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Structural implications of a G170R mutation of alanine:glyoxylate aminotransferase that is associated with peroxisome-to-mitochondrion mistargeting.

ABSTRACT: In a subset of patients with the hereditary kidney-stone disease primary hyperoxaluria type 1 (PH1), the liver-specific enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria. This is a consequence of the combined presence of the common P11L polymorphism and a disease-specific G170R mutation. In this paper, the crystal structure of mutant human AGT containing the G170R replacement determined at a resolution of 2.6 A is reported. The crystal structure of AGT consists of an intimate dimer in which an extended N-terminal segment of 21 amino acids from one subunit wraps as an elongated irregular coil around the outside of the crystallographic symmetry-related subunit. In addition to the N-terminal segment, the monomer structure contains a large domain of 261 amino acids and a small C-terminal domain of 110 amino acids. Comparison of the mutant AGT structure and that of wild-type normal AGT shows that the two structures are almost identical, with a backbone-atom r.m.s. deviation of 0.34 A. However, evidence of significant local structural changes in the vicinity of the G170R mutation might be linked to the apparent decrease in protein stability.

SUBMITTER: Djordjevic S

PROVIDER: S-EPMC2833026 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Structural implications of a G170R mutation of alanine:glyoxylate aminotransferase that is associated with peroxisome-to-mitochondrion mistargeting.

Djordjevic Snezana S Zhang Xiaoxuan X Bartlam Mark M Ye Sheng S Rao Zihe Z Danpure Christopher J CJ

Acta crystallographica. Section F, Structural biology and crystallization communications 20100223 Pt 3

In a subset of patients with the hereditary kidney-stone disease primary hyperoxaluria type 1 (PH1), the liver-specific enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria. This is a consequence of the combined presence of the common P11L polymorphism and a disease-specific G170R mutation. In this paper, the crystal structure of mutant human AGT containing the G170R replacement determined at a resolution of 2.6 A is reported. The crystal structure of ...[more]

PMID: 20208150

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Project description: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.

Dataset Information

Structural implications of a G170R mutation of alanine:glyoxylate aminotransferase that is associated with peroxisome-to-mitochondrion mistargeting.

Publications

Structural implications of a G170R mutation of alanine:glyoxylate aminotransferase that is associated with peroxisome-to-mitochondrion mistargeting.

Similar Datasets

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