A Unique β-1,2-Mannosyltransferase of Thermotoga maritima That Uses Di-myo-Inositol Phosphate as the Mannosyl Acceptor▿

In addition to di- myo -inositol-1,3′-phosphate (DIP), a compatible solute widespread in hyperthermophiles, the organic solute pool of Thermotoga maritima comprises 2-( O -β- d- mannosyl)-di- myo -inositol-1,3′-phosphate (MDIP) and 2-( O -β- d- mannosyl-1,2- O -β- d- mannosyl)-di- myo -inositol-1,3′-phosphate (MMDIP), two newly identified β-1,2-mannosides. In cells grown under heat stress, MDIP was the major solute, accounting for 43% of the total pool; MMDIP and DIP accumulated to similar levels, each corresponding to 11.5% of the total pool. The synthesis of MDIP involved the transfer of the mannosyl group from GDP-mannose to DIP in a single-step reaction catalyzed by MDIP synthase. This enzyme used MDIP as an acceptor of a second mannose residue, yielding the di-mannosylated compound. Minor amounts of the tri-mannosylated form were also detected. With a genomic approach, putative genes for MDIP synthase were identified in the genome of T. maritima , and the assignment was confirmed by functional expression in Escherichia coli . Genes with significant sequence identity were found only in the genomes of Thermotoga spp., Aquifex aeolicus , and Archaeoglobus profundus . MDIP synthase of T. maritima had maximal activity at 95°C and apparent K m values of 16 mM and 0.7 mM for DIP and GDP-mannose, respectively. The stereochemistry of MDIP was characterized by isotopic labeling and nuclear magnetic resonance (NMR): DIP selectively labeled with carbon 13 at position C1 of the l- inositol moiety was synthesized and used as a substrate for MDIP synthase. This β-1,2-mannosyltransferase is unrelated to known glycosyltransferases, and within the domain Bacteria , it is restricted to members of the two deepest lineages, i.e., the Thermotogales and the Aquificales . To our knowledge, this is the first β-1,2-mannosyltransferase characterized thus far.