MOLECULAR ANALYSIS OF SURFACE-ASSOCIATED ENZYMES OF PORPHYROMONAS-GINGIVALIS

There is now increasing evidence that surface-associated enzymes, prev iously considered to be involved in intermediary metabolism or virulen ce, play a significant role in physiological reactions such as signal transduction, transport systems, and metabolic processes. Herein we re port the molecular aspects of two such enzymes, the cysteine proteinas e gingivain and NAD-dependent glutamate dehydrogenase of Porphyromonas gingivalis. The gdh gene comprises an open reading frame of 1,335 bas e pairs that encodes a 49,000-M(r) protein of 445 amino acids. The gdh gene showed high homology (78.3%) with that of Clostridium symbiosum. Optimal codons accounted for 35.9% of the total codon usage, indicati ng high expression of this enzyme, These data are currently being used to carry out targeted mutagenesis, which was established here for gin givain. Conditions for targeted mutagenesis within the histidine domai n of the catalytic site of gingivain using Tn 4351 was successfully ac hieved. Consequently, the catalytic functions, such as gingivain's cap acity to hydrolyze the synthetic substrate alpha-benzoyI-L-arginine-4- nitroanilide, were disrupted.