The structures of the modified folates present in Pyrococcus furiosus have been determined. This was accomplished largely by the characterization of the arylamines resulting from the air oxidative cleavage of the reduced modified folates present in these cells, using both chemical and enzymatic methods. Cell extracts separated on DEAE-Sephadex columns showed one major peak containing the arylamines derived from the modified folates. These arylamines were not retained on the DEAE-Sephadex columns, indicating that they contained no net negative charge. Purification of the azo dye derivatives of these arylamines on a Bio-Gel P-6 column showed the presence of three different compounds (compounds 1, 2, and 3) in an average amount of 4.1, 7.6, and 22 nmol/g dry weight of cells, respectively. Each of these compounds readily underwent mild acid hydrolysis (0.1 M HCl, 110-degrees-C, 1 min) to produce the azo dye derivative of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane(pAPT). The structure and stereochemistry (ribo) of the pAPT was the same as the PAPT present in methanopterin. In addition, compounds 1, 2, and 3 were each shown to contain 1 mol equiv of ribose and 1, 2, and 3 mol equiv of N-acetylglucosamine (gluNAc), respectively, and were designated as the azo dye derivatives of pAPT-ribose-gluNAc, pAPT-ribose-(gluNAc)2, and pAPT-ribose-(gluNAc)3. Each of these compounds was readily cleaved to the azo dye derivative of pAPT-ribose by the enzymatic action of beta-N-acetylglucosaminidase, indicating that all the gluNAc residues were beta-linked. The linkage between the ribose and the pAPT in the pAPT-ribose was assigned as alpha(1-->5) because of its identity with pAPT-ribose isolated from methaniline (1-(4-aminophenyl)-1-deoxy-5-O-[5-O-[(1,3-dicarboxypropoxy)hydroxyphosphinyl]-alpha-D-ribofuranosyl]ribitol) after enzymatic cleavage with the phosphodiesterase I from Crotalus atrox venom. pAPT-ribose-(gluNAc)3 and pAPT-ribose-(gluNAc)2 were found to be readily cleaved by chitinase to pAPT-ribose-gluNAc and pAPT-ribose, whereas pAPT-ribose-gluNAc was not cleaved by chitinase. These observations indicated that all linkages between gluNAc residues were beta(1-->4) and that the oxidized modified folates present in P. furiosus consist of three compounds each containing a core structure of 1-[4-[[1-(2-amino-7-methyl-4-oxo-6-pteridinyl)-ethyl]amino]phenyl]-1-deoxy-[1-alpha-D-ribofuranosyl]ribitol linked to 1, 2, and 3 gluNAc. In each of these compounds the first gluNAc of the chain is beta-linked to the 5-position of the ribose. Thus, the modified folates in P. furiosus are in the same family of modified folates as is methanopterin. For a coenzyme, the structures of these modified folates are very useful in that they contain no charged groups.
