Structural studies of duck δ2 crystallin mutants provide insight into the role of Thr161 and the 280s loop in catalysis

Sampaleanu, L.M.; Codding, Penelope W.; Lobsanov, Y.D.; Tsai, M.; Smith, G. D.; Horvatin, C.; Howell, P. Lynne

doi:10.1042/bj20040656

Cited by 12 publications

(14 citation statements)

References 49 publications

(87 reference statements)

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“…Across this family, three sequence domains show high conservation (6,22,26,27). These domains are also found in the IDS-lyases (Fig.…”

Section: Discussionmentioning

confidence: 77%

“…(25) alignment of duck ␦II-crystallin (P24058), E. coli argininosuccinate lyase (AAC76942), and the amino acid sequences AAZ80811 (BY6-lyase) and AAZ80812 (SLRS7-lyase). Three domains with high sequence similarities within the fumarase II enzyme family (6,22,26,27) are boxed. Positions of ␦II-crystallin amino acid residues involved in binding of argininosuccinate (21) are marked with arrowheads.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Sequencing and Heterologous Expression of an Epimerase and Two Lyases from Iminodisuccinate-Degrading Bacteria

Bäuerle

Cokesa

Hofmann

et al. 2006

Appl Environ Microbiol

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Sodium iminodisuccinate (IDS) is a widely used synthetic, medium-strong chelator belonging to the group of aminopolycarboxylates. IDS can be used as a substitute for EDTA, which is not biodegradable according to OECD tests 301A, 301F, and 302B (19). In contrast, all three epimers of IDS (R,S-, S,S-, and R,R-IDS), are readily biodegradable (9).The bacterial strains Ralstonia sp. strain SLRS7 and Agrobacterium tumefaciens BY6 grow on IDS as the sole source of carbon and nitrogen. Cofactor-independent C-N lyases catalyze the cleavage of IDS, generating D-aspartic acid and fumaric acid from R,S-IDS as well as L-aspartic acid and fumaric acid from S,S-IDS (9, 10). R,R-IDS metabolism is preceded by epimerization. A cofactor-independent epimerase from A. tumefaciens BY6 catalyzes the conversion of all three epimers into each other (9). For the lyases and the epimerase, the natural function is still unknown.Here, we report sequences, cloning, recombinant expression, and purification of the IDS-converting enzymes. MATERIALS AND METHODSStrains, plasmids, and growth conditions. The isolation and growth conditions of A. tumefaciens BY6 and of Ralstonia sp. strain SLRS7 have been reported previously (9, 10). Escherichia coli strains were grown aerobically in LuriaBertani (LB) or 2ϫ tryptone-yeast extract medium (1) supplemented with final concentrations of 100 g ml Ϫ1 ampicillin, 25 g ml Ϫ1 chloramphenicol, and 50 g ml Ϫ1 kanamycin according to the requirements. A list of strains and plasmids used and constructed is given in Table 1.Protein purification from wild-type strains. The purification of IDS-epimerase and IDS-lyase has been described previously (9, 10). IDS-lyase from Ralstonia sp. strain SLRS7 was purified with slight modifications of the original protocol. An initial (NH 4 ) 2 SO 4 (40% [wt/vol]) precipitation of the lyase was applied. As an additional purification step, hydroxyapatite chromatography was performed with a Bio-Scale cHT5-1 (Bio-Rad Laboratories, Germany) column, using a linear gradient comprising 2.5 mM potassium phosphate, pH 7.5 (buffer A), and 500 mM potassium phosphate, pH 7.5 (buffer B), with ϩ0.4% (vol/vol) buffer B per ml. Elution of IDS-lyase started with 12% (vol/vol) buffer B. A specific activity of up to 10 mol IDS min Ϫ1 mg Ϫ1 was obtained for SLRS7-lyase.Protein cleavage, peptide separation, and sequencing. Trypsin digestion of 200 g protein was based on the method of Stone et al. (24). Separation of tryptic peptides of epimerase and SLRS7-lyase by reversed-phase high-pressure liquid chromatography was performed using a 250-by 4-mm Grom-Sil ODS-5 column (where ODS is octyldecyl silane) (Grom, Germany) and an aqueous solution of 0.1% (vol/vol) trifluoroacetic acid (solvent A) and 0.085% (vol/vol) trifluoroacetic acid in 80% (vol/vol) acetonitrile (solvent B). The following gradients were applied: 0 to 7.5 ml, 7% B; 7.5 to 57.5 ml, 7 to 50% B; and 57.5 to 77.5 ml, 50 to 98% B (0.5 ml min Ϫ1 ; detection at 210 nm). The BY6-lyase fragments were fractionated using a 100-by 2-mm TSK-Gel Super...

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“…Across this family, three sequence domains show high conservation (6,22,26,27). These domains are also found in the IDS-lyases (Fig.…”

Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Sequencing and Heterologous Expression of an Epimerase and Two Lyases from Iminodisuccinate-Degrading Bacteria

Bäuerle

Cokesa

Hofmann

et al. 2006

Appl Environ Microbiol

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“…1C,D). These residues are conserved within the sequences of homologous proteins, and also participate in hydrogen bonding at the subunit interface within the tertiary structure of duck δ‐crystallin and ASL [7,9,17].…”

Section: Discussionmentioning

confidence: 99%

Substitution of residues at the double dimer interface affects the stability and oligomerization of goose δ‐crystallin

et al. 2009

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δ‐Crystallin is the major structural protein in avian and reptilian eye lenses, and confers special refractive properties. The protein is a homotetramer arranged as a dimer of dimers. In the present study, the roles of the side chains of Glu267, Lys315, and Glu327, which provide hydrogen bonds at the double dimer interface, were investigated. Hydrophobic side chain substitution led to all mutant proteins having an unstable dimer interface. The E267L/E327L mutant had the greatest sensitivity to temperature, urea and guanidinium hydrochloride denaturation, and the most extensive exposure of hydrophobic patches, as judged by 1‐anilinonaphthalene‐8‐sulfonic acid fluorescence, CD, and tryptophan fluorescence. In contrast, the E267L/K315L/E327L mutant showed higher stability than the E267L/E327L mutant. Some level of the dissociated dimeric form was observed in the K315L mutant, but it was not observed for the K315A and E267L/K315L mutants. The E327L mutant was partially in the dissociated dimeric form, whereas the E267/E327L mutant was predominantly dissociated into dimers. In contrast, the triple mutant of E267L/K315L/E327L retained a tetrameric structure. In the presence of urea, a stable monomeric intermediate with higher stability than the wild type was identified for the K315A mutant. Disruption of interfacial interactions at Glu267 led to polymerization of partly unfolded intermediates in the presence of 3 m urea. However, these polymeric forms were not observed with combinations of the E267L mutation with other mutations. These results indicate that these hydrogen bonds, which are present at different contact surfaces in the dimer–dimer interface, perform distinct functions in double dimer assembly. The coordination of these interactions is critical for the stability and tetramer formation of δ‐crystallin.

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“…Argininosuccinate lyase (ASL) is the final enzyme in the arginine biosynthesis pathway and catalyses the reversible breakdown of argininosuccinate into arginine and fumarate . The most extensively studied homologue of ASL is an eye lens protein from duck, δ‐crystallin . Lens proteins are not renewed and are among the longest‐lived proteins in vertebrates, which resist aggregation and degradation.…”

Section: Introductionmentioning

confidence: 99%

Structural studies on M. tuberculosis argininosuccinate lyase and its liganded complex: Insights into catalytic mechanism

et al. 2019

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Argininosuccinate lyase catalyses the reversible breakdown of argininosuccinate into arginine and fumarate and is known to form tetramers in its quaternary association. The absence of structures involving competent enzymes bound to substrate/products came in the way of the precise elucidation of the catalytic mechanism of this family of proteins. Crystal structures of the enzyme from Mycobacterium tuberculosis in an unliganded form and its complex with the substrate/products have now been determined at 2.2 and 2.7 Å, respectively. The refinement of the structure of the complex was bedevilled by the presence of a lattice translocation defect. The two tetramers in the apo‐crystals and the one in the crystals of the liganded protein, have the same structure except for the movements associated with enzyme action. Each molecule consists of an N‐domain, an M‐domain, and a C‐domain. The molecule consists of four binding sites, each made up of peptide stretches from three subunits. Three binding sites appear to be occupied by the ligand in the transition state, while the products occupy the fourth site. The structure exhibits the movement of a loop in the M‐domain and parts of the C‐domain. This is the first instance when the appropriate movements are observed in a complex with bound substrate/product. The detailed picture of the binding site, active site residues and the movements associated with catalysis thus obtained, enabled a revisit of the mechanism of action of the enzyme. © 2019 IUBMB Life, 71(5):643–652, 2019

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Structural studies of duck δ2 crystallin mutants provide insight into the role of Thr161 and the 280s loop in catalysis

Cited by 12 publications

References 49 publications

Sequencing and Heterologous Expression of an Epimerase and Two Lyases from Iminodisuccinate-Degrading Bacteria

Sequencing and Heterologous Expression of an Epimerase and Two Lyases from Iminodisuccinate-Degrading Bacteria

Substitution of residues at the double dimer interface affects the stability and oligomerization of goose δ‐crystallin

Structural studies on M. tuberculosis argininosuccinate lyase and its liganded complex: Insights into catalytic mechanism

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