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.
Tippelt
A
A
University of Tübingen, Mikrobiologie/Biotechnologie, Auf der Morgenstelle 28, D-72076 Tübingen, Germany.
Jahnke
L
L
Poralla
K
K
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D019751:Intramolecular Transferases / Q000235:genetics* / Q000378:metabolism*; D008772:Methylococcaceae / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013256:Steroids / Q000096:biosynthesis; D014315:Triterpenes / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D013256:Steroids; D014315:Triterpenes; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase; C021924:lanosterol synthase