Sialic Acid Synthase from Escherichia coli and Streptococcus agalactiae: Structural Characterization and Identification of Essential Catalytic Residues
Date Issued
2005
Date
2005
Author(s)
Hwang, Tzann-Shun
DOI
zh-TW
Abstract
The capsular polysaccharide of Escherichia coli K1 containing alpha2,8-linked polysialic acid has been recognized as an important virulent determinant to cause invasive infections such as neonatal meningitis and septicemia. The capsular polysaccharide of Streptococcus agalactiae (Group B Streptococci, GBS) containing alpha2,3-sialylated oligosaccharide is also a critical virulence factor for causing neonatal septicemia, pyogenic meningitis and pneumonia. The sialic acid presented in GBS capsule has been found enhancing resistance to phagocytosis by inhibiting the alternative pathway of complement cascade to evade host defenses. In E. coli and S. agalactiae, the gene responsible for the biosynthesis of sialic acid is the neuB gene in kps gene cluster. Sialic acid synthase (NeuB), encoded by neuB gene, can catalyze the condensing reaction of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) to form N-acetylneuraminic acid (NeuAc). The product of NeuB is relative to the infection of pathogens; therefore, the investigation on the active site of NeuB is a good approach for drug design and enzyme’s application.
In this study, neuB genes from E. coli and S. agalactiae were cloned from genomic DNA and over-expressed as EcNeuB and SaNeuB, respectively. Optimal conditions for enzyme reaction, including pH, temperature, stability and metal requirement, were established. Characterization of EcNeuB and SaNeuB was also conducted. In the preparation of EcNeuB, a specific cleavage by endogenous protease(s) was found at Lys280 of sialic acid synthase (40 kDa). The cleavage results in the formation of two inactive fragments of 33 kDa and 7 kDa. The CD, MALDI-TOF-MS and chemical cross-linking studies demonstrated that the fragmentation is associated with a significant change of the enzyme from a tetrameric to trimeric form. Further studies by nano-spray ESI-MS and electron microscopy demonstrated NeuB existed in a tetrameric form by dimer-dimer interaction.
Sulfhydryl-modifying reagents were able to completely inactivate the enzyme activity. The iodoacetic acid (IAA) inactivation of EcNeuB and SaNeuB was in a time- and dose-dependent manner, which revealed that Cys was important for enzyme activity. Study of the sulfhydryl group by 5,5-dithiobis-2-nitrobenzoate (DTNB) titration showed no disulfide bond in both EcNeuB and SaNeuB, suggesting the activity loss was caused by the modification of Cys residue. Site-directed mutagenesis, enzyme assay and kinetic analysis showed that C12A and C176A of EcNeuB and C10A and C169A of SaNeuB were important for enzyme activity. The substrate protection experiments indicated that aforementioned Cys residues were located in the active site and involved in the binding of PEP and Mn2+, since the substrate binding could prevent NeuB from the inactivation of IAA and bromopyruvate. Further studies on substrate protection of SaNeuB mutants and molecular modeling of SaNeuB showed that Cys169 was a residue involved in the binding of PEP and Mn2+. On the other hand, Cys10 was proposed to interact with Gln37 that is essential for the binding of PEP. Chemical modification and site-directed mutagenesis showed Arg301 and Arg277 of SaNeuB were essential in the substrate binding. Molecular modeling of SaNeuB showed that Arg301 and Arg277 were involved in the binding of ManNAc and PEP, respectively.
Subjects
唾液酸合成酶
Sialic Acid Synthase
Escherichia coli
Streptococcus agalactiae
Type
other
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