Chemical Modification and Site-directed Mutagenesis of Cys and Arg of the Sialic Acid Synthase from Streptococcus agalactiae

• Main Authors: Jui-Chuan Chen, 陳瑞娟
• Other Authors: Chun-Hung Lin
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/68666321788700792948

碩士 === 國立臺灣大學 === 生化科學研究所 === 92 === Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. The production of a type-specific capsular polysaccharide (CPS) is essential to the virulence of this pathogen and enables the bacteria to evade host immue defenses. As a key component of the capsular polysaccharide, sialic acid (or N-acetylneuraminic acid) is ��2,3-linked to galactose to form a disaccharide that is pending from the repeating trisaccharyl backbone. Sialic acid synthase (E.C. 4.1.3.19, NeuB) is the important enzyme which is responsible for sialic acid biosynthesis. The sialic acid synthase from Streptococcus agalactiae (abbreviated as SaNeuB) consists of 341 amino acids corresponding to the molecular mass of 39 kDa. The enzyme catalyzes the irreversible aldol condensation of PEP and ManNAc to form sialic acid and is active only in the presence of a divalent metal ion, such as Mg2+, Mn2+, Co2+ or Ni2+. Understanding the residues involved in catalysis or substrate binding is a prerequisite to design enzyme inhibitors and develop anti-bacterial reagents. This work presents the modification of Cys and Arg residues by using specific chemical modifiers and site-directed mutagenesis as the two residues probably participate in the metal coordination and the binding of PEP, respectively. IAA (a cysteine modifier) and PGO (an arginine modifier) were utilized to inactivate SaNeuB in a time- and dose-dependent manner, revealing that Cys and Arg are important to enzyme activity. The substrate protection with PEP and Mn2+, remained around 60% (for IAA inactivation) and 40% (for PGO inactivation) of the control, respectively, supporting that Cys and Arg are in or near the active site. DTNB was used to carry out the titration of sulfhydryl group. No disulfide bond was found in SaNeuB, based on the DTNB titration study of the sulfhydryl group. As a consequence, the activity loss due to the cysteine modifier is not associated with the disruption of disalfide bond. Furthermore, three Cys�_Ala and nine Arg �_ Ala mutants were generated by site-directed mutagenesis. The mutants C10A, C169A and R83A retain 20% activity, whereas R277A and R301A mutants were virtually inactive. A comparison of the circular dichroism spectra of the wild-type and all the mutants indicated that there was no significant difference. Although the kinetics studies showed no obvious changes in the Km of PEP and ManNAc, the results obtained from the chemical modification and site-directed mutagenesis studies implied that some of the Cys and Arg residues are essential to enzyme catalysis. According to the comparison of the conserved sequence with KDO8P synthase and DAH7P synthase, we suggest that Cys10 and C169 may play an important role in metal coordination, Arg277 and Arg301 may involve in the binding of phosphate group of PEP.