Purification and some properties of the squalene-tetrahymanol cyclase from Tetrahymena thermophila

Saar, Jörg; Kader, Jean‐Claude; Poralla, Karl; Ourisson, Guy

doi:10.1016/0304-4165(91)90080-z

Cited by 22 publications

(18 citation statements)

References 42 publications

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“…Direct plotting of the initial velocity versus the substrate concentration and double-reciprocal plots according to the method of Lineweaver-Burk [18] were used to determine the K,,, and V,,,, values ( Table 3). The K, value of 17 pM for the recombinant His,-SHC is in agreement with the values determined for purified Tetruhymenu thermophilus squalene-tetrahymanol cyclase [20] and Zyrnornonas mohilis SHC [21] (18 pM and 12 pM, respectively); it is about 5.5-fold higher than the previous described K, of 3 pM of the partially purified wildtype SHC from Alicyclobacillus acidocaldarius [22]. This may be due to the stabilizing effects of residual cellular proteins in this preparation.…”

Section: Structural Comparison Of Wild-type and Mutant Enzymessupporting

confidence: 86%

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

Feil

Süßmuth

Jung

et al. 1996

European Journal of Biochemistry

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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...

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Section: Structural Comparison Of Wild-type and Mutant Enzymessupporting

confidence: 86%

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

Feil

Süßmuth

Jung

et al. 1996

European Journal of Biochemistry

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“…This enzyme forms, from squalene yet another cyclic compound, the gammaceran derivative tetrahymanol (Fig. 1; which biogenetically is a hopanoid isomer and can be considered to be a quasi‐hopanoid) and smaller amounts of the hopanoid diplopterol (Bouvier et al ., 1980; Ourisson et al ., 1987; Saar et al ., 1991). It has been shown that the biosynthesis of tetrahymanol proceeds via a non‐oxidative proton‐initiated, enzyme‐catalysed cyclization of the squalene in an all‐chair confirmation (Zander et al ., 1970; Aberhart and Caspi, 1979).…”

Section: Discussionmentioning

confidence: 99%

Phylogenetic analysis of the triterpene cyclase protein family in prokaryotes and eukaryotes suggests bidirectional lateral gene transfer

Frickey

Kannenberg

2009

Environmental Microbiology

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Functional constraints to modifications in triterpene cyclase amino acid sequences make them good candidates for evolutionary studies on the phylogenetic relatedness of these enzymes in prokaryotes as well as in eukaryotes. In this study, we used a set of identified triterpene cyclases, a group of mainly bacterial squalene cyclases and a group of predominantly eukaryotic oxidosqualene cyclases, as seed sequences to identify 5288 putative triterpene cyclase homologues in publicly available databases. The Cluster Analysis of Sequences software was used to detect groups of sequences with increased pairwise sequence similarity. The sequences fall into two main clusters, a bacterial and a eukaryotic. The conserved, informative regions of a multiple sequence alignment of the family were used to construct a neighbour-joining phylogenetic tree using the AsaturA and maximum likelihood phylogenetic tree using the PhyML software. Both analyses showed that most of the triterpene cyclase sequences were similarly grouped to the accepted taxonomic relationships of the organism the sequences originated from, supporting the idea of vertical transfer of cyclase genes from parent to offspring as the main evolutionary driving force in this protein family. However, a small group of sequences from three bacterial species (Stigmatella, Gemmata and Methylococcus) grouped with an otherwise purely eukaryotic cluster of oxidosqualene cyclases, while a small group of sequences from seven fungal species and a sequence from the fern Adiantum grouped consistently with a cluster of otherwise purely bacterial squalene cyclases. This suggests that lateral gene transfer may have taken place, entailing a transfer of oxidosqualene cyclases from eukaryotes to bacteria and a transfer of squalene cyclase from bacteria to an ancestor of the group of Pezizomycotina fungi.

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“…Further, the biochemical mechanism of tetrahymanol synthesis in bacteria is unclear. In ciliates, squalene-tetrahymanol cyclase (Stc) catalyzes the cyclization of squalene directly to tetrahymanol (24), but neither of the two known bacterial tetrahymanol producers harbor a copy of Stc (10,24). R. palustris and B. japonicum do possess an evolutionarily related cyclase, squalene-hopene cyclase (Shc), whose main function is the cyclization of squalene to the hopanoid diploptene (25).…”

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A distinct pathway for tetrahymanol synthesis in bacteria

Banta

Wei

Welander

2015

Proc. Natl. Acad. Sci. U.S.A.

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Tetrahymanol is a polycyclic triterpenoid lipid first discovered in the ciliate Tetrahymena pyriformis whose potential diagenetic product, gammacerane, is often used as a biomarker for water column stratification in ancient ecosystems. Bacteria are also a potential source of tetrahymanol, but neither the distribution of this lipid in extant bacteria nor the significance of bacterial tetrahymanol synthesis for interpreting gammacerane biosignatures is known. Here we couple comparative genomics with genetic and lipid analyses to link a protein of unknown function to tetrahymanol synthesis in bacteria. This tetrahymanol synthase (Ths) is found in a variety of bacterial genomes, including aerobic methanotrophs, nitrite-oxidizers, and sulfate-reducers, and in a subset of aquatic and terrestrial metagenomes. Thus, the potential to produce tetrahymanol is more widespread in the bacterial domain than previously thought. However, Ths is not encoded in any eukaryotic genomes, nor is it homologous to eukaryotic squalene-tetrahymanol cyclase, which catalyzes the cyclization of squalene directly to tetrahymanol. Rather, heterologous expression studies suggest that bacteria couple the cyclization of squalene to a hopene molecule by squalene-hopene cyclase with a subsequent Ths-dependent ring expansion to form tetrahymanol. Thus, bacteria and eukaryotes have evolved distinct biochemical mechanisms for producing tetrahymanol.gammacerane | tetrahymanol | biomarkers | methanotrophs | sterols

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Purification and some properties of the squalene-tetrahymanol cyclase from Tetrahymena thermophila

Cited by 22 publications

References 42 publications

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

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

Phylogenetic analysis of the triterpene cyclase protein family in prokaryotes and eukaryotes suggests bidirectional lateral gene transfer

A distinct pathway for tetrahymanol synthesis in bacteria

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