Molecular analysis of surface-associated enzymes of Porphyromonas gingivalis.
There is now increasing evidence that surface-associated enzymes, previously considered to be involved in intermediary metabolism or virulence, play a role in physiological reactions such as signal transduction, transport systems, and metabolic processes. Herein we report the molecular aspects of two such enzymes, the cysteine proteinase gingivain and NAD-dependent glutamate dehydrogenase of Porphyromonas gingivalis. The gdh gene comprises an open reading frame of 1,335 base 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, indicating high expression of this enzyme. These data are currently being used to carry out targeted mutagenesis, which was established here for gingivain. Conditions for targeted mutagenesis within the histidine domain of the catalytic site of gingivain using Tn 4351 was successfully achieved. Consequently, the catalytic functions, such as gingivain's capacity to hydrolyze the synthetic substrate alpha-benzoyl-arginine-4-nitroanilide, were disrupted.
Gharbia
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Department of Microbiology, Eastman Dental Institute, University of London, United Kingdom.
Shah
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HN
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics*; D004269:DNA, Bacterial; D018086:Glutamate Dehydrogenase (NADP+) / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016966:Porphyromonas gingivalis / Q000201:enzymology*
D000596:Amino Acids; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D018086:Glutamate Dehydrogenase (NADP+); D003546:Cysteine Endopeptidases; C067046:gingivain