Temperature Dependence of the IMPase K m Correlates with DIP Accumulation in Archaeoglobus fulgidus

Di‐myo‐inositol‐1,1′‐phosphate (DIP) is a compatible solute in many hyperthermophilic archaea (e.g., Archaeoglobus fulgidus ) when the cells are grown above 75°C. The biosynthetic pathway proposed for DIP requires (i) myo ‐inositol‐1‐phosphate synthase (mIPS) to convert D‐glucose‐6‐phosphate to L‐inositol‐1‐phosphate (L‐I‐1‐P), (ii) inositol monophosphatase (IMPase) to generate myo ‐inositol, (iii) CTP:L‐I‐1‐P cytidylyltransferase to form CDP‐inositol, and (iv) DIP synthase to condense CDP‐inositol and myo ‐inositol. What controls this pathway so that DIP is accumulated only above 75°C is unclear. The A. fulgidus mIPS is quite active between 75 and 90°C. However, the temperature dependence of K m for the A. fulgidus IMPase suggests this enzyme contributes to regulating DIP synthesis. With both L‐ and D‐inositol‐1‐phosphates, the IMPase K m varied slightly from 50 to 75°C, then dramatically decreased at 85°C, while k cat followed Arrhenius behavior. 31 P NMR studies confirmed that substrate binding to the enzyme was indeed weak below 80°C. In contrast, the IMPase from Methanococcus jannaschii , an organism that does not accumulate DIP, had a low K m over the entire temperature range. A structural comparison of the two archaeal IMPases identified a hydrogen bonding network present in the active site of the A. fulgidus enzyme and not in the M. jannaschii IMPase, whose disruption (e.g., mutations S171A or T174L) prevented the drop in K m at high temperatures. We suggest that the temperature‐dependent synthesis and accumulation of DIP in A. fulgidus is regulated in part by the temperature dependence of K m of the IMPase activity in the cells. Supported by DOE DE‐FG02‐91ER20025.