Structural and thermodynamic insight into phenylalanine hydroxylase from the human pathogen Legionella pneumophila

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.

Unpacking the Pathogen Box—An Open Source Tool for Fighting Neglected Tropical Disease

“…MMV560185 ( 596 ) was identified as an inhibitor of phenylalanine hydroxylase in Legionella pneumophila , as well as human MetAP‐1 . Phenylalanine hydroxylase catalyses the biosynthesis of pyomelanin, which is a melanin analogue responsible for iron acquisition required for bacterial growth . While this target has not been explored for the development of antimalarials, Fuchs et al.…”

Discovery of a Specific Inhibitor of Pyomelanin Synthesis in Legionella pneumophila

“…PAH (EC 1.14.16.1) is a non-heme iron-dependent enzyme that catalyzes the conversion from l -Phe to l -Tyr, using the cofactor (6 R )- l - erythro -5,6,7,8-tetrahydrobiopterin (BH 4 ) and molecular oxygen as additional substrate. Noticeably, while mammalian PAHs are tetrameric and other bacterial PAHs studied so far are monomeric, , our recently determined crystal structure of L. pneumophila PAH (lpPAH) revealed that it is a homodimer . In-depth characterization of recombinant lpPAH showed that the enzyme is dependent on iron for catalytic activity and has low affinity for the substrate l -Phe and the cofactor BH 4 , high activity at 30–50 °C, and a high conformational thermal stability with a midpoint denaturation temperature ( T m ) of 79 °C .…”

Structural studies of a novel auxiliary-domain-containing phenylalanine hydroxylase from Bacillus cereus ATCC 14579

“…When the gene encoding PAH is mutated on both alleles, resulting in an inactive enzyme, the disease is known as phenylketonuria (PKU; Scriver, 1995;Williams et al, 2008;Ronau et al, 2014). In bacteria, PAH allows bacterial growth in tyrosine-free media and assists in the production of pyomelanin by participating in the homogentisic acid synthesis pathway (Leiros et al, 2013). Pyomelanin is a red-brown pigment that has various functions such as in the virulence of pathogenic bacteria, antioxidation and iron assimilation (Leiros et al, 2013).…”

“…In bacteria, PAH allows bacterial growth in tyrosine-free media and assists in the production of pyomelanin by participating in the homogentisic acid synthesis pathway (Leiros et al, 2013). Pyomelanin is a red-brown pigment that has various functions such as in the virulence of pathogenic bacteria, antioxidation and iron assimilation (Leiros et al, 2013).…”

“…The overall structures of prokaryotic and eukaryotic PAHs differ from each other (Leiros et al, 2013;Ronau et al, 2014;Arturo et al, 2016). In prokaryotes, PAHs are known to consist of a single domain and exist as monomers (Ronau et al, 2014).…”