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Crystallization and X-ray analysis of a bacterial non-haem iron-containing phenylalanine hydroxylase from the Gram-negative opportunistic pathogen Pseudomonas aeruginosa.
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
2003 (English)In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 59, no Pt 7, 1310-2 p.Article in journal (Refereed) Published
Abstract [en]

Monooxygenases are frequently involved in the pathways that mediate the pivotal role of microorganisms in recycling carbon from the environment. A structural study of a monooxygenase from Pseudomonas aeruginosa that was identified as a phenylalanine hydroxylase has been initiated. The single-domain monomeric protein harbours a non-haem iron at the active site. The sequence identity to the catalytic domains of tyrosine and tryptophan hydroxylases suggests that the enzyme is not restricted to the substrate phenylalanine alone. Here, the cloning, purification and crystallization of native and SeMet-labelled P. aeruginosa phenylalanine hydroxylase are reported. Crystals grew in space group P6(1), with unit-cell parameters a = b = 210.5, c = 100.7 A, and diffracted to a d spacing of 2.0 A. Crystals of SeMet-labelled protein were used to collect a three-wavelength multiple anomalous dispersion (MAD) data set around the Se K edge.

Place, publisher, year, edition, pages
2003. Vol. 59, no Pt 7, 1310-2 p.
Keyword [en]
Bacterial Proteins/chemistry, Cloning; Molecular, Crystallization/methods, Iron, Phenylalanine Hydroxylase/*chemistry/genetics/isolation & purification, Pseudomonas aeruginosa/*enzymology, Selenomethionine, X-Ray Diffraction/methods
URN: urn:nbn:se:umu:diva-13967PubMedID: 12832796OAI: diva2:153638
Available from: 2007-11-23 Created: 2007-11-23 Last updated: 2016-02-19Bibliographically approved
In thesis
1. X-ray characterization of PaPheOH, a bacterial phenylalanine hydroxylase
Open this publication in new window or tab >>X-ray characterization of PaPheOH, a bacterial phenylalanine hydroxylase
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many human diseases are associated with the malfunction of enzymes in the aromatic amino acid hydroxylase family, e.g. phenylketonuria (PKU), hyperphenylalaninemia (HPA), schizophrenia and Parkinson's disease. The family of aromatic aminoacid hydroxylases comprises the enzymes phenylalanine hydroxylase (PheOH), tyrosine hydroxylase (TyrOH) and tryptophane hydroxylase (TrpOH). These enzymes require the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and atomic oxygen. In eukaryotes, the aromatic amino acid hydroxylases share the same organization with a N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain. Aromatic amino acid hydroxylases that correspond to the core catalytic domain of the eukaryotic enzymes are found in bacteria. The main focus of this thesis is the structural characterization of a phenylalanine hydroxylase from the bacterium Pseudomonas aeruginosa (PaPheOH).

To initiate the structural characterization, the active site environment was investigated with X-ray absorption spectroscopy (XAS). The experimental data support a model where the active site iron is coordinated by four oxygen atoms and two nitrogen atoms. We suggest that two water molecules, His121, His126 and Glu166 coordinates the active site iron. In this model, Glu166 provides two of the oxygen atoms in a bidentate binding geometry. EXAFS and XANES studies indicate that structural rearrangements are induced in the second and third coordination shells in samples of PaPheOH with BH4 and/or L-Phe.

The 1.6 Å X-ray structure of PaPheOH shows a catalytic core that is composed of helices and strands in a bowl-like arrangement. The iron is octahedrally coordinated, by two water molecules and the evolutionary conserved His121, His126 and Glu166 that coordinates the iron with bidentate geometry. The pterin binding loop of PaPheOH (residue 81-86) adopts a conformation that is displaced by 5-6 Å from the expected pterin binding site. Consistent with the unfavourable position of the pterin binding loop is the observation that PaPheOH has a low specific activity compared to the enzymes from human and Chromobacterium violaceum.

The second part of this thesis focus on the crystallization and structure determination of the actin binding domain of a-actinin (ABD). a-Actinin is located in the Z-disc of skeletal muscle were it crosslinks actin filaments to the filamentous protein titin. The ABD domain of a-actinin crystallizes in space group P21 with four molecules in the asymmetric unit. The structure of the ABD domain has been solved to a d-spacing of 2.0 Å. The two CH-domains of ABD is composed of 5 a-helices each. The a-helices fold into a closed compact conformation with extensive intramolecular contacts between the two domains.

Place, publisher, year, edition, pages
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet), 2003. 49 p.
Biochemistry, PaPheOH, PheOH, PAH, phhA, phenylalanine hydroxylase, protein crystallography, a-actinin, microcrystal, ABD, CH-domain, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:umu:diva-100 (URN)
Public defence
2003-10-03, D, 1D, 9 trappor, Umeå, 10:00
Available from: 2003-09-11 Created: 2003-09-11 Last updated: 2015-01-16Bibliographically approved

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Stier, GunterSauer, Uwe
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