Unexpected Reactivity of 2‑Fluorolinalyl Diphosphate
in the Active Site of Crystalline 2‑Methylisoborneol Synthase
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Abstract
The
crystal structure of 2-methylisoborneol synthase (MIBS) from <i>Streptomyces coelicolor</i> A3(2) has been determined in its
unliganded state and in complex with two Mg<sup>2+</sup> ions and
2-fluoroneryl diphosphate at 1.85 and 2.00 Å resolution, respectively.
Under normal circumstances, MIBS catalyzes the cyclization of the
naturally occurring, noncanonical 11-carbon isoprenoid substrate,
2-methylgeranyl diphosphate, which first undergoes an ionization–isomerization–ionization
sequence through the tertiary diphosphate intermediate 2-methyllinalyl
diphosphate to enable subsequent cyclization chemistry. MIBS does
not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and
we recently reported the crystal structure of MIBS complexed with
this unreactive substrate analogue [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D.
W. (2012) Biochemistry 51, 3011–3020]. However, cocrystallization of MIBS with the fluorinated analogue
of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl
diphosphate, reveals unexpected reactivity for the intermediate analogue
and yields the crystal structure of the complex with the primary allylic
diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure
of the unliganded enzyme reveals that the crystalline enzyme active
site remains partially open, presumably due to the binding of only
two Mg<sup>2+</sup> ions. Assays in solution indicate that MIBS catalyzes
the generation of (1<i>R</i>)-(+)-camphor from the substrate
2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl
diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization
mechanism leading to 2-substituted isoborneol products; however, the
initially generated 2-fluoroisoborneol cyclization product is unstable
and undergoes elimination of hydrogen fluoride to yield (1<i>R</i>)-(+)-camphor