are the same as those found in the lipids of extant organisms and no demonstrably extinct structures have been reported. Furthermore, their patterns of occurrence over billion year time-scales correlate strongly with environments of deposition. Accordingly, biomarkers are excellent indicators of environmental conditions even though the taxonomic affinities of all biomarkers cannot be precisely specified. Biomarkers are ultimately tied to biochemicals with very specific functional properties, and interpretations of the biomarker record will benefit from increased understanding of the biological roles of geologically durable molecules.

Figure 1

Generalized synthetic pathway of sterols. Sterol precursor squalene is oxidatively converted to oxidosqualene, which is cyclased to one of two protosterols: cycloartenol or lanosterol. The protosterol undergoes subsequent modifications including oxidative demethylations and desaturations to result in the terminal sterol product. Enzymes are labelled with EC number where available, or gene abbreviation. Terminal sterols yield derived steranes after burial and diagenesis. Enzymes labelled in bold are discussed in the text. Those requiring molecular oxygen are noted.

Figure 2

Mechanism for the epoxidation of squalene by squalene monooxygenase (SQMO).

Figure 3

(a) Alignment of oxidosqualene cyclase (OSC) protein sequences. The catalytic acid, D455, is highlighted in red. Positions 381, 449 and 453 are differentially conserved between lanosterol synthases (yellow) and cycloartenol synthases (green). Highly conserved residues that are substituted in bacterial OSCs are highlighted in blue. Residues that are shown in the structures in (b) and (c) are indicated by asterisks. Numbering (throughout this figure and in text) refers to Homo sapiens OSC. (b) View of the active site of H. sapiens OSC with lanosterol shown in light blue. The hydrogen-bonding network around the catalytic acid D455 is indicated by dashed lines. H232 and Y503 constitute the catalytic base dyad and effect the final deprotonation. (c) View of the active site in the molecular plane of lanosterol. Note the distance of second-sphere residue C449 from the substrate. (b) and (c) are drawn from the crystal structure determined by Thoma et al. (2004).

Figure 4

Steps in the oxidation of the 14α angular methyl group of sterols by the CYP51 active site and molecular oxygen. The methyl group is oxidized three times by the cycle (a–h), each using one molecule of O₂. Note that the Fe (IV) + Por+ complex is written Fe(V) for simplicity. After the third oxidation the methyl group is removed.

Figure 5

Total ion chromatograms for showing sterols in the total lipid extracts of four of the investigated cyanobacterial cultures (a–d). Phormidium sp. RCO4 and OSS4 are isolates from Yellowstone National Park. All organisms were grown for a biomarker and isotopic investigation of mat-forming communities (Jahnke et al. 2004). RI, relative intensity; I.S., internal standard.

Figure 6

Gas chromatography–mass spectrometry data, depicted as the m/z 129 ion which is diagnostic for trimethylsilyl (TMS) sterols, for some cyanobacteria cultured in the presence of cycloheximide (a–d). The only detectable sterol is ergosterol which was also present in blank analyses of the BG-11 culture medium and attributable to that source.