2ggl

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spinqualitylabelsall models 2ggl, resolution 2.40Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The mutant A222C of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase

Overview

N-Acylamino acid racemase (NAAAR) and N-carbamoyl-D-amino-acid, amidohydrolase (D-NCAase) are important biocatalysts for producing, enantiopure alpha-amino acids. NAAAR forms an octameric assembly and, displays induced fit movements upon substrate binding, while D-NCAase is a, tetramer that does not change conformation in the presence of a ligand. To, investigate the effects of introducing potentially stabilizing S-S bridges, in these different multimeric enzymes, cysteine residues predicted to form, inter or intra-subunit disulfide bonds were introduced by site-directed, mutagenesis. Inter-subunit S-S bonds were formed in two NAAAR variants, (A68C-D72C and P60C-Y100C) and two d-NCAase variants (A302C and, P295C-F304C). Intra-subunit S-S bonds were formed in two additional NAAAR, variants (E149C-A182C and V265C). Crystal structures of NAAARs variants, show limited deviations from the wild-type overall tertiary structure. An, apo A68C-D72C subunit differs from the wild-type enzyme, in which it has, an ordered lid loop, resembling ligand-bound NAAAR. The structures of, A222C and A302C D-NCAases are nearly identical to the wild-type enzyme., All mutants with inter-subunit bridges had increases in thermostability., Compared with the wild-type enzyme, A68C-D72C NAAAR showed similar kcat/Km, ratios, whereas mutant D-NCAases demonstrated increased kcat/Km ratios at, high temperatures (A302C: 4.2-fold at 65 degrees C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the, fine-tuned catalytic site as temperature increases, achieving enhanced, activity.

About this Structure

2GGL is a Single protein structure of sequence from Agrobacterium tumefaciens. This structure superseeds the now removed PDB entries 2FKU and 2BA4. Active as N-carbamoyl-D-amino acid hydrolase, with EC number 3.5.1.77 Full crystallographic information is available from OCA.

Reference

Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase., Chiu WC, You JY, Liu JS, Hsu SK, Hsu WH, Shih CH, Hwang JK, Wang WC, J Mol Biol. 2006 Jun 9;359(3):741-53. Epub 2006 Apr 18. PMID:16650857

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