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Volume 155, Issue 3

Identification and molecular characterization of tryptophanase encoded by in Free

Abstract

Indole produced via the -elimination reaction of -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 . A database search showed that the amino acid sequence deduced from of W83 is 45 % identical with that from of K-12, which encodes tryptophanase. Replacement of the gene in the chromosomal DNA with the chloramphenicol-resistance gene abolished indole production. The production of indole was restored by the introduction of , demonstrating that the gene is functionally equivalent to . However, RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends analyses showed that, unlike , is expressed alone in and that the nucleotide sequence of the transcription start site is different, suggesting that the expression of is controlled by a unique mechanism. Purified recombinant tryptophanase exhibited the Michaelis–Menten kinetics values =0.20±0.01 mM and =1.37±0.06 s 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 -ethyl--cysteine and -methyl--cysteine, but not -alanine, -serine or -cysteine.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): CRP, cAMP receptor protein , GST, glutathionine S-transferase , PLP, pyridoxal 5′-phosphate and RLM-RACE, RNA ligase-mediated rapid amplification of cDNA ends
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2009-03-01
2024-04-23
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Identification and molecular characterization of tryptophanase encoded by tnaA in Porphyromonas gingivalis
Microbiology 155, 968 (2009); https://doi.org/10.1099/mic.0.024174-0
/content/journal/micro/10.1099/mic.0.024174-0
/content/journal/micro/10.1099/mic.0.024174-0
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