Identification and molecular characterization of tryptophanase encoded by tnaA in Porphyromonas gingivalis

Abstract

Indole produced via theβ-elimination reaction ofl-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 bacteriumPorphyromonas gingivalis. A database search showed that the amino acid sequence deduced frompg1401ofP. gingivalisW83 is 45 % identical with that fromtnaAofEscherichia coliK-12, which encodes tryptophanase. Replacement of thepg1401gene in the chromosomal DNA with the chloramphenicol-resistance gene abolished indole production. The production of indole was restored by the introduction ofpg1401, demonstrating that the gene is functionally equivalent totnaA. However, RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends analyses showed that, unlikeE. coli tnaA,pg1401is expressed alone inP. gingivalisand that the nucleotide sequence of the transcription start site is different, suggesting that the expression ofP. gingivalis tnaAis controlled by a unique mechanism. Purified recombinantP. gingivalistryptophanase exhibited the Michaelis–Menten kinetics valuesKm=0.20±0.01 mM andkcat=1.37±0.06 s−1in 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 degradedS-ethyl-l-cysteine andS-methyl-l-cysteine, but notl-alanine,l-serine orl-cysteine.