Human hepatic peroxisomal AGT (alanine:glyoxylate aminotransferase) is a PLP (pyridoxal 5'-phosphate)-dependent enzyme whose deficiency causes primary hyperoxaluria Type I, a rare autosomal recessive disorder. To acquire experimental evidence for the physiological function of AGT, the K(eq),(overall) of the reaction, the steady-state kinetic parameters of the forward and reverse reactions, and the pre-steady-state kinetics of the half-reactions of the PLP form of AGT with L-alanine or glycine and the PMP (pyridoxamine 5'-phosphate) form with pyruvate or glyoxylate have been measured. The results indicate that the enzyme is highly specific for catalysing glyoxylate to glycine processing, thereby playing a key role in glyoxylate detoxification. Analysis of the reaction course also reveals that PMP remains bound to the enzyme during the catalytic cycle and that the AGT-PMP complex displays a reactivity towards oxo acids higher than that of apoAGT in the presence of PMP. These findings are tentatively related to possible subtle rearrangements at the active site also indicated by the putative binding mode of catalytic intermediates. Additionally, the catalytic and spectroscopic features of the naturally occurring G82E variant have been analysed. Although, like the wild-type, the G82E variant is able to bind 2 mol PLP/dimer, it exhibits a significant reduced affinity for PLP and even more for PMP compared with wild-type, and an altered conformational state of the bound PLP. The striking molecular defect of the mutant, consisting in the dramatic decrease of the overall catalytic activity (approximately 0.1% of that of normal AGT), appears to be related to the inability to undergo an efficient transaldimination of the PLP form of the enzyme with amino acids as well as an efficient conversion of AGT-PMP into AGT-PLP. Overall, careful biochemical analyses have allowed elucidation of the mechanism of action of AGT and the way in which the disease causing G82E mutation affects it.

No abstract

DOPA decarboxylase, the dimeric enzyme responsible for the synthesis of neurotransmitters dopamine and serotonin, is involved in severe neurological diseases such as Parkinson disease, schizophrenia, and depression. Binding of the pyridoxal-5′-phosphate (PLP) cofactor to the apoenzyme is thought to represent a central mechanism for the regulation of its activity. We solved the structure of the human apoenzyme and found it exists in an unexpected open conformation: compared to the pig kidney holoenzyme, the dimer subunits move 20 Å apart and the two active sites become solvent exposed. Moreover, by tuning the PLP concentration in the crystals, we obtained two more structures with different conformations of the active site. Analysis of three-dimensional data coupled to a kinetic study allows to identify the structural determinants of the open/close conformational change occurring upon PLP binding and thereby propose a model for the preferential degradation of the apoenzymes of Group II decarboxylases.apoprotein | Shiff base | stability | kinetics | FRET

G41 is an interfacial residue located within the α-helix 34-42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (∼5; 000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 → R mutation causes a partial unwinding of the 34-42 α-helix and a displacement of the first 44 N-terminal residues including the active site loop 24-32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.dimer interface | pathogenic variant | protein aggregation | pyridoxal 5'-phosphate

No abstract