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

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Abstract
With the help of a PCR-based screening method, the gene encoding squalene-hopene cyclase (SHC) ofBradyrhizobium japonicumUSDA 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-formingBradyrhizobiumbacteria. Hybridization experiments showed that the gene is present in bacteria of allBradyrhizobiumstrains tested and in photosynthetic bacteria forming stem nodules on tropical legumes of the genusAeschynomene.TheBradyrhizobium shcgene 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-negativeZymomonas mobilisand lower similarity (48%) to the SHCs from the Gram-positiveAlicyclobacillus acidocaldariusandAlicyclobacillus acidoterrestris. BradyrhizobiumSHC also showed similarity (38-43%) to eukaryotic oxidosqualene cyclases. TheB. japonicum shcgene was expressed inEscherichia coli.The recombinant SHC catalysed the cyclization of squalene to the hopanoids hopene and diplopterolin vitro.However, the formation of the gammacerane derivative tetrahymanol, which is produced in addition to hopanoids inB. japonicumstrainsin vivo,could not be detectedin vitro.Therefore, the presence of a second squalene cyclase inB. japonicumcan be assumed. Sequence analysis of 0.5 kb upstream from theshcgene identified a partial ORF with significant similarity to the C-terminus of an ORF located immediately upstream from theshcgene inZ. mobilis.Both ORFs also showed similarity to phytoene desaturases from cyanobacteria and plants. The 3'-end of this ORF fromB. japonicumoverlaps with 13 bp at the 5'-end ofshc.The close proximity of this ORF toshcsuggests thatshcand this ORF may be part of an operon.