When incubated with their substrates, human phosphomannomutase and L-3-phosphoserine phosphatase are known to form phosphoenzymes with chemical characteristics of an acyl-phosphate. The phosphorylated residue in phosphomannomutase has now been identified by mass spectrometry after reduction of the phosphoenzyme with tritiated borohydride and trypsin digestion. It is the first aspartate in a conserved DVDGT motif. Replacement of either aspartate of this motif by asparagine or glutamate resulted in complete inactivation of the enzyme. The same mutations performed in the DXDST motif of L-3-phosphoserine phosphatase also resulted in complete inactivation of the enzyme, except for the replacement of the second aspartate by glutamate, which reduced the activity by only about 40%. This suggests that the first aspartate of the motif is also the phosphorylated residue in L-3-phosphoserine phosphatase. Data banks contained seven other phosphomutases or phosphatases sharing a similar, totally conserved DXDX(T/V) motif at their amino terminus. One of these (beta-phosphoglucomutase) is shown to form a phosphoenzyme with the characteristics of an acyl-phosphate. In conclusion, phosphomannomutase and L-3-phosphoserine phosphatase belong to a new phosphotransferase family with an amino-terminal DXDX(T/V) motif that serves as an intermediate phosphoryl acceptor.

Escherichia coli was found to grow on fructoselysine as an energetic substrate at a rate of about one-third of that observed with glucose. Extracts of cells grown on fructoselysine catalyzed in the presence of ATP the phosphorylation of fructoselysine and a delayed formation of glucose 6-phosphate from this substrate. Data base searches allowed us to identify an operon containing a putative kinase (YhfQ) belonging to the PfkB/ ribokinase family, a putative deglycase (YhfN), homologous to the isomerase domain of glucosamine-6-phosphate synthase, and a putative cationic amino acid transporter (YhfM). The proteins encoded by YhfQ and YhfN were overexpressed in E. coli, purified, and shown to catalyze the ATP-dependent phosphorylation of fructoselysine to a product identified as fructoselysine 6-phosphate by 31 P NMR (YhfQ), and the reversible conversion of fructoselysine 6-phosphate and water to lysine and glucose 6-phosphate (YhfN). The K m of the kinase for fructoselysine amounted to 18 M, and the K m of the deglycase for fructoselysine 6-phosphate, to 0.4 mM. A value of 0.15 M was found for the equilibrium constant of the deglycase reaction. The kinase and the deglycase were both induced when E. coli was grown on fructoselysine and then reached activities sufficient to account for the rate of fructoselysine utilization.Fructosamines are the products of a non-enzymatic reaction of glucose with primary amines followed by an Amadori rearrangement. These reactions, known as glycation (to be distinguished from glycosylation, which is enzymatically catalyzed), typically modify the amino terminus and the lysine side-chains of proteins (reviewed in Refs.

Fructosamines are thought to play an important role in the development of diabetic complications. Little is known about reactions that could metabolize these compounds in mammalian tissues, except for recent indications that they can be converted to fructosamine 3-phosphates. The purpose of the present work was to identify and characterize the enzyme responsible for this conversion. Erythrocyte extracts were found to catalyze the ATP-dependent phosphorylation of 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine. The enzyme responsible for this conversion was purified approximately 2,500-fold by chromatography on Blue Sepharose, Q Sepharose, and Sephacryl S-200 and shown to copurify with a 35,000-M(r) protein. Partial sequences of tryptic peptides were derived from the protein by nanoelectrospray-ionization mass spectrometry, which allowed for the identification of the corresponding human and mouse cDNAs. Both cDNAs encode proteins of 309 amino acids, showing 89% identity with each other and homologous to proteins of unknown function predicted from the sequences of several bacterial genomes. Both proteins were expressed in Escherichia coli and purified. They were shown to catalyze the phosphorylation of DMF, fructoselysine, fructoseglycine, and fructose in order of decreasing affinity. They also phosphorylated glycated lysozyme, though not unmodified lysozyme. Nuclear magnetic resonance analysis of phosphorylated DMF and phosphorylated fructoseglycine showed that the phosphate was bound to the third carbon of the 1-deoxyfructose moiety. The physiological function of fructosamine-3-kinase may be to initiate a process leading to the deglycation of fructoselysine and of glycated proteins.

Fructosamine 3-kinase, which phosphorylates low-molecular-mass and protein-bound fructosamines on the third carbon of their deoxyfructose moiety, is quite active in erythrocytes, and was proposed to initiate a process removing fructosamine residues from proteins. In the present study, we show that incubation of human erythrocytes with 200 mM glucose not only caused the progressive formation of glycated haemoglobin, but also increased the level of an anionic form of haemoglobin containing alkali-labile phosphate, to approx. 5% of total haemoglobin. 1-Deoxy-1-morpholinofructose (DMF), a substrate and competitive inhibitor of fructosamine 3-kinase, doubled the rate of accumulation of glycated haemoglobin, but markedly decreased the amount of haemoglobin containing alkali-labile phosphate. The latter corresponds therefore to haemoglobin bound to a fructosamine 3-phosphate group (FN3P-Hb). Returning erythrocytes incubated with 200 mM glucose and DMF to a low-glucose medium devoid of DMF caused a decrease in the amount of glycated haemoglobin, a transient increase in FN3P-Hb and a net decrease in the sum (glycated haemoglobin+FN3P-Hb). These effects were prevented by DMF, indicating that fructosamine 3-kinase is involved in the removal of fructosamine residues. The second step of this 'deglycation' process is most likely a spontaneous decomposition of the fructosamine 3-phosphate residues to a free amine, 3-deoxyglucosone and P(i). This is consistent with the findings that 2-oxo-3-deoxygluconate, the product of 3-deoxyglucosone oxidation, is formed in erythrocytes incubated for 2 days with 200 mM glucose in a sufficient amount to account for the removal of fructosamine residues from proteins, and that DMF appears to inhibit the formation of 2-oxo-3-deoxygluconate from elevated glucose concentrations.

Fructosamine-3-kinase (FN3K) is an enzyme that appears to be responsible for the removal of fructosamines from proteins. In this study, we report the sequence of human and mouse cDNAs encoding proteins sharing 65% sequence identity with FN3K. The genes encoding FN3K and FN3K-related protein (FN3K-RP) are present next to each other on human chromosome 17q25, and they both have a similar 6-exon structure. Northern blots of mouse tissues RNAs indicate a high level of expression of both genes in bone marrow, brain, kidneys, and spleen. Human FN3K-RP was transfected in human embryonic kidney (HEK) cells, and the expressed protein was partially purified by chromatography on Blue Sepharose. Unlike FN3K, FN3K-RP did not phosphorylate fructoselysine, 1-deoxy-1-morpholino-fructose, or lysozyme glycated with glucose. In a more systematic screening for potential substrates for FN3K-RP, we found, however, that both enzymes phosphorylated ketosamines with a D-configuration in C3 (psicoselysine, 1-deoxy-1-morpholino-psicose, 1-deoxy-1-morpholino-ribulose, lysozyme glycated with allose-the C3 epimer of glucose, or with ribose). Tandem mass spectrometry and nuclear magnetic resonance analysis of the product of phosphorylation of 1-deoxy-1-morpholino-psicose by FN3K-RP indicated that this enzyme phosphorylates the third carbon of the sugar moiety. These results indicate that FN3K-RP is a ketosamine-3-kinase (ketosamine-3-kinase 2). This enzyme presumably plays a role in freeing proteins from ribulosamines or psicosamines, which might arise in a several step process, from the reaction of amines with glucose and/or glycolytic intermediates. This role is shared by fructosamine-3-kinase (ketosamine-3-kinase 1), which has, in addition, the unique capacity to phosphorylate fructosamines.