Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI2) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the precursors to more than 65,000 natural products in the isoprenoid biosynthetic pathway. Recently, we crystallized the homotetrameric structure of IDI2 (~160 kDa) from Streptococcus pneumoniae (wt-sp-IDI2) to 1.4 �. In conjunction with studies of the catalytic mechanism for IDI2, we created a catalytically active monomeric form of the enzyme (m-sp-IDI2, ~40 kDa) that contained a single point mutation and a 6-residue C-terminal deletion (Figure 1). The oligomerization state of m-sp-IDI2 was confirmed by size-exclusion chromatography and equilibrium sedimentation. UV-Vis spectra of enzyme bound flavin mononucleotide (FMN) cofactor in FMNox, FMNred, and FMNred�IPP states are the same for both m-sp-IDI2 and wt-sp-IDI2, which is consistent with other IDI2 homologs. Although the m-sp-IDI2 has reduced substrate binding affinity and thermal stability (?Tm ~20 oC) relative to the wt enzyme, this variant has better dispersion of 1H/15N crosspeaks in its 2D NMR in preliminary studies with the 15N-labeled protein and is expected to show less NMR peak broadening, enabling the investigation of the catalytic mechanism of IDI2.

Construction of monomeric type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase with catalytic activity

248th National Meeting (2014)

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