title: Identification and molecular characterization of tryptophanase encoded by tnaA in Porphyromonas gingivalis.
abstract: Indole produced via the beta-elimination reaction of l-tryptophan by pyridoxal 5'-phosphate-dependent tryptophanase (EC 4.1.99.1) has recently been shown to be an extracellular and intercellular signalling molecule in bacteria, and controls bacterial biofilm formation and virulence factors. In the present study, we determined the molecular basis of indole production in the periodontopathogenic bacterium Porphyromonas gingivalis. A database search showed that the amino acid sequence deduced from pg1401 of P. gingivalis W83 is 45 % identical with that from tnaA of Escherichia coli K-12, which encodes tryptophanase. Replacement of the pg1401 gene in the chromosomal DNA with the chloramphenicol-resistance gene abolished indole production. The production of indole was restored by the introduction of pg1401, demonstrating that the gene is functionally equivalent to tnaA. However, RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends analyses showed that, unlike E. coli tnaA, pg1401 is expressed alone in P. gingivalis and that the nucleotide sequence of the transcription start site is different, suggesting that the expression of P. gingivalis tnaA is controlled by a unique mechanism. Purified recombinant P. gingivalis tryptophanase exhibited the Michaelis-Menten kinetics values K(m)=0.20+/-0.01 mM and k(cat)=1.37+/-0.06 s(-1) in potassium phosphate buffer, but in sodium phosphate buffer, the enzyme showed lower activity. However, the cation in the buffer, K(+) or Na(+), did not appear to affect the quaternary structure of the enzyme or the binding of pyridoxal 5'-phosphate to the enzyme. The enzyme also degraded S-ethyl-l-cysteine and S-methyl-l-cysteine, but not l-alanine, l-serine or l-cysteine.
authors.affiliation: Department of Dental Pharmacology, Iwate Medical University School of Dentistry, Morioka, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yoshida
authors.affiliation: Department of Dental Pharmacology, Iwate Medical University School of Dentistry, Morioka, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Sasaki
authors.affiliation: Department of Periodontology, Iwate Medical University School of Dentistry, Morioka, Japan.|Department of Dental Pharmacology, Iwate Medical University School of Dentistry, Morioka, Japan.
authors.forename: Shuntaro
authors.initials: S
authors.lastname: Ito
authors.affiliation: Department of Dental Pharmacology, Iwate Medical University School of Dentistry, Morioka, Japan.
authors.forename: Haruki
authors.initials: H
authors.lastname: Tamura
authors.affiliation: Department of Periodontology, Iwate Medical University School of Dentistry, Morioka, Japan.
authors.forename: Kazushi
authors.initials: K
authors.lastname: Kunimatsu
authors.affiliation: Department of Dental Pharmacology, Iwate Medical University School of Dentistry, Morioka, Japan.
authors.forename: Hirohisa
authors.initials: H
authors.lastname: Kato
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007211:Indoles / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D016966:Porphyromonas gingivalis / Q000201:enzymology / Q000235:genetics*; D020836:Protein Structure, Quaternary; D012329:RNA, Bacterial / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D024363:Transcription Initiation Site; D014364:Tryptophan / Q000378:metabolism; D014368:Tryptophanase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007211:Indoles; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D014364:Tryptophan; D014368:Tryptophanase