Cation−π Interaction in the Polyolefin Cyclization Cascade Uncovered by Incorporating Unnatural Amino Acids into the Catalytic Sites of Squalene Cyclase

Abstract

It has been assumed that the pi-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-pi 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-pi 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-pi 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-pi 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 pi-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-pi interaction in triterpene biosynthesis.

Squalene-Hopene Cyclases

“…The exact reaction mechanism of these diterpene cyclases without any more similarities to the complex structure of TTCs still remains unclear. The intricate structure of SHC correlates with its very low turnover rate (1.98 turnovers/s) (50). Taking into account that SHCs first channel the substrate into the catalytic cave, fold the compound correctly, promote five cyclization reactions, and export the bulky product, this is conceivable.…”

“…The authors suggested a barrier-free ring closure of the A to D rings followed by a pause at the 6-6-6-5 tetracyclic tertiary carbocation before concerted ring expansion of ring D and formation of the E ring. In contrast to this, Hoshino and Sato analyzed SHC muteins and isolated not only tricyclic but also bicyclic and monocyclic products, which are postulated to represent intermediates occurring during the formation of the A to C rings (Table 3) (37,50). However, there is some agreement that the 6-6-5 carbocation is transiently formed in the overall squalene cyclization process.…”

“…Even slightly different van der Waals radii of active-site-lining amino acids lead to a modified product pattern. The use of unnatural amino acids avoids this problem: Morikubo and coworkers showed that cation-interactions occupy a key position in the catalytic mechanism by the use of mono-, di-, and trifluorophenylalanines (50). These unnatural amino acids have van der Waals radii similar to those of phenylalanine but have an extremely higher electronegativity, anticipating a slower reaction rate with a normal bell-shaped profile.…”

“…Measurements of the polycyclization activity of muteins harboring corresponding unnatural amino acids corroborated this assumption. The cationinteractions in conjunction with the meandering conformation of the substrate effectively foster the propagation of carbocyclic rings and are crucial for an efficient termination reaction and ring enlargement process in cyclic triterpene biosynthesis (50).…”

Activation-Independent Cyclization of Monoterpenoids

“…Thus, it is reasonable to assume that activation-independent monoterpene cyclases exist or can be evolved from existing enzymes. Consequently, we decided to investigate the diversity of currently known SHCs for their potential to catalyze the cyclization of monoterpenoids, and we started with the SHC from Alicyclobacillus acidocaldarius (formerly Bacillus acidocaldarius) (Shc Aac ), which is the best-studied triterpene cyclase so far: its structure and details of its reaction mechanism have been determined in the last decades (11,14,31,32). Interestingly, Shc Aac is rather unspecific with respect to carbon atom backbone lengths of accepted substrates and can cyclize a variety of alternative substrates, such as, e.g., homofarnesol to ambroxane, an important aroma chemical (11,18; for a summary, see Table 2 in reference 28).…”