PURIFICATION AND CHARACTERIZATION OF ALLANTOATE AMIDOHYDROLASE FROM BACILLUS-FASTIDIOSUS

Allantoate amidohydrolase from Bacillus fastidiosus was purified 170-f old to homogeneity as judged by isoelectric focusing and nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoresis, The mo lecular mass was estimated to be 128 kDa, The enzyme appeared to be a homodimer with a subunit molecular mass of 66 kDa. The enzyme has an i soelectric point of 5.6. Allantoate amidohydrolase is a Mn2+-dependent enzyme exhibiting a pH optimum around 8.8. Its K-m value for allantoa te was estimated to be 9 mM. Similar to other microbial allantoate ami dohydrolases the enzyme can be reversibly activated and inactivated. N o indication for the involvement of arginine, lysine, and cysteine res idues in the catalytic action of the enzyme was obtained. Diethylpyroc arbonate strongly inhibited the enzyme activity, indicating the involv ement of histidine or tyrosine residues in catalytic action. However, no recovery was obtained by treatment with hydroxylamine as would be e xpected if such residues were modified. The enzyme could be reversibly denatured by urea, guanidine, and sodium dodecyl sulfate. (C) 1995 Ac ademic Press, Inc.