標題: Structure-stability-activity relationship in covalently cross-linked N-carbamoyl D-amino acid amidohydrolase and N-acylamino acid racemase
作者: Chiu, WC
You, JY
Liu, JS
Hsu, SK
Hsu, WH
Shih, CH
Hwang, JK
Wang, WC
生物科技學系
生物資訊及系統生物研究所
Department of Biological Science and Technology
Institude of Bioinformatics and Systems Biology
關鍵字: NAAAR;N-acylamino acid racemase;D-NCAase;N-carbamoyl D-amino acid amidohydrolase;disulfide bond;thermostability;activity
公開日期: 9-Jun-2006
摘要: N-Acylamino acid racemase (NAAAR) and N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) are important biocatalysts for producing enantiopure a-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 k(cat)/K-m ratios, whereas mutant D-NCAases demonstrated increased k(cat)/K-m 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. (c) 2006 Elsevier Ltd. All rights reserved.
URI: http://dx.doi.org/10.1016/j.jmb.2006.03.063
http://hdl.handle.net/11536/12161
ISSN: 0022-2836
DOI: 10.1016/j.jmb.2006.03.063
期刊: JOURNAL OF MOLECULAR BIOLOGY
Volume: 359
Issue: 3
起始頁: 741
結束頁: 753
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