Crystal Structure of Alanine:Glyoxylate Aminotransferase and the Relationship Between Genotype and Enzymatic Phenotype in Primary Hyperoxaluria Type 1

Xx, Zhang; Roe, S. Mark; Hou, YW; Bartlam, Mark; Rao, ZH; Pearl, L.H.; Danpure, Christopher J.

doi:10.1016/s0022-2836(03)00791-5

Cited by 129 publications

(157 citation statements)

References 41 publications

Supporting

Mentioning

148

Contrasting

Order By: Relevance

“…The three-dimensional structural model of AGT was as- sembled using the MolFeat program on the basis of X-ray analysis and structure models 6) (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

Characterization of Peroxisomal Targeting Signals on Alanine : Glyoxylate Aminotransferase

Ikeda

Kanouchi

Minatogawa

2008

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

NotesProteins destined for the peroxisome matrix or membrane possess distinct targeting signals that engage signal sequence receptors to drive their transport to their final subcellular destination. Peroxisomal targeting signal 1 (PTS1) and 2 (PTS2) are well characterized as signaling the import from the cytosol into the peroxisomal matrix of soluble peroxisomal enzymes. PTS1 is a peptide with a three-amino acid sequence of serine (S), lysine (K), and leucine (L) (SKL motif) in the extreme COOH terminus of the protein. However, recent studies revealed that PTS1 has a wide range of variants with respect to the length, sequences, and position of the amino acid residues. The membrane protein is considered to be localized through a different mechanism because the SKL motif has never been found in peroxisomal membrane proteins. The soluble protein is thought to have the main signal for localization because the SKL motif exists in most soluble peroxisomal proteins. PTS1 signals bind to their receptor, Pex5p, for the protein to be targeted to peroxisome. PTS2 is located at or near the NH 2 termini of some peroxisomal matrix proteins with a wide range of consensus sequences. PTS2 interacts with their import receptor, Pex7p, and the proteins with PTS2 signals are targeted to peroxisome.Alanine : glyoxylate aminotransferase (AGT) is a liver-specific peroxisomal enzyme. Deficiency in AGT causes primary hyperoxaluria type I (PH1), which is a rare autosomal recessive disease. AGT is a protein composed of 392 amino acids (a.a.), and it has a similar lysine (K), lysine (K), and leucine (L) (KKL) amino acid sequence to that of the SKL motif in the extreme COOH terminus. It was reported that the COOH-terminal tripeptide KKL of human AGT is necessary as the peroxisomal targeting sequence. However, the peroxisomal targeting signal of AGT and the mechanism of targeting to peroxisome are still unclear. [1][2][3][4][5] In the present paper, human-origin AGT was targeted into peroxisome at specific regions of the AGT (59-66 a.a. and 389-392 a.a.) in HeLa cells using enhanced green fluorescent protein (eGFP)-tagged AGT and deletion mutants. In the three-dimensional structural model their sites were separately located on the surface of the AGT protein. MATERIALS AND METHODS Construction of PlasmidsThe expression vector for the eGFP-tagged full-length AGT1-392 a.a. (AGT fused to the COOH terminus of eGFP), pEGFPAGT1-392, was constructed by inserting the cDNA fragment of AGT PCR-amplified from the expression vector of the human-origin AGT, pTrcHis2AGT (a gift from Dr. S. Hirose, Fukuoka University, Fukuoka, Japan) into the EcoRI site of the multi-cloning site (MCS) of the expression vector of eGFP, pEGFP-C1 (Clontech Co., Palo Alto, CA, U.S.A.). The expression vectors of the eGFP-tagged AGT deletion mutants were constructed by modifying pEGFPAGT1-392. The full-length AGT and deletion mutants used in the paper are schematically shown in Fig. 1.Cell Culture and Transfection HeLa cells and COS-7 cells were purchased from the Institute of Phy...

show abstract

“…The three-dimensional structural model of AGT was as- sembled using the MolFeat program on the basis of X-ray analysis and structure models 6) (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

Characterization of Peroxisomal Targeting Signals on Alanine : Glyoxylate Aminotransferase

Ikeda

Kanouchi

Minatogawa

2008

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

show abstract

“…Structural comparison with several other aminotransferases allowed us to propose further chemical manipulation of 4-(2-aminophenyl)-4-oxobutyric acid, aimed at improving its potency and specificity. A DALI search (40) identified human alanine-glyoxylate aminotransferase (AGT) (41) as the closest structural ortholog of Ag-HKT. Human AGT and Ag-HKT belong to the same subgroup of PLP-dependent aminotransferases, show 43% sequence identity, and can be superposed with a rms deviation of 1.2 Å for 382 C␣ pairs.…”

Section: Resultsmentioning

confidence: 99%

“…Statistics for data collection are listed in Table 1. The structure determination of the PLP form of the enzyme was carried out by means of the molecular replacement technique using the coordinates of a monomer of human AGT as the search model (Protein Data Bank ID code 1H0C) (41). The program AMORE (46) was used to calculate both crossrotation and translation functions in the 10 -4 -Å resolution range, allowing for the identification of a clear solution of both functions for all four molecules present in the asymmetric unit.…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of the Anopheles gambiae 3-hydroxykynurenine transaminase

Rossi

Garavaglia²,

Giovenzana³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

In Anopheles gambiae, the vector for the most deadly malaria parasite Plasmodium falciparum, xanthurenic acid (XA) plays a key role in parasite gametogenesis and fertility. In mosquitoes, XA is produced by transamination of 3-hydroxykynurenine (3-HK), a reaction that represents the main route to prevent the accumulation of the potentially toxic 3-HK excess. Interfering with XA metabolism in A. gambiae therefore appears an attractive avenue for the development of malaria transmission-blocking drugs and insecticides. We have determined the crystal structure of A. gambiae 3-HK transaminase in its pyridoxal 5 -phosphate form and in complex with a newly synthesized competitive enzyme inhibitor. Structural inspection of the enzyme active site reveals the key molecular determinants for ligand recognition and catalysis. Major contributions toward inhibitor binding are provided by a salt bridge between the inhibitor carboxylate and Arg-356 and by a remarkable hydrogen bond network involving the anthranilic moiety of the inhibitor and backbone atoms of residues Gly-25 and Asn-44. This study may be useful for the structurebased design of specific enzyme inhibitors of potential interest as antimalarial agents.kynurenine pathway ͉ xanthurenic acid

show abstract

“…The enzyme crystal structure, complexed with the competitive inhibitor amino-oxyacetic acid, was determined at a resolution of 2.5 Å. Each subunit includes a N-terminal extension (residues 1-21), a large N-terminal domain (residues 22-282) containing the PLP-binding lysine (K209), and a smaller C-terminal domain (residues 283-392) (1). Steady-state and pre-steady-state kinetic studies featuring the AGT transamination revealed high specificity for glyoxylate to glycine processing, consistent with a key role of AGT in glyoxylate detoxification (2).…”

mentioning

confidence: 99%

Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I

Cellini

Montioli

Paiardini³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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

show abstract

Crystal Structure of Alanine:Glyoxylate Aminotransferase and the Relationship Between Genotype and Enzymatic Phenotype in Primary Hyperoxaluria Type 1

Cited by 129 publications

References 41 publications

Characterization of Peroxisomal Targeting Signals on Alanine : Glyoxylate Aminotransferase

Characterization of Peroxisomal Targeting Signals on Alanine : Glyoxylate Aminotransferase

Crystal structure of the Anopheles gambiae 3-hydroxykynurenine transaminase

Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I

Contact Info

Product

Resources

About