Site‐Directed Mutagenesis of Putative Active‐Site Residues in Squalene‐Hopene Cyclase

Abstract

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...

Functional Analysis of the Two Interacting Cyclase Domains in ent-Kaurene Synthase from the FungusPhaeosphaeria sp. L487 and a Comparison with Cyclases from Higher Plants

“…It is believed that the second aspartate residue acts as the proton donor. This hypothesis is supported by loss of activity in SHC mutants in which the aspartate-rich motif was exchanged by site-directed mutagenesis (19). In contrast to CPS, cyclization of KS, type A cyclization, is initiated by elimination of the diphosphate group of CDP and is accompanied by rearrangement of the carbon skeleton (Fig.…”

Squalene-Hopene Cyclases

“…The carboxyl groups of Asp374 and Asp377 accommodate the positive charge of the D376-H451 pair prior to proton transfer. After proton transfer to the 2,3-double bond of squalene, the D376-H451 pair loses its charge, leaving the remaining negative charge on the D374-D377 pair for stabilization of the initial cationic intermediates (24,101). Reprotonation of D376 occurs through a water molecule bound to Y495-OH, which can transfer protons from disordered water in the solvent-accessible upper cavity of SHC (100).…”

“…D374, D376, and D377 represent this motif in A. acidocaldarius. D376 and D377 are essential for SHC activity (24), whereas mutation of Asp374 only lowers polycyclization activity (83). The polar hydrogen bonding network around E45, consisting of Q262, E45, E93, and R127, and a water molecule at the bottom of the active-site cavity are involved in the final deprotonation step (101).…”

Distribution of microbial terpenoid lipid cyclases in the global ocean metagenome

“…SHCs can be identified readily based on their numerous conserved amino acids (AAs; Hoshino and Sato, 2002;Summons et al, 2006;Fischer and Pearson, 2007). Specific functional motifs (Feil et al, 1996;Wendt et al, 2000) and analysis of secondary structures (Wendt et al, 1997;SchulzGasch and Stahl, 2003) show that the two classes can be distinguished by sequence differences alone. There are no abiotic processes or other enzymes known to perform the functions of SHCs (Ourisson et al, 1987;Wendt et al, 2000).…”