Abstract
Phenylalanine hydroxylase from Legionella pneumophila (lpPAH) has a major functional role in the synthesis of the pigment pyomelanin, which is a potential virulence factor. We present here the crystal structure of lpPAH, which is a dimeric enzyme that shows high thermostability, with a midpoint denaturation temperature of 79 °C, and low substrate affinity. The structure revealed a dimerization motif that includes ionic interactions and a hydrophobic core, composed of both β-structure and a C-terminal region, with the specific residues (P255, P256, Y257 and F258) interacting with the same residues from the adjacent subunit within the dimer. This unique dimerization interface, together with a number of aromatic clusters, appears to contribute to the high thermal stability of lpPAH. The crystal structure also explains the increased aggregation of the enzyme in the presence of salt. Moreover, the low affinity for substrate l-Phe could be explained from three consecutive glycine residues (G181, 182, 183) located at the substrate-binding site. This is the first structure of a dimeric bacterial PAH and provides a framework for interpreting the molecular and kinetic properties of lpPAH and for further investigating the regulation of the enzyme.
Highlights
The gram-negative bacterium Legionella pneumophila belongs to the γ-proteobacteria
The crystallization of L. pneumophila PAH (lpPAH) was challenging since the crystals grew as plates with some disorder found in the final models, and due to the low symmetry spacegroup P21 a decent rotation range of X-ray data was needed to obtain more that 90% completeness
The final lpPAH model refined to an R-factor of 27.6% and an R-free value of 30.2% (Table 1), which are slightly high but can be explained by the disorder found for chains C and D
Summary
The gram-negative bacterium Legionella pneumophila belongs to the γ-proteobacteria. In nature, L. pneumophila is an inhabitant of warm freshwater habitats where its multiplication is mainly restricted to intracellular niches inside amoebal hosts and after infection it continues this intracellular life-style within the human host by multiplying inside macrophages [1]. Humans are a dead-end host for this pathogen, but it still causes outbreaks of Legionnaires’ disease, a potentially fatal form of pneumonia, when it multiplies in warm, stagnant water that are spread in aerosols through human-made installations such as fountains and showers [2,3,4] In such aqueous environments L. pneumophila grows well at temperatures in the range 20–48 ◦C, but its tolerance to higher temperatures can lead to regrowth after heat-disinfection [5]. The number of reported cases of Legionnaires’ disease has increased in the last decade [6], making eradication of the bacterium from infection sources an important task. There is a need for research on possible targets, notably on putative virulence factors
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