Squalene-hopene cyclase (SHC) catalyzes the complex polycylization of squalene to hopene, similar to the cyclization of oxidosqualene to sterols. Sequence analysis of SHC revealed a highly conserved aspartate-rich motif (DDTA), comparable to the DCTA motif of oxidosqualene cyclases, which is supposed to be part of the active site. In order to determine the importance of the motif in squalene cyclization, the conserved residues Asp376 and Asp377 in the DDTA motif of SHC from Alicyclobacillus acidocaldarius were individually replaced by glutamate, glutamine, glycine, and arginine. With the exception of the [Glu376]SHC mutant, all other substitutions resulted in almost or complete loss of enzyme activity. Compared to that of the wild-type enzyme, the specific activity of the [Glu376]SHC mutant enzyme was reduced to lo%, accompanied by a significant decrease in the apparent V, , , , whereas the apparent K,,, remained unchanged. CD measurements indicated that mutations did not affect the secondary structure. It is proposed that Asp376 and Asp377 are crucial for catalysis and may act as point charges to stabilize intermediate cations. Moreover, for squalene-hopene cyclase, a high content of a-helical conformation could be found, providing the first structural information for a triterpene cyclase.Keywords: squalene-hopene cyclase ; Alicyclobacillus acidocaldarius ; mutagenesis ; active site ; circular dichroism.Hopanoids are pentacyclic triterpenoids. These membrane condensing lipids are widely distributed in the domain Bacteria [l]. Squalene-hopene cyclase (SHC) catalyzes the crucial step in the hopanoid biosynthetic pathway (Scheme 1). This enzyme performs a similar reaction to that of oxidosqualene-lanosterol cyclases from animals and fungi and oxidosqualene-cycloartenol cyclase from plants [2]. Together with the oxidosqualene cyclases, SHC catalyzes the most complex one-step reaction in biochemistry; 12 bonds are altered, 5 cycles formed, and 9 stereocenters established [3]. This complex reaction can be dissected into four basic steps as follow: binding of the particular conformation of the substrate ; initiation by protonation and generation of the first carbocation; propagation of the reaction and stabilization of the intermediate carbocations ; termination of the reaction and final deprotonation. In the case of oxidosqualenelanosterol cyclase, the termination is accompanied by a sequence of 1,2-hydride and methyl shifts prior to proton abstraction. In this context, our aim was to obtain a first hint to the role of certain amino acids of the SHC in cyclization reaction.All known triterpene cyclases are similar in their amino acid sequences. Analysis of the primary structures revealed interesting features. All cyclases contain a non-tandem repeat, the so- called QW motif [4]. This motif is discussed to be involved in carbocation stabilization by cation-71-interactions via aromatic side chains of amino acids [5, 61. Additionally, a conserved aspartate-containing domain was found with the consensus sequences DDTA in...