Molecular identification of hydroxylysine kinase and of ammoniophospholyases acting on 5-phosphohydroxy-L-lysine and phosphoethanolamine

Abstract

The purpose of the present work was to identify the catalytic activity of AGXT2L1 and AGXT2L2, two closely related, putative pyridoxal-phosphate-dependent enzymes encoded by vertebrate genomes. The existence of bacterial homologues (40-50% identity with AGXT2L1 and AGXT2L2) forming bi- or tri-functional proteins with a putative kinase belonging to the family of aminoglycoside phosphotransferases suggested that AGXT2L1 and AGXT2L2 acted on phosphorylated and aminated compounds. Vertebrate genomes were found to encode a homologue (AGPHD1) of these putative bacterial kinases, which was therefore likely to phosphorylate an amino compound bearing a hydroxyl group. These and other considerations led us to hypothesize that AGPHD1 corresponded to 5-hydroxy-L-lysine kinase and that AGXT2L1 and AGXT2L2 catalyzed the pyridoxal-phosphate-dependent breakdown of phosphoethanolamine and 5-phosphohydroxy-L-lysine. The three recombinant human proteins were produced and purified to homogeneity. AGPHD1 was indeed found to catalyze the GTP-dependent phosphorylation of 5-hydroxy-L-lysine. The phosphorylation product made by this enzyme was metabolized by AGXT2L2, which converted it to ammonia, inorganic phosphate, and 2-aminoadipate semialdehyde. AGXT2L1 catalyzed a similar reaction on phosphoethanolamine, converting it to ammonia, inorganic phosphate, and acetaldehyde. AGPHD1 and AGXT2L2 are likely to be the mutated enzymes in 5-hydroxylysinuria and 5-phosphohydroxylysinuria, respectively. The high level of expression of AGXT2L1 in human brain, as well as data in the literature linking AGXT2L1 to schizophrenia and bipolar disorders, suggest that these diseases may involve a perturbation of brain phosphoethanolamine metabolism. AGXT2L1 and AGXT2L2, the first ammoniophospholyases to be identified, belong to a family of aminotransferases acting on ω-amines.

FIGURE 1.

Evolutionary tree of AGXT2L1 and AGXT2L2 and related sequences. The phylogenetic tree was inferred from the aligned protein sequences using Bayesian analysis (MrBayes) methods. The horizontal bar represents 50 substitutions per 100 amino acid residues. The following sequences were used to construct the tree: AGXT2 (alanine-glyoxylate aminotransferase 2) from Homo sapiens (NP_114106), Mus musculus (Q3UEG6), and Monodelphis domestica (XP_001373137); AGXT2L1 (AGXT2-like 1) from H. sapiens (NP_112569), M. musculus (NP_082183), and M. domestica (XP_001364959); AGXT2L2 (AGXT2-like 2) from H. sapiens (NP_699204), M. musculus (EDL33664), and Ornithorhynchus anatinus (XP_001518367); GABAT (γ-aminobutyrate aminotransferase) from H. sapiens (NP_065737), M. musculus (NP_766549), and M. domestica (XP_001376874); ornithine aminotransferase (OAT) from H. sapiens (NP_000265), M. musculus ( BAE26718), and M. domestica (XP_001363028); bacterial proteins from Flavobacterium psychrophilum JIP02/86 (YP_001295781), P. atrosepticum SCRI1043 (YP_050155), and Polaromonas sp. JS666 (YP_547276); and two proteins from Sinorhizobium meliloti 1021 (NP_437638, protein 1; NP_436219, protein 2).

FIGURE 2.

Association of bacterial homologues of AGXT2L1/L2 with putative aminoglycoside phosphotransferases in bacterial genomes. The figure shows representative examples of the genomic region comprising the bacterial homologues of AGXT2L1/L2 shown in Fig. 1. In some cases, the homologue is a bifunctional protein (YP_001295781) fused with a putative phosphotransferase (as seen in F. psychrophilum JIP02/86 contig NC_009613.1) or a trifunctional protein (NP_437638) with an additional putative peptidase of the M23/M37 family (“region 1” of the genome of S. meliloti 1021 contig NC_003078). In other cases, it is encoded by a gene neighboring another gene encoding a protein of the aminoglycoside phosphotransferase family (YP_050155 and YP_050154 in P. atrosepticum SCRI1043, contig NC_004547.2; YP_547276 and YP_547277 in Polaromonas sp. JS666, contig NC_007948.1; and NP_436219 and NP_436218 in region 2 of the genome of S. meliloti 1021 contig NC_003037.1). Note in some cases the proximity to genes putatively involved in polyamine metabolism (PotA, PotB, PotC, and PotD, which are different components of the spermidine/putrescine transporter, as well as acetyl-polyamine deacetylase).

FIGURE 3.

PEtN phospholyase activity of recombinant human AGXT2L1. AGXT2L1 was incubated with 5 mm PEtN for 10 and 20 min at 30 °C, before stopping the reaction. Acetaldehyde (CH₃CHO), ammonia (NH₃), and P_i were measured in the incubation medium.

FIGURE 4.

5PHyl phospholyase activity of recombinant human AGXT2L2. A, AGXT2L2 was incubated with 1 mm 5PHyl for 30 min at 30 °C, as described under “Experimental Procedures.” After stopping the reaction, 2-AASA, ammonia (NH₃), and P_i were measured in the incubation medium. B, the saturation curve of AGXT2L2 was obtained by measuring the production of [³²P]P_i from [³²P]5PHyl in the presence of variable concentrations of unlabeled 5PHyl.

FIGURE 5.

Contribution of AGPHD1, AGXT2L2, and AGXT2L1 to the metabolism of 5PHyl and ethanolamine in vertebrates.