Methodology for the Preparation of Pure Recombinant S. cerevisiae Lanosterol Synthase Using a Baculovirus Expression System. Evidence That Oxirane Cleavage and A-Ring Formation Are Concerted in the Biosynthesis of Lanosterol from 2,3-Oxidosqualene

Abstract

Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme from Saccharomyces cerevisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis, was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps. Using pure enzyme the kinetics of cyclization were determined using Michaelis−Menten analysis for 2,3-oxidosqualene (1) and two analogs in which the C−6 methyl was replaced by H (3) or Cl (4). The measured V max/K M ratios for 1, 3, and 4 were found to be 138, 9.4, and 21.9, respectively, a clear indication that oxirane cleavage and cyclization to form the A-ring are concerted, since the nucleophilicity of the proximate double bond influences the rate of oxirane cleavage. No catalytic metal ions could be detected in purified lanosterol synthase by atomic absorption analysis. Site-directed mutagenesis studies of each of the six strongly conserved aspartic acid residues (D → N mutation) and each of the nine conserved glutamic acid residues (E → Q) revealed that only one, D456, is essential for catalytic function of the enzyme. The essential D456 residue is a likely candidate for electrophilic (specifically protic) activation of the oxirane function.

Enzyme Mechanisms for Triterpene Cyclization: New Pieces of the Puzzle

“…[9,11] As predicted for this structure, Asp 455 OSC is not polarized by a positively charged histidine, and consequently the countercharged Asp 377 SHC :Asp 374 SHC pair is missing as well. [3,5,9] Surprisingly, Asp 455 OSC is hydrogen-bonded to two cysteine residues, and it directs its less acidic syn proton toward the active-site cavity.…”

“…OSC is the only basic residue in proximity to the termination site, [3,11] and they demonstrate that His-232 OSC forms a hydrogen bond with the Tyr 503 OSC hydroxy group, which is in a better position to accept the proton from C9 of the lanosterol cation than His 232 OCS itself (Figure 1). [9] The assignment of His 232 OSC :Tyr 503 OSC as the catalytic base dyad of OSC is supported by additional data: 1) The essential character of the corresponding His 234 of the S. cerevisiae OSC [11] and 2) the finding that the Tyr 510Ala mutant of S. cerevisiae forms parkeol (23), [22] a product that corresponds to that of the nonenzymatic rearrangement of 21 to 22.…”

β‐Amyrin synthase

“…Since all attempts to express OSC in E. coli have been unsuccessful in the past [13], we tried to express them in the yeast mutant GIL77 [34], an ergosterol auxotroph lacking lanosterol synthase activity, which accumulates oxidosqualene inside the cell. As this mutant eliminates the background production of lanosterol, we sought to detect cyclization products in a yeast transformed with either PNXa or PNYa clones.…”

Functiona l Analysis of Eubacterial Diterpene Cyclases Responsible for Biosynthesis of a Diterpene Antibiotic, Terpentecin

“…The cyclization mechanisms of their products were extensively studied by using recombinant enzymes. Triterpene cyclases from either eukaryotes (3)(4)(5)(6)(7)(8)(9)(10) or prokaryotes (11)(12)(13)(14)(15)(16)(17) are representatives. As for diterpene cyclases, reaction mechanisms of enzymes from eukaryotes such as abietadiene synthase (18 -23), (Ϫ)-copalyl diphosphate synthase (24), and ent-kaurene synthase (25) have been recently and extensively studied.…”