Squalene-hopene cyclase from Bradyrhizobium japonicum: cloning, expression, sequence analysis and comparison to other triterpenoid cyclases

Abstract

With the help of a PCR-based screening method, the gene encoding squalene-hopene cyclase (SHC) of Bradyrhizobium japonicum USDA 110 was isolated from a cosmid library. The SHC catalyses the cyclization of squalene to hopanoids, a class of triterpenoid lipids recently discovered in nitrogen-fixing, root-nodule-forming Bradyrhizobium bacteria. Hybridization experiments showed that the gene is present in bacteria of all Bradyrhizobium strains tested and in photosynthetic bacteria forming stem nodules on tropical legumes of the genus Aeschynomene. The Bradyrhizobium shc gene is 1983 bp in length and encodes a protein of 660 amino acid residues with a calculated molecular mass of 73671 Da. Comparison of the deduced amino acid sequence with the sequences of other SHCs revealed highest similarity (70%) to the SHC from the Gram-negative Zymomonas mobilis and lower similarity (48%) to the SHCs from the Gram-positive Alicyclobacillus acidocaldarius and Alicyclobacillus acidoterrestris. Bradyrhizobium SHC also showed similarity (38-43%) to eukaryotic oxidosqualene cyclases. The B. japonicum shc gene was expressed in Escherichia coli. The recombinant SHC catalysed the cyclization of squalene to the hopanoids hopene and diplopterol in vitro. However, the formation of the gammacerane derivative tetrahymanol, which is produced in addition to hopanoids in B. japonicum strains in vivo, could not be detected in vitro. Therefore, the presence of a second squalene cyclase in B. japonicum can be assumed. Sequence analysis of 0.5 kb upstream from the shc gene identified a partial ORF with significant similarity to the C-terminus of an ORF located immediately upstream from the shc gene in Z. mobilis. Both ORFs also showed similarity to phytoene desaturases from cyanobacteria and plants. The 3'-end of this ORF from B. japonicum overlaps with 13 bp at the 5'-end of shc. The close proximity of this ORF to shc suggests that shc and this ORF may be part of an operon.