Identification of enzymes acting on α‐glycated amino acids in Bacillus subtilis

Abstract

We have characterized the Bacillus subtilis homologs of fructoselysine 6-kinase and fructoselysine-6-phosphate deglycase, two enzymes that specifically metabolize the Amadori compound fructose-e-lysine in Escherichia coli. The B. subtilis enzymes also catalyzed the phosphorylation of fructosamines to fructosamine 6-phosphates (YurL) and the conversion of the latter to glucose 6-phosphate and a free amino acid (YurP). However, their specificity was totally different from that of the E. coli enzymes, since they acted on fructoseglycine, fructosevaline (YurL) or their 6-phosphoderivatives (YurP) with more than 30-fold higher catalytic efficiencies than on fructose-e-lysine (6-phosphate). These enzymes are therefore involved in the metabolism of a-glycated amino acids.

Substrate Specificity of theEscherichia ColiFRLB Amadoriase

“…The result also implies that in the forward reaction the FrlB enzyme might recognize and remove an Amadori product formed not only at the ɛ-but also at the α-NH 2 group of lysine. The reason why we detected activity of FrlB with other amino acids except with Lys in contrast to others (21,22) may be explained by the higher substrate concentrations we used in the enzyme assay.…”

“…Published procedure for measurement of the FrlB enzyme activity is based on the reversal of the reaction at high, non-physiological concentrations of glucose 6-phosphate and lysine (21,22). We applied the same approach with some modifications.…”

“…5 shows the relative FrlB activity with the twenty amino acids allocated to three characteristics groups (charged, polar and hydrophobic). The enzyme activity on α-Boc-Lys was taken for 100%, because this is the only amino acid reported to be a substrate for the FrlB amadoriase (21,22). However, Fig.…”

“…The FrlB amadoriase acts in concert with the FrlD kinase, which first phoshporylates the fructose moiety in N ɛ -fructoselysine at position C-6. In a later publication, Van Schaftingen and co-workers reported that, in contrast to the Bacillus subtilis ortholog, the E. coli FrlB amadoriase is highly specific for AP bound to the ɛ-amino group of lysine but not to the α-amino group of other amino acids including Gly, Val, Gln, Met, Arg and Ile (22). The authors proposed that the role of the FrlB enzyme is to allow bacteria in the human hind gut to catabolize fructoselisine, which is released during digestion of glycated food proteins.…”

Mutations in yhiT enable utilization of exogenous pyrimidine intermediates in Salmonella enterica serovar Typhimurium

“…The first ORF (yhiV; STM3601) of the putative four-gene operon is deduced to encode a 36 kDa hydrophobic polypeptide with a conserved phosphosugar isomerase domain commonly observed in glucose phosphate isomerases. YhiV shows similarity to gene products involved in the catabolism of sugar-amino acid conjugates, specifically, the conversion of fructosamine 6-phosphates to glucose 6-phosphate and free amino acids in Bacillus subtilis (YurP; 58 %) (Wiame et al, 2004) and the conversion of mannopine to glutamine and mannose in Agrobacterium tumefaciens (MocD; 46 %) (Kim & Farrand, 1996). ORF2 (yhiU, STM3600) is predicted to encode a 31 kDa hydrophobic polypeptide, which displays 48 % similarity to the putative kinase MocE from A. tumefaciens (Kim & Farrand, 1996) and 54 % similarity to the kinase YurL of B. subtilis (Fujita et al, 1986;Wiame et al, 2004).…”