SITE-DIRECTED MUTATIONS OF THE CATALYTIC AND CONSERVED AMINO-ACIDS OFTHE NEURAMINIDASE GENE, NANH, OF CLOSTRIDIUM-PERFRINGENS ATCC-10543

The small nanH gene encoding the neuraminidase from Clostridium perfri ngens ATCC 10543 was cloned in JM109 using pUC19 as a vector. Sequence analysis revealed an ORF encoding 382 amino acids without a signal pe ptide sequence. Four regions of amino-acid sequence, 71-82, 140-151, 2 08-219, and 255-266 constituted four repeated and conserved sequence m otifs,-Ser-X-Asp-X-Gly-X-Thr-Trp-, the ''Asp boxes.'' When compared th e nanH polypeptides of C. perfringens ATCC 10543 and Salmonella typhim urium LT12 shared 33% sequence identity and 60% similarity if conserva tive replacements were included. The homology-modeled structure of C. perfringens NanH showed the same folding topology as the x-ray three-d imensional structure of NanH in S. typhimurium LT12. Amino acid residu es Arg(37), Arg(56), Asp(62), His(63), Asp(100), Glu(230), Asp(247), T yr(347), and Glu(362) located around the pocket of modeled C. perfring ens small nanH were superimposed with the active-site pocket of S. typ himurium LT12, nanH. The catalytic amino-acid residues as well as the role of the ''Asp boxes'' have not been characterized for C. perfringe ns and S. typhimurium. In this study, Asp(100), Glu(230), and Asp(62) were found to be involved in the catalytic activity of C. perfringens small nanH with immunoreactive properties and site-directed mutagenesi s analysis. Four ''Asp-box'' motifs were found remote from the active- site pocket. Mutational and immunoreactive analysis of the highly cons erved amino acids located in the ''Asp boxes'' suggest that these high ly conserved residues are important in maintaining the tertiary struct ure of NanH. The results of this study provide some knowledge for the design of new inhibitors of small neuraminidase.