2ggj: Difference between revisions

[[Image:2ggj.gif|left|200px]]<br /><applet load="2ggj" size="350" color="white" frame="true" align="right" spinBox="true"  

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.

2GGJ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Deinococcus_radiodurans Deinococcus radiodurans]. This structure supersedes the now removed PDB entries 2FKT and 2BAE. Active as [http://en.wikipedia.org/wiki/Amino-acid_racemase Amino-acid racemase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.1.1.10 5.1.1.10] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2GGJ OCA].  

[[Category: Chiu, W C.]]

[[Category: Wang, W C.]]

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