Glycerol Phosphate Cytidylyltransferase Stereospecificity Is Key to Understanding the Distinct Stereochemical Compositions of Glycerophosphoinositol in Bacteria and Archaea

Glycerophosphoinositol (GPI) is a compatible solute present in a few hyperthermophiles. Interestingly, different GPI stereoisomers accumulate inBacteria andArchaea , and the basis for this domain-dependent specificity was investigated herein. The archaeonArchaeoglobus fulgidus and the bacteriumAquifex aeolicus were used as model organisms. The synthesis of GPI involves glycerol phosphate cytidylyltransferase (GCT), which catalyzes the production of CDP-glycerol from CTP and glycerol phosphate, and di-myo -inositol phosphate-phosphate synthase (DIPPS), catalyzing the formation of phosphorylated GPI from CDP-glycerol andl -myo -inositol 1-phosphate. DIPPS ofA. fulgidus recognized the two CDP-glycerol stereoisomers similarly. This feature and the ability of31 P nuclear magnetic resonance (NMR) to distinguish the GPI diastereomers provided a means to study the stereospecificity of GCTs. The AF1418 gene and genes aq_185 and aq_1368 are annotated as putative GCT genes in the genomes ofA. fulgidus andAq. aeolicus , respectively. The functions of these genes were determined by assaying the activity of the respective recombinant proteins: AQ1368 and AQ185 are GCTs, while AF1418 has flavin adenine dinucleotide (FAD) synthetase activity. AQ185 is absolutely specific forsn -glycerol 3-phosphate, while AQ1368 recognizes the two enantiomers but has a 2:1 preference forsn -glycerol 3-phosphate. In contrast, the partially purifiedA. fulgidus GCT usessn -glycerol 1-phosphate preferentially (4:1). Significantly, the predominant GPI stereoforms found in the bacterium and the archaeon reflect the distinct stereospecificities of the respective GCTs: i.e.,A. fulgidu s accumulates predominantlysn -glycero-1-phospho-3-l -myo -inositol, whileAq. aeolicus accumulatessn -glycero-3-phospho-3-l-myo -inositol.IMPORTANCE Compatible solutes of hyperthermophiles show high efficacy in thermal protection of proteins in comparison with solutes typical of mesophiles; therefore, they are potentially useful in several biotechnological applications. Glycerophosphoinositol (GPI) is synthesized from CDP-glycerol andl -myo -inositol 1-phosphate in a few hyperthermophiles. In this study, the molecular configuration of the GPI stereoisomers accumulated by members of theBacteria andArchaea was established. The stereospecificity of glycerol phosphate cytidylyltransferase (GCT), the enzyme catalyzing the synthesis of CDP-glycerol, is crucial to the stereochemistry of GPI. However, the stereospecific properties of GCTs have not been investigated thus far. We devised a method to characterize GCT stereospecificity which does not requiresn -glycerol 1-phosphate, a commercially unavailable substrate. This led us to understand the biochemical basis for the distinct GPI stereoisomer composition observed in archaea and bacteria.