Site-directed Mutagenesis of Sialic Acid Synthase in E. coli K1: to search for the active site residues

• Main Authors: Chiung-Fang Chang, 張瓊方
• Other Authors: Chun-Hung Lin
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/38638376297303417969

碩士 === 國立臺灣大學 === 生化科學研究所 === 91 === Polysialic acid exists as a bacterial capsular homopolysaccharide of N-acetylneuraminic acid (NeuAc) with -(2,8) ketosidic linkages in Escherichia coli K1. This polysaccharyl capsule has attracted much attention because it is a virulent determinant of neonatal meningitis in human. The gene neuB, which is responsible for the sialic acid biosynthesis in E. coli K1, is located in the kps gene cluster. Its gene product, N-acetylneuraminic acid synthase (NeuB), consists of 346 amino acids corresponding to the molecular mass of 40 kDa. In presence of divalent metal cation Mg2+ or Mn2+, NeuB catalyzes the condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) to form NeuAc. The information of the NeuB active site can be utilized to design inhibitors as well as to improve the enzymatic synthesis of sialic acids. According to the sequence alignment of NeuB among seven bacterial strains, several highly conserved residues were selected for site-directed mutagenesis. In addition, mutants were also designed by comparing NeuB with other PEP-dependent enzymes such as KDO8P synthase and DAH7P synthase because some conserved motifs have been proposed to participate in the catalysis and PEP binding. Fourteen amino acid residues were selected as the targets of site-directed mutagenesis. These amino acids were replaced with alanine in the preliminary screening. The enzymatic activities and kinetic parameters of all purified mutants were determined. Four mutants NeuB*E228A, H230A, D241A and R308A were found to be catalytically inactive, indicating the importance of these amino acids to preserve the enzyme function. Circular dichroism analyses showed that the secondary structure of each mutant was consistent with that of the wild-type NeuB, indicating no structural changes. Accordingly, the loss of activity may probably result from the absence of these important active-site residues during catalysis. The Km values of NeuB*E122A and K229A for PEP were 7 and 9 times, respectively, higher than that of the wild-type NeuB, which revealed that the lower affinity with PEP. Additional mutants focusing on H230 and R308 were constructed in the secondary screening. Mutants NeuB*H230Q, H230F completely lost their activity, proving that H230 is a critical residue for the catalysis. Meanwhile, NeuB*R308K and NeuB*R308E were constructed for further study. Compared with the wild-type NeuB, the activity of NeuB*R308K decreased to only 1.6% and the Km value for PEP was 7 times higher. NeuB*R308E cannot be purified by Ni2+-affinity chromatography. These observations demonstrated that the positively charged R308 was significant in maintaining protein structure and function. Based on the results of site-directed mutants and the implications from PEP-dependent enzymes, some active site residues were proposed, including PEP-binding residues K229, H230, R308 and possible metal ligands C12, E228, H230 and D241. Finally, the conditions for the crystallization of NeuB proteins (from E. coli and S. agalactiae) were optimized. The information obtained from X-ray crystallography as well as site-directed mutagenesis will shed light on the active-site structure and detailed reaction mechanism of NeuB.