The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product

Abstract

The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.

Keywords: Saprolegnia parasitica; lanosterol biosynthesis; oomycete; oxidosqualene cyclase; sterols.

FIGURE 1

Enzymatic cyclisation of 2,3-oxidosqualene (OS) to lanosterol (LA) and cycloartenol (CA). Oxidosqualene cyclase enzymes (OSC) protonate the OS substrate, leading to the production of LA or CA.

FIGURE 2

Predicted domains of the two OSC enzymes from S. parasitica (SpLASA and SpLASB) and sequence alignment with similar enzymes from different taxa. (A) SpLASA (SPRG_11783) contains two separate Pfam squalene-hopene cyclase domains. SpLASB (SPRG_17895) contains the C-terminal domain only. (B) Protein sequence alignment of diverse OSC enzymes highlighting a key amino acid triad involved in enzyme. The numbers given for the positions of the three amino acids are from the human OSC. Variations in these positions are highlighted with different colors and the enzymes are grouped into LAS and CAS. The S. parasitica sequence shown is from SpLASA. Arabidopsis thaliana CA and A. thaliana LA refer to the CAS and LAS enzymes from A. thaliana, respectively.

FIGURE 3

Phylogenetic analysis of 63 characterized and predicted OSC sequences. Species group into four clades, numbered 1–4. The outer colored ring denotes the taxonomic groups of the species from which the sequences originate, as indicated by the color key. The small colored hexagons each correspond to a different triad amino acid pattern. The names of the species for which enzyme specificity has been biochemically demonstrated are underlined. Bootstrap values from 100 replicate analyses are shown at each root branch. Full organism names and sequence identification numbers are listed in Supplementary Table S1.

FIGURE 4

SDS-PAGE analysis and identity confirmation of the recombinant SpLASA protein. (A) The recombinant protein expressed in E. coli was purified by immobilized metal ion affinity chromatography (IMAC). (B) The identity of the Coomassie blue-stained SDS-PAGE band was confirmed by mass spectrometry analysis after in-gel partial proteolysis with trypsin. The peptides identified unequivocally confirmed that the purified recombinant protein corresponds to SpLASA. The corresponding spectra are presented in Supplementary Figure S2.

FIGURE 5

GC-MS analysis of the product formed by E. coli cells expressing SpLASA and grown in the presence of the OS substrate. (A) Control E. coli BL21 cells producing green fluorescent protein showed no conversion of the OS substrate. (B) E. coli cells that expressed SpLASA but that were not induced by IPTG for protein expression showed no conversion of OS. (C) E. coli cells that expressed SpLASA and were induced by IPTG for protein expression specifically converted a portion of the OS substrate to LA. (D) Negative control performed on E. coli cells producing SpLASA and grown in the absence of the OS substrate shows no conversion of OS. The major peak detected in all samples is an unidentified compound that does not correspond to any sterol as judged by MS analysis (not shown). The identity of the OS and LA GC peaks were assigned by comparison to standards and confirmed by inspection of their MS fragmentation patterns.

FIGURE 6

GC-MS analysis of the product formed in vitro by the purified recombinant SpLASA protein expressed in E. coli. (A,B) Gas chromatogram and corresponding MS fragmentation pattern of OS and LA standards, respectively. (C) GC-MS analysis of the reaction mixture recovered after incubation of 112.5 μg of the purified recombinant SpLASA protein with 10.3 μg OS showing conversion of OS to LA. (D) Incubation of increasing amounts of the purified recombinant SpLASA with a constant amount of OS (10.3 μg). The amount of LA formed increased linearly in the presence of increasing amounts of SpLASA. Reciprocally, the amount of OS recovered at the end of the reaction decreased linearly.