Squalene-hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids

We report the cloning and characterisation of the Methylococcus capsulatus shc gene, which encodes the squalene-hopene cyclase (SHC). This enzyme catalyses the complex cyclization of squalene to the pentacyclic triterpene skeleton of hopanoids and represents the key reaction in this biosynthesis. Using a combination of PCR amplification and DNA hybridization, two overlapping 2.6 kb PstI and 3.3 kb SalI DNA fragments were cloned bearing a 1962 bp open reading frame encoding a 74 kDa protein with 654 amino acids and a predicted isoelectric point at about pH 6.3. The deduced amino acid sequence of the M. capsulatus shc gene showed significant similarity to known prokaryotic SHCs and to a lesser degree to the related eukaryotic oxidosqualene cyclases (OSCs). Like other triterpene cyclases, the M. capsulatus SHC contains seven so-called QW-motifs as well as an aspartate-rich domain. The recombinant M. capsulatus SHC was expressed in Escherichia coli and in vitro activity of the recombinant cyclase was demonstrated using crude cell-free lysate or solubilized membrane preparation. The cyclization products hop-22-ene and hopan-22-ol (diplopterol) were identified by GC and GC-MS.