Plant ribulosamine/erythrulosamine 3-kinase, a putative protein-repair enzyme

Abstract

FN3K (fructosamine 3-kinase) is a mammalian enzyme that catalyses the phosphorylation of fructosamines, which thereby becomes unstable and detaches from proteins. The homologous mammalian enzyme, FN3K-RP (FN3K-related protein), does not phosphorylate fructosamines but ribulosamines, which are probably formed through a spontaneous reaction of amines with ribose 5-phosphate, an intermediate of the pentose-phosphate pathway and the Calvin cycle. We show in the present study that spinach leaf extracts display a substantial ribulosamine kinase activity (approx. 700 times higher than the specific activity of FN3K in erythrocytes). The ribulosamine kinase was purified approx. 400 times and shown to phosphorylate ribulose-epsilon-lysine, protein-bound ribulosamines and also, with higher affinity, erythrulose-epsilon-lysine and protein-bound erythrulosamines. Evidence is presented for the fact that the third carbon of the sugar portion is phosphorylated by this enzyme and that this leads to the formation of unstable compounds decomposing with half-lives of approx. 30 min at 37 degrees C (ribulosamine 3-phosphates) and 5 min at 30 degrees C (erythrulosamine 3-phosphates). This decomposition results in the formation of a 2-oxo-3-deoxyaldose and inorganic phosphate, with regeneration of the free amino group. The Arabidopsis thaliana homologue of FN3K/FN3K-RP was overexpressed in Escherichia coli and shown to have properties similar to those of the enzyme purified from spinach leaves. These results indicate that the plant FN3K/FN3K-RP homologue, which appears to be targeted to the chloroplast in many species, is a ribulosamine/erythrulosamine 3-kinase. This enzyme may participate in a protein deglycation process removing Amadori products derived from ribose 5-phosphate and erythrose 4-phosphate, two Calvin cycle intermediates that are potent glycating agents.

Figure 1. Structures of some ketoamines and of the decomposition product of their 3-phospho-derivatives

Psicosamines are the C-3-epimers of fructosamines.

Figure 2. Chromatography of a spinach leaf extract on DEAE-Sepharose

A 25% PEG pellet resuspended in buffer B was applied to a DEAE-Sepharose column. Protein was eluted with a linear NaCl gradient and fractions of 5 ml (1–25) or 3 ml (26–80) were collected. Ketoamine kinase activity was assayed using lysozyme glycated with glucose (▲) or ribose (○) and [γ-³²P]ATP, or with [¹⁴C]ribuloselysine and unlabelled ATP (■). A₂₈₀ was also measured.

Figure 3. Inhibition of the phosphorylation of [¹⁴C]ribuloselysine by different unlabelled ketoamines

The phosphorylation of [¹⁴C]ribuloselysine by spinach leaf ribulosamine kinase was tested in the presence of different concentrations of the indicated compounds. Deoxymorpholinofructose, ribuloseglycine, ribuloseleucine, ribulose and erythrulose were also devoid of effect (results not shown). Results expressed are means for two experimental values.

Figure 4. Tandem MS analysis of the phosphorylation product of ribuloselysine by spinach leaf ribulosamine-kinase

The fragmentation spectrum of the deprotonated, monosodium molecular ion at m/z 379 is shown. The structure of ribuloselysine 3-phosphate and the assignment of the main peaks are also shown.

Figure 5. Stability of the putative ribuloselysine 3-phosphate and erythruloselysine 3-phosphate

Ribulose-lysine was phosphorylated with the spinach leaf enzyme and [γ-³²P]ATP. After 10 min, the reaction was stopped by adding 10 mM EDTA and the incubation was prolonged at 37 °C for the indicated times. The products (ribuloselysine 3-phosphate and inorganic phosphate) were analysed by anion-exchange chromatography. The same experiment was performed with erythruloselysine, but at 30 °C and using shorter incubation times. The inset shows the results expressed as the decimal logarithm of the ratio of the concentration of ketoamine-phosphates at the indicated times on their concentration when EDTA was added.

Figure 6. Tandem MS analysis of the quinoxaline derivative of the erythruloselysine-phosphate degradation product

Erythruloselysine was incubated for 4 h at 30 °C with spinach leaf ketoamine 3-kinase and ATP, and the resulting mixture was treated with o-phenylenediamine. The quinoxaline derivative was purified by HPLC and analysed by tandem MS. The fragmentation spectrum of the protonated molecular ion at m/z 175 and the structure of hydroxyethylquinoxaline are shown.

Figure 7. Alignment of human FN3K and FN3K-RP with homologous proteins from A. thaliana and O. sativa

Conserved residues are indicated in bold. The following sequences are shown: A. thaliana (At) NP_191667; O. sativa (Os) AK066948; human FN3K (FHs) Q9H479; human FN3K-RP (RHs) Q9HA64.