Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di- myo -inositol-phosphate

Abstract

Di-myo-inositol 1,1 -phosphate (DIP) is a major osmoprotecting metabolite in a number of hyperthermophilic species of archaea and bacteria. Although the DIP biosynthesis pathway was previously proposed, genes encoding only two of the four required enzymes, inositol-1-phosphate synthase and inositol monophosphatase, were identified. In this study we used a comparative genomic analysis to predict two additional genes of this pathway (termed dipA and dipB) that remained missing. In Thermotoga maritima both candidate genes (in an originally misannotated locus TM1418) form an operon with the inositol-1-phosphate synthase encoding gene (TM1419). A predicted inositol-monophosphate cytidylyltransferase activity was directly confirmed for the purified product of T. maritima gene dipA cloned and expressed in Escherichia coli. The entire DIP pathway was reconstituted in E. coli by cloning of the TM1418 -TM1419 operon in pBAD expression vector and confirmed to function in the crude lysate. 31 P NMR and MS analysis revealed that DIP synthesis proceeds via a phosphorylated DIP intermediate, P-DIP, which is generated by the dipBencoded enzyme, now termed P-DIP synthase. This previously unknown intermediate is apparently converted to the final product, DIP, by an inositol monophosphatase-like phosphatase. These findings allowed us to revise the previously proposed DIP pathway. The genomic survey confirmed its presence in the species known to use DIP for osmoprotection. Among several newly identified species with a postulated DIP pathway, Aeropyrum pernix was directly proven to produce this osmolyte.comparative genomics ͉ di-myo-inositol-1,3-phosphate biosynthesis ͉ Thermotoga maritima T he most common mechanism of osmoadaptation in microorganisms involves the accumulation of specific organic osmolytes, so-called compatible solutes, amino acids, sugars, and polyols, that can be taken up from the environment or synthesized de novo (1-3). In thermophiles and hyperthermophiles, compatible solutes are generally different from those found in mesophiles (4), and many of them additionally contribute to thermoprotection.Di-myo-inositol 1,1Ј-phosphate (DIP) is one of the major compatible solutes in a number of thermophilic archaea of the genera Pyrococcus, Methanococcus, Thermococcus, and Archaeoglobus and bacteria belonging to the genera Aquifex, Rubrobacter, and Thermotoga, including Thermotoga maritima and Thermotoga neapolitana (4-7). DIP levels are highly increased at supraoptimal growth temperatures in both Methanococcus igneus and Thermotogales, suggesting that this solute also plays a thermoprotective role (6,8).Based on the study in M. igneus (9) and the reported stereochemistry of DIP (10), the pathway of DIP biosynthesis was originally proposed to occur in four steps: (i) synthesis of L-myoinositol-1-phosphate (L-I-1-P) from glucose-6-phosphate by NAD ϩ -dependent L-I-1-P synthase [inositol-1-phosphate synthase (IPS)]; (ii) hydrolysis of some of the L-I-1-P by inositol monophosphatase (IMP); (iii) coupling of the L-I-1-P with ...

Crystal Structure of Archaeoglobus fulgidus CTP:Inositol-1-Phosphate Cytidylyltransferase, a Key Enzyme for Di- myo -Inositol-Phosphate Synthesis in (Hyper)Thermophiles

“…A typical Michaelis-Menten kinetics was observed for the two substrates. The V max (62.9 mol ⅐ min Ϫ1 ⅐ mg Ϫ1 ) was 2.5-fold lower than that of the T. maritima IPCT (29). With respect to substrate affinity, the truncated A. fulgidus IPCT showed K m s of 0.87 mM and 0.58 mM for inositol-1P and CTP, respectively, which are in the same range of magnitude as those of the homofunctional enzyme from T. maritima (29).…”

“…In most organisms known to accumulate DIP, the two activities are present in a single polypeptide chain, constituting the bifunctional enzyme IPCT/DIPPS; however, in Thermotoga maritima, the two activities are encoded by separate, consecutive genes and the same gene organization was predicted in other members of hyperthermophilic Bacteria and in a few Archaea (29).…”

Mobile loop mutations in an archaeal inositol monophosphatase: Modulating three‐metal ion assisted catalysis and lithium inhibition

“…1,2 However, in some hyperthermophilic microorganisms, this enzyme is critical for the synthesis of di-myo-inositol-1,1 0 -phosphate, a solute accumulated in response to salt and temperature stress. [3][4][5][6] The enzymes from hyperthermophiles have also been shown to catalyze the specific cleavage of the C-1 phosphate of fructose-1,6-bisphosphate (FBP) as well as the NADP(H) phosphate. [7][8][9] These IMPase enzymes, part of a larger IMPase superfamily, 10 are usually symmetric homodimers (although the enzyme from T. maritima exists as a tetramer 11 ) with each subunit organized in a five-layered ababa sandwich.…”

Thermococcus kodakar ensis Mutants Deficient in Di- myo -Inositol Phosphate Use Aspartate To Cope with Heat Stress

“…T. kodakarensis is the only marine hyperthermophile for which a number of genetic tools have been developed, including Escherichia coli-T. kodakarensis shuttle vectors and a reliable gene disruption system (19,29,32,34). The genome of T. kodakarensis possesses a gene encoding CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase (IPCT/DIPPS), a key enzyme in DIP synthesis (2,25,26). This enzyme catalyzes the synthesis of CDP-inositol from CTP and inositol-1-phosphate as well as the transfer of the inositol group from CDP-inositol to a second molecule of inositol-1-phosphate to yield a phosphorylated form of DIP (2).…”