Phenylalanine Hydroxylase from Legionella pneumophila Is a Thermostable Enzyme with a Major Functional Role in Pyomelanin Synthesis

Abstract

Background Legionella pneumophila is a pathogenic bacterium that can cause Legionnaires’ disease and other non-pneumonic infections in humans. This bacterium produces a pyomelanin pigment, a potential virulence factor with ferric reductase activity. In this work, we have investigated the role of phenylalanine hydroxylase from L. pneumophila (lpPAH), the product of the phhA gene, in the synthesis of the pyomelanin pigment and the growth of the bacterium in defined compositions.Methodology/Principal FindingsComparative studies of wild-type and phhA mutant corroborate that lpPAH provides the excess tyrosine for pigment synthesis. phhA and letA (gacA) appear transcriptionally linked when bacteria were grown in buffered yeast extract medium at 37°C. phhA is expressed in L. pneumophila growing in macrophages. We also cloned and characterized lpPAH, which showed many characteristics of other PAHs studied so far, including Fe(II) requirement for activity. However, it also showed many particular properties such as dimerization, a high conformational thermal stability, with a midpoint denaturation temperature (T m) = 79±0.5°C, a high specific activity at 37°C (10.2±0.3 µmol L-Tyr/mg/min) and low affinity for the substrate (K m (L-Phe) = 735±50 µM.Conclusions/SignificancelpPAH has a major functional role in the synthesis of pyomelanin and promotes growth in low-tyrosine media. The high thermal stability of lpPAH might reflect the adaptation of the enzyme to withstand relatively high survival temperatures.

Selective Requirement of the Shikimate Pathway of Legionella pneumophila for Intravacuolar Growth within Human Macrophages but Not within Acanthamoeba

“…1) (46-49). It has been shown that an abundance of either tyrosine or phenylalanine in the medium increases pigment production (50) and that conversion of Phe into Tyr by the phenylalanine hydroxylase (PAH; Fig. 1) provides the excess Tyr needed to produce the pigment (50).…”

Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

“…are expressed in L. pneumophila biofilms and in Legionella-infected macrophages and the pigment is seen in L. pneumophilainfected lungs and sputum (6,31,(45)(46)(47). That nonpigmented mutants are still able to grow in amoebae and macrophage host cells (28,30,36) is compatible with the fact that the bacterium has multiple means of iron uptake.…”

“…In order to construct the lbtA lly double mutant NU424, plasmid pUClly::GNT (31) was introduced into the lbtA mutant NU302 by transformation. Transformants were selected on antibiotic-containing agar medium, and insertion of the gentamicin resistance cassette into the lly gene was confirmed by PCR using primers llyLpgFBcl1 and llyLpgXho1, as previously described (31). Escherichia coli strain DH5␣ (Invitrogen, Carlsbad, CA) was used as the host for recombinant plasmids.…”

“…It was later shown that pigment results from oxidative polymerization of the homogentisic acid (HGA) that is released by the bacteria into the culture supernatant (26). HGA is a product of phenylalanine/tyrosine catabolism; i.e., L-phenylalanine is converted to L-tyrosine by the phenylalanine hydroxylase encoded by phhA, L-tyrosine is then converted to 4-hydroxyphenylpyruvate by an amino acid transferase encoded by hisC2, and 4-hydroxyphenylpyruvate is finally converted to HGA by the 4-hydroxyphenylpyruvate dioxygenase encoded by lly (26)(27)(28)(29)(30)(31). The HGA that is made can be either secreted, as noted above, or broken down to 4-maleyl-acetoacetate by the homogentisate 1,2-dioxygenase, encoded by hmgA (31,32).…”

“…HGA is a product of phenylalanine/tyrosine catabolism; i.e., L-phenylalanine is converted to L-tyrosine by the phenylalanine hydroxylase encoded by phhA, L-tyrosine is then converted to 4-hydroxyphenylpyruvate by an amino acid transferase encoded by hisC2, and 4-hydroxyphenylpyruvate is finally converted to HGA by the 4-hydroxyphenylpyruvate dioxygenase encoded by lly (26)(27)(28)(29)(30)(31). The HGA that is made can be either secreted, as noted above, or broken down to 4-maleyl-acetoacetate by the homogentisate 1,2-dioxygenase, encoded by hmgA (31,32). Thus, the L. pneumophila pigment is HGA-melanin, a type of pyomelanin that is also made by various other environmental bacteria as well as some other pathogens (33)(34)(35).…”