Structure-Function Relationship and Protein Engineering of Deinococcus radiodurans N-acylamino Acid Racemase

• Main Authors: Wei-Chun Chiu, 邱偉鈞
• Other Authors: Wen-Ching Wang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/85828616695588564363

博士 === 國立清華大學 === 生命科學系 === 94 === N-acylamino acid racemase (NAAAR) that catalyzes racemization of N-acylamino acid is valuable to produce enantiopure α-amino acids in couple with an aminoacylase. The pupose of this investigation is to study the structure-function relationship of NAAAR and to increase the industrial value by protein engineering. Chapter 1 describes the significance and background of the thesis. Chapter 2 focused on results and discussion of crystal structural determination, the overall structural descriptions, and structural comparisons. The crystal structure of NAAAR was solved to 1.3 Å using multiwavelength anomalous diffraction (MAD) method. The structure consist of a homooctamer in which each subunit has an architecture characteristic of enolases with a capping domain and a (β/α)7β barrel domain. In chapter 3, we solved the lignaded structures of NAAAR-Mg2+, NAAAR-N-acetyl-L-glutamine-Mg2+ and NAAAR-N-acetyl-D-methionine-Mg2+. Based on these structures, the active site region contains Lys170, Asp195, Glu220, Asp245, Lys269 are identified critical residues: Lys170 and Lys269 are two catalysts to abstract an α-proton of a carboxylate, while Asp195, Glu220, and Asp245 are Mg2+-binding residues. Four subsites for substrate binding are also identified: catalytic site (C), metal-binding site (M), side-chain-binding region (S), and a flexible lid region (L). Structural comparisons of various members of the enolase superfamily reveal a highly conserved catalytic and metal-binding sites among enolases. Moreover, L region and S site involved in the substrate recognition are less conserved, suggesting a consequence of divergence into functionally distinct enzymes. In Chpater 4, variants of NAAAR (S142A, K170A, K170R, K269A, K269R, M298A, L299A, D322N, D322A and Y329A) were prepared by site directed mutagenesis, and then tested their catalytic efficiency to determine the importance for efficient catalysis. Four variants (K170A, K170R, K269A and K269R) had no enzyme activity, confirming the important roles of Lys170 and Lys269 in catalysis. In Chapter 5, we futher investigated the effects of introducing potentially stabilizing S-S bridges in these different multimeric enzymes. Cysteines 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). 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. 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 °C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the fine-tuned catalytic site as temperature increases, achieving enhanced activity. In the last chapter, we focused on increasing enzyme activity. According the analysis of prior chapters, the hydrophobic property in the catalytic cavity is very important to enzyme catalyst and substrate binding. We replaced residues of Thr28, Gln33 and Tyr62 by phenylalanine in the L1 and L2 regions that are highly hydrophobic in catalytic cavity. The results showed Q33F mutant was 4-fold more activity than wild-type, and Y62F mutant catalyzed the single way of L form → D form racemization only.