title: Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB.
abstract: Pseudomonas putida GPo1 alkane hydroxylase (AlkB) is an integral membrane protein that catalyses the hydroxylation of medium-chain alkanes (C3-C12). 1-Octyne irreversibly inhibits this non-haem di-iron mono-oxygenase under turnover conditions, suggesting that it acts as a mechanism-based inactivator. Upon binding to the active site, 1-octyne is postulated to be oxidized to an oxirene that rapidly rearranges to a reactive ketene which covalently acylates nearby residues, resulting in enzyme inactivation. In analysis of inactivated AlkB by LC-MS/MS, several residues exhibited a mass increase of 126.1 Da, corresponding to the octanoyl moiety derived from oxidative activation of 1-octyne. Mutagenesis studies of conserved acylated residues showed that Lys18 plays a critical role in enzyme function, as a single-point mutation of Lys18 to alanine (K18A) completely abolished enzymatic activity. Finally, we present a computational 3D model structure of the transmembrane domain of AlkB, which revealed the overall packing arrangement of the transmembrane helices within the lipid bilayer and the location of the active site mapped by the 1-octyne modifications.
authors.affiliation: *Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Hernan
authors.initials: H
authors.lastname: Alonso
authors.affiliation: †Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Oded
authors.initials: O
authors.lastname: Kleifeld
authors.affiliation: ‡Bioinformatics Unit, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Adva
authors.initials: A
authors.lastname: Yeheskel
authors.affiliation: *Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Poh C
authors.initials: PC
authors.lastname: Ong
authors.affiliation: *Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Yu C
authors.initials: YC
authors.lastname: Liu
authors.affiliation: §School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Jeanette E
authors.initials: JE
authors.lastname: Stok
authors.affiliation: §School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: James J
authors.initials: JJ
authors.lastname: De Voss
authors.forename: Anna
authors.initials: A
authors.lastname: Roujeinikova
mesh_terms: D000473:Alkanes / Q000378:metabolism*; D000480:Alkynes / Q000378:metabolism / Q000494:pharmacology; D020134:Catalytic Domain; D042926:Cytochrome P-450 CYP4A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D006900:Hydroxylation; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D000473:Alkanes; D000480:Alkynes; D008565:Membrane Proteins; C042001:1-octyne; D042926:Cytochrome P-450 CYP4A; D008239:Lysine