Altered product pattern of a squalene-hopene cyclase by mutagenesis of active site residues

Merkofer, Thorsten; Pale-Grosdemange, Catherine; Wendt, K. Ulrich; Rohmer, Michel; Poralla, Karl

doi:10.1016/s0040-4039(99)00145-8

Cited by 45 publications

(45 citation statements)

References 6 publications

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“…These SHC active-site residues are within 8 Å (measured using the Rasmol program) of the ligand in the 2 Å resolution A. acidocaldarius crystal structure. Mutagenic studies have already shown that ScERG7 residue D456 (7) and AaSHC residues W312 (40), D376 (41), and W489 (42) are catalytically important. A comprehensive list of conserved residues that map to the active site could not be compiled because the enzyme sequences did not align reliably.…”

Section: Resultsmentioning

confidence: 99%

Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA

Godzina

Lovato

Meyer

et al. 2000

Lipids

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Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50-52% identical to known higher plant cycloartenol synthases and 40-49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.

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Section: Resultsmentioning

confidence: 99%

Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA

Godzina

Lovato

Meyer

et al. 2000

Lipids

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“…However, B. subtilis SqhC contains functional motifs responsible for some critical steps of cyclase activity: a domain rich in aspartic acid initiates the protonation of a polyprene precursor, and aromatic residues are present that would stabilize the first two cyclohexyl rings (5) (SI Text). In contrast to most known squalene-hopene cyclases, however, SqhC does not contain residue F601 that would enable formation of the third cyclohexyl ring from a squalene precursor (7,8). Thus, B. subtilis would be expected to produce cyclic polyprenoids, but not hopanoids.…”

Section: Biosynthesis Of Polycyclic Terpenoids By Bacillus Subtilismentioning

confidence: 97%

“…Finally, spores of a strain whose sqhC had been mutated to abolish the hypothesized initiation site of cyclization (7,17) had an equally reduced resistance to H 2 O 2 as the sqhC-lacking mutant TB10 (Fig. S5).…”

Section: Protective Role Of Sporulenesmentioning

confidence: 99%

A polycyclic terpenoid that alleviates oxidative stress

Bosak

Losick

Pearson

2008

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

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Polycyclic terpenoid lipids such as hopanes and steranes have been widely used to understand ancient biology, Earth history, and the oxygenation of the ocean-atmosphere system. Some of these lipids are believed to be produced only by aerobic organisms, whereas others actually require molecular oxygen for their biosynthesis. A persistent question remains: Did some polycyclic lipids initially evolve in response to certain environmental or metabolic stresses, including the presence of oxygen? Here, we identify tetracyclic isoprenoids in spores of the bacterium Bacillus subtilis. We call them sporulenes. They are produced by cyclization of regular polyprenes, a reaction that is more favorable chemically than the formation of terpenoids such as hopanoids and steroids from squalene. The simplicity of the reaction suggests that the B. subtilis cyclase may be analogous to evolutionarily ancient cyclases. We show that these molecules increase the resistance of spores to a reactive oxygen species, demonstrating a specific physiological role for a nonpigment bacterial lipid biomarker. Geostable derivatives of these compounds in sediments could thus be used as direct indicators of oxidative stress and aerobic environments.biomarker ͉ spores ͉ squalene-hopene cyclase ͉ lipid ͉ peroxide resistance P olycyclic terpenoids are a class of organic molecules that includes cholesterol and numerous other related lipids. Their molecular fossils, which are ubiquitous in the sedimentary rock record, are used as records of ancient bacterial and eukaryotic life. For example, the timing of the onset of oxygenic photosynthesis has been reported to be as early as 2.7 billion years ago (1) based on the detection of polycyclic terpenoids presumably derived from Cyanobacteria (2). However, recent studies have questioned this assumption and have urged caution about making taxonomic associations of specific lipid biomarkers with specific microbial groups (3, 4).Here, we take a different approach to this debate by showing definitively that some polycyclic terpenoids reflect a specific physiological function. The association of molecular fossils with physiological functions reduces the dependence of molecular fossils on specific organisms and taxonomic groups. Significantly, we demonstrate in vivo the protective role of a class of polycyclic terpenoids against a reactive oxygen species. Geostable derivatives of these compounds, called sporulenes, could become more definitive geologic markers for aerobic bacteria and environments. Future studies of the specific biological roles of other polycyclic terpenoids may greatly inform the interpretations of the past, including the oxygenation of the Earth. Biosynthesis of Polycyclic Terpenoids by Bacillus subtilisThe best-studied polycyclic terpenoids, tetracyclic sterols and pentacyclic hopanoids, are produced enzymatically from acyclic 30-carbon (C 30 ) precursors by a family of squalene and oxidosqualene cyclases (5, 6). A gene (sqhC) encoding a putative squalene cyclase enzyme (SqhC) is present in a two...

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“…It is often replaced by Arg in other cyclases and believed to function analogously. 58 Hoshino used site-directed mutagenesis to clarify the existence of a secondary support mechanism that provides activation of residues along the front line. For example, substitution of Tyr495 by Phe resulted in complete loss of activity due simply to removal of the hydroxyl group.…”

Section: Site-directed Mutagenesis and Crystallographymentioning

confidence: 99%

A Case Study in Biomimetic Total Synthesis: Polyolefin Carbocyclizations to Terpenes and Steroids

2005

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Altered product pattern of a squalene-hopene cyclase by mutagenesis of active site residues

Cited by 45 publications

References 6 publications

Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA

Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA

A polycyclic terpenoid that alleviates oxidative stress

A Case Study in Biomimetic Total Synthesis: Polyolefin Carbocyclizations to Terpenes and Steroids

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