The temperature dependence of the inositol monophosphatase Km correlates with accumulation of di-myo-inositol 1,1'-phosphate in Archaeoglobus fulgidus.
Di-myo-inositol 1,1'-phosphate (DIP) accumulates as a compatible solute in many hyperthermophilic archaea (e.g., Archaeoglobus fulgidus) when the cells are grown above 80 degrees C. Recent microarray analysis of A. fulgidus transcripts [Rohlin, L., et al. (2005) J. Bacteriol. 187, 6046] indicates that neither the myo-inositol-1-phosphate synthase, the first step in inositol biosynthesis, nor the inositol monophosphatase (IMPase), which generates myo-inositol, are significantly upregulated upon thermal stress. Although other factors could contribute to regulation of DIP synthesis in cells, there is an 8-10-fold decrease in the K(m) of the IMPase for inositol phosphates between 75 and 85 degrees C (for l-I-1-P, the K(m) decreased from 13.2 to 1.67 mM) that correlates with the observed accumulation of DIP in cells. Between 55 and 75 degrees C, K(m) values decreased 2.3-fold at most. The enzyme also exhibits fructose bisphosphatase activity. However, the K(m) for fructose 1,6-bisphosphate was low and the same (0.15 +/- 0.01 mM) at 55 and 70 degrees C. This indicates that the unusual temperature dependence of K(m) is specific for I-1-P substrates. (31)P NMR studies confirmed that the affinity of inositol 1-phosphate for the enzyme was indeed weak (K(D) >or= 5 mM) below but increased significantly at 80 degrees C. In contrast, the IMPase from Methanococcus jannaschii, an organism in which DIP does not accumulate, had a low K(m) for I-1-P 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, the disruption (e.g., A. fulgidus IMPase S171A or T174L) of which prevented the drop in K(m) at high temperatures. We suggest that the temperature-dependent synthesis and accumulation of DIP in A. fulgidus are regulated in part by the temperature dependence of the K(m) of the IMPase activity in the cells.
Wang
Yanling Karen
YK
Boston College, Chestnut Hill, Massachusetts 02467, USA.
Morgan
Adam
A
Stieglitz
Kimberly
K
Stec
Boguslaw
B
Thompson
Benjamin
B
Miller
Scott J
SJ
Roberts
Mary F
MF
D000596:Amino Acids / Q000378:metabolism; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000378:metabolism; D001665:Binding Sites; D004795:Enzyme Stability / Q000235:genetics / Q000502:physiology; D006860:Hydrogen Bonding; D007295:Inositol Phosphates / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology / Q000378:metabolism; D008958:Models, Molecular; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D007295:Inositol Phosphates; C075843:di-myo-inositol-1,1'-phosphate; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase