SITE-DIRECTED MUTAGENESIS OF PUTATIVE ACTIVE-SITE RESIDUES IN SQUALENE-HOPENE CYCLASE

Squalene-hopene cyclase (SHC) catalyzes the complex polycylization of squalene to hopene, similar to the cyclization of oxidosqualene to ste rols. Sequence analysis of SHC revealed a highly conserved aspartate-r ich motif (DDTA), comparable to the DCTA motif of oxidosqualene cyclas es, which is supposed to be part of the active site. In order to deter mine the importance of the motif in squalene cyclization, the conserve d residues Asp376 and Asp377 in the DDTA motif of SHC from Alicyclabac illus acidocaldarius were individually replaced by glutamate, glutamin e, glycine, and arginine. With the exception of the [Glu376]SHC mutant , all other substitutions resulted in almost or complete loss of enzym e activity. Compared to that of the wild-type enzyme, the specific act ivity of the [Glu376]SHC mutant enzyme was reduced to 10%, accompanied by a significant decrease in the apparent V-max, whereas the apparent K-m remained unchanged. CD measurements indicated that mutations did not affect the secondary structure. It is proposed that Asp376 and Asp 377 are crucial for catalysis and may act as point charges to stabiliz e intermediate cations. Moreover, for squalene-hopene cyclase, a high content of alpha-helical conformation could be found, providing the fi rst structural information for a triterpene cyclase.