Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB

Alonso, Hernan, Kleifeld, Oded, Yeheskel, Adva, Ong, Poh C., Liu, Yu C., Stok, Jeanette E., De Voss, James J. and Roujeinikova, Anna (2014) Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB. Biochemical Journal, 460 2: 283-293. doi:10.1042/BJ20131648


Author Alonso, Hernan
Kleifeld, Oded
Yeheskel, Adva
Ong, Poh C.
Liu, Yu C.
Stok, Jeanette E.
De Voss, James J.
Roujeinikova, Anna
Title Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB
Formatted title
Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB
Journal name Biochemical Journal   Check publisher's open access policy
ISSN 1470-8728
0264-6021
Publication date 2014-06-01
Year available 2014
Sub-type Article (original research)
DOI 10.1042/BJ20131648
Open Access Status
Volume 460
Issue 2
Start page 283
End page 293
Total pages 11
Place of publication London, United Kingdom
Publisher Portland Press
Collection year 2015
Language eng
Formatted 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.
Keyword Alkane hydroxylase (AlkB)
Alkyne
Enzyme inactivation
Mono-oxygenase
Pseudomonas putida
Suicide inhibitor
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2015 Collection
School of Chemistry and Molecular Biosciences
 
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