2GGK

The mutant A302C of Agrobacterium radiobacter N-carbamoyl-D-amino-acid amidohydrolase

Replaces:  2FKVReplaces:  2BA5

Summary for 2GGK

• Entry DOI: 10.2210/pdb2ggk/pdb
• Related: 1FO6 2GGL
• Descriptor: N-carbamoyl-D-amino acid amidohydrolase (2 entities in total)
• Functional Keywords: n-carbamoyl-d-amino-acid amidohydrolase, hydrolase
• Biological source: Agrobacterium tumefaciens
• Total number of polymer chains: 4
• Total formula weight: 136964.50

Authors

Wang, W.C.,Chiu, W.C.,You, J.Y. (deposition date: 2006-03-24, release date: 2006-04-11, Last modification date: 2024-10-23)

Primary citation

Chiu, W.C.,You, J.Y.,Liu, J.S.,Hsu, S.K.,Hsu, W.H.,Shih, C.H.,Hwang, J.K.,Wang, W.C.
Structure-Stability-Activity Relationship in Covalently Cross-linked N-Carbamoyl d-Amino acid Amidohydrolase and N-Acylamino acid Racemase.
J.Mol.Biol., 359:741-753, 2006

PubMed Abstract: 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.

PubMed: 16650857
DOI: 10.1016/j.jmb.2006.03.063

Experimental method

X-RAY DIFFRACTION (2.3 Å)