1615-91-4Relevant articles and documents
Squalene-Hopene Cyclase: Mechanistic Insights into the Polycyclization Cascades of Squalene Analogs Bearing Ethyl and Hydroxymethyl Groups at the C-2 and C-23 Positions
Kaneko, Ikki,Terasawa, Yuri,Hoshino, Tsutomu
, p. 11139 - 11157 (2018)
Squalene-hopene cyclase (SHC) catalyzes the conversion of squalene 1 into 6,6,6,6,5-fused pentacyclic hopene 2 and hopanol 3. To elucidate the binding sites for the terminal positions of 1, four analogs, having the larger ethyl (Et) and the hydrophilic CH2OH groups at the 23E or 23Z positions of 1, were incubated with SHC. The analog with the Et group at the 23E position (23E-Et-1) yielded two tetra- and three pentacyclic products; however, the analog possessing the Et group at the 23Z position (23Z-Et-1) gave two hopene homologs and the neohopane skeleton, but no hopanol homologs. Hopene homolog (C31) was generated from 23E-Et-1 by deprotonation from 23Z-Me (normal cyclization cascade). Intriguingly, the same homolog was also generated from the geometrical isomer 23Z-Et-1, indicating C?C bond rotation about the C-21?C-22 axis of the hopanyl cation and the more compact nature of the binding domain at 23Z compared with 23E. On the other hand, analogs with the CH2OH group gave novel hopane skeletons having 1-formylethyl and 1-hydroxyprop-2-en-2-yl residues at C-21. Products bearing an aldehyde group were generated in higher yield from 23Z-CH2OH-1 (89 %), than from 23E-CH2OH-1 (26 %). The significant yield (26 %) of the aldehyde products from 23E-CH2OH-1 indicated that C?C bond rotation had occurred owing to the absence of hydrophobic interactions between the hydrophilic 23E-CH2OH and its binding site. The polycyclization mechanisms of the four different analogs are discussed.
Overexpression of Squalene-Hopene Cyclase by the pET Vector in Escherich Coli and First Identification of Tryptophan and Aspartic Acid Residues inside the QW Motif as Active Sites
Sato, Tsutomu,Kanai, Yoshinori,Hoshino, Tsutomu
, p. 407 - 411 (1998)
An overexpression system for squalene-hopene cyclase (SHC) was constructed by using the pET3a vector, which is responsible for high expression with help from the strong T7 promoter when incorporated into E. coli BL21(DE3). Site-directed mutagenesis experiments prove that two amino acid residues of tryptophan and aspartic acid inside the QW-motif 5 resided as active sites.
Cation-π interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase
Morikubo, Noriko,Fukuda, Yoriyuki,Ohtake, Kazumasa,Shinya, Naoko,Kiga, Daisuke,Sakamoto, Kensaku,Asanuma, Miwako,Hirota, Hiroshi,Yokoyama, Shigeyuki,Hoshino, Tsutomu
, p. 13184 - 13194 (2006)
It has been assumed that the π-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-π interaction, natural and unnatural aromatic amino acids were site-specifically incorporated into squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-π binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the protein architecture caused by the introduction of the amino acids more bulky than phenylalanine. Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these amino acids reduce cation-π binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-π binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the π-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-π interaction in triterpene biosynthesis.
Kinetic studies on the function of all the conserved tryptophans involved inside and outside the QW motifs of squalene-hopene cyclase: Stabilizing effect of the protein structure against thermal denaturation
Sato, Tsutomu,Hoshino, Tsutomu
, p. 1171 - 1180 (1999)
Site-directed mutagenesis experiments were carried out to identify the responsibility of the eight QW motifs for the reaction catalyzed by squalene-hopene cyclase (SHC). Alterations of the conserved tryptophans, which are responsible for the stacking structure with glutamine, into aliphatic amino acids gave a significantly lower temperature for the catalytic optimum as for the mutageneses of QW motifs 4, 5a and 5b, which are specifically present in SHCs. However, there was no change in the optimal temperatures of the mutated SHCs targeted at the other five motifs 1, 2, 3, 5c and 6. Thus, reinforcement against heat denaturation can be proposed as a function of the three QW motifs 4, 5a and 5b, but no function could be identified for the QW motifs 1, 2, 3, 5c and 6, although they are commonly found in all the families of prokaryotic SHCs and eukaryotic oxidosqualene cyclases. On the other hand, the three conserved tryptophans of W169, W312 and W489, which are located inside the putative central cavity and outside the QW motifs, were identified as components of the active sites, but also had a function against thermal denaturation. The other two tryptophan residues of W142 and W558, which are located outside the QW motifs, were found not to be active sites, but also had a role for stabilizing the protein structure. It is noteworthy that the mutants replaced by phenylalanine had higher temperatures for the catalytic optimum than those replaced by aliphatic amino acids. The catalytic optimal pH values for all the mutants remained unchanged with an identical value of 6.0.
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Huneck,S.
, p. 3282 - 3283 (1971)
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Radical SAM-Dependent Adenosylation Involved in Bacteriohopanepolyol Biosynthesis?
Zhong, Yuting,Ji, Xinjian,Zhang, Qi
, p. 39 - 42 (2020)
Bacteriohopanepolyols are a group of triterpenoids that play important roles in regulating bacterial cell membrane function. As an intermediate in bacteriohopanepolyol biosynthesis, adenosylhopane production is related to a putative Fe-S protein HpnH, but the exact role of this enzyme remains unsolved. Here we report characterization of HpnH as a novel radical S-adenosylmethionine (SAM) superfamily enzyme. In contrast to almost all the members in the superfamily, HpnH does not initiate the reaction by a hydrogen atom abstraction process. Instead, it catalyzes the adenosylation of hopene via a radical addition reaction to produce adenosylhopane, representing the second example of radical SAM-dependent adenosylation involved in natural product biosynthesis.
Characterization of Radical SAM Adenosylhopane Synthase, HpnH, which Catalyzes the 5′-Deoxyadenosyl Radical Addition to Diploptene in the Biosynthesis of C35 Bacteriohopanepolyols
Sato, Shusuke,Kudo, Fumitaka,Rohmer, Michel,Eguchi, Tadashi
, p. 237 - 241 (2019/11/28)
Adenosylhopane is a crucial intermediate in the biosynthesis of bacteriohopanepolyols, which are widespread prokaryotic membrane lipids. Herein, it is demonstrated that reconstituted HpnH, a putative radical S-adenosyl-l-methionine (SAM) enzyme, commonly encoded in the hopanoid biosynthetic gene cluster, converts diploptene into adenosylhopane in the presence of SAM, flavodoxin, flavodoxin reductase, and NADPH. NMR spectra of the enzymatic reaction product were identical to those of synthetic (22R)-adenosylhopane, indicating that HpnH catalyzes stereoselective C?C formation between C29 of diploptene and C5′ of 5′-deoxyadenosine. Further, the HpnH reaction in D2O-containing buffer revealed that a D atom was incorporated at the C22 position of adenosylhopane. Based on these results, we propose a radical addition reaction mechanism catalyzed by HpnH for the formation of the C35 bacteriohopane skeleton.
