Site-directed mutations of the catalytic and conserved amino acids of the neuraminidase gene, nanH, of Clostridium perfringens ATCC 10543.
The small nanH gene encoding the neuraminidase from Clostridium perfringens ATCC 10543 was cloned in JM109 using pUC19 as a vector. Sequence analysis revealed an ORF encoding 382 amino acids without a signal peptide sequence. Four regions of amino-acid sequence, 71-82, 140-151, 208-219, and 255-266 constituted four repeated and conserved sequence motifs-Ser-X-Asp-X-Gly-X-Thr-Trp-, the "Asp boxes." When compared, the nanH polypeptides of C. perfringens ATCC 10543 and Salmonella typhimurium LT12 shared 33% sequence identity and 60% similarity if conservative replacements were included. The homology-modeled structure of C. perfringens NanH showed the same folding topology as the x-ray three-dimensional structure of NanH in S. typhimurium LT12. Amino acid residues Arg37, Arg56, Asp62, His63, Asp100, Glu230, Asp247, Tyr347, and Glu362 located around the pocket of modeled C. perfringens small nanH were superimposed with the active-site pocket of S. typhimurium LT12, nanH. The catalytic amino-acid residues as well as the role of the "Asp boxes" have not been characterized for C. perfringens and S. typhimurium. In this study, Asp100, Glu230, and Asp62 were found to be involved in the catalytic activity of C. perfringens small nanH with immunoreactive properties and site-directed mutagenesis analysis. Four "Asp-box" motifs were found remote from the active-site pocket. Mutational and immunoreactive analysis of the highly conserved amino acids located in the "Asp boxes" suggest that these highly conserved residues are important in maintaining the tertiary structure of NanH. The results of this study provide some knowledge for the design of new inhibitors of small neuraminidase.
Chien
C H
CH
Institute of Biochemistry, School of Life Science, National Yang-Ming University, Taipei, Taiwan, Republic of China.
Shann
Y J
YJ
Sheu
S Y
SY
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D015153:Blotting, Western; D055598:Chemical Phenomena; D002621:Chemistry; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology*; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers; D005798:Genes, Bacterial / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000235:genetics*; D011233:Precipitin Tests; D011487:Protein Conformation; D017421:Sequence Analysis
D017931:DNA Primers; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid