Prenyl transferases from Micrococcus luteus: Characterization of undecaprenyl pyrophosphate synthetase

Three prenyl transferases in Micrococcus luteus were recovered in the soluble fraction following cell disruption. Undecaprenyl pyrophosphate (C₅₅-PP) synthetase chromatographed on DEAE-cellulose independently from geranylgeranyl-PP and octaprenyl-PP synthetases. Further purification of C₅₅-PP synthetase resulted in an approximate 250-fold purification over the crude lysate. The molecular weight of the synthetase was estimated to be between 47,000 and 49,000 by Sephadex G-100 chromatography. The enzyme had a broad specificity toward the allylic pyrophosphate substrate. The reactivities of the allylic substrates increased with chain length, C₁₀ < C₁₅ < C₂₀, except for trans-solanesyl-PP, which was unreactive. Moreover, the enzyme was active on allylic substrates having both cis- and trans-stereochemistry. Although C₅₅-PP and C₅₀-PP were the major products, some shorter chain products were also produced, when t,t-farnesyl pyrophosphate and Δ³sopentenyl pyrophosphate (IPP) were used as substrates. The stereochemistries of the products formed with C₅₅-PP synthetase were established, using ¹⁴C]IPP and 2R-2-³H] and 2S-2-³H]IPP. Each new isoprene unit added had a cis-configuration. The enzyme was inactive in the absence of added effectors. It was stimulated by Triton X-100, egg lecithin, and a whole phospholipid extract from M. luteus. Cardiolipin and deoxycholate were poor activators of the enzyme. The product chain length distribution observed with the phospholipid-activated enzyme showed highly favored production of the C₅₅-PP product over the C₅₀-PP product.