Phenylalanine hydroxylase from Legionella pneumophila is a thermostable enzyme with a major functional role in pyomelanin synthesis.
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.


RESULTS
Comparative 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
lpPAH 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.
Department of Biomedicine, University of Bergen, Bergen, Norway.
Marte I
MI
Flydal
Christa H
CH
Chatfield
Huaixin
H
Zheng
Felizza F
FF
Gunderson
Oscar
O
Aubi
Nicholas P
NP
Cianciotto
Aurora
A
Martinez
D001426:Bacterial Proteins; D004795:Enzyme Stability; D016952:Legionella pneumophila / Q000201:enzymology*; D008543:Melanins / Q000096:biosynthesis*; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D013696:Temperature; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; D008543:Melanins; C023793:pyomelanin; D014443:Tyrosine; D010651:Phenylalanine Hydroxylase