Incubation of 2-Methylisoborneol Synthase with the Intermediate Analogue 2-Methylneryl Diphosphate

Incubation of synthetic 2-methylneryl diphosphate (2-MeNPP, 10) with 2-methylisoborneol synthase (MIBS) gave a mixture of products that differed significantly from that derived from the natural substrate (E)-2-methylgeranyl disphosphate (3, 2-MeGPP. The proportion of (−)-2-methylisoborneol (1) decreased from 89% to 17% while that of 2-methylenebornane (4) increased from 10% to 26%, with the relative yields of the isomeric homo-monoterpenes 2-methyl-2-bornene (5) and 1-methylcamphene (6) remaining essentially unchanged (<1% each), as determined by chiral gas chromatographic-mass spectrometric (GC-MS) analysis. The majority of the product mixture resulting from the MIBS-catalyzed cyclization of 2-MeNPP (10) consisted of the anomalous monocyclic homo-monoterpenes (±)-2-methylllimonene (15, 39%) and 2-methyl-α-terpineol (13, 10%), as well as the acylic derivatives 2-methylnerol (11, 7%) and 2-methyllinalool (14, <1%). The steady state kinetic parameters of the MIBS-catalyzed reaction, determined using [1-(3)H]-2-MeNPP, were k(cat) 0.0046 ± 0.0003 s(−1), K(m) 18 ± 6 μM, and k(cat)/K(m) 2.55 × 10(2) M(−1)s(−1). By comparison, the natural substrate 2-MeGPP had a k(cat) 0.105 ± 0.007 s(−1), K(m) 95 ± 49 μM, and k(cat)/K(m) 1.11 × 10(3) M(−1)s(−1). Taken together with earlier X-ray crystallographic studies of MIBS, as well as previous investigations of the mechanistically related plant monoterpene cyclase, bornyl diphosphate synthase, these results provide important insights into the binding and cyclization of both native substrates and intermediates and their analogues.