Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB.

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.

Vector-based RNA interference of cathepsin B1 in Schistosoma mansoni.

In helminth parasites, proteolytic enzymes have been implicated in facilitating host invasion, moulting, feeding, and evasion of the host immune response. These key functions render them potential targets for anti-parasite chemotherapy and immunotherapy. Schistosomes feed on host blood and the digested haemoglobin is their major source of amino acids. Haemoglobin digestion is essential for parasite development, growth, and reproduction. We recently reported the use of pseudotyped Moloney murine leukaemia virus to accomplish transformation of Schistosoma mansoni. Here, we report the design of a viral vector expressing a dsRNA hairpin to silence expression of the schistosome cathepsin B1 (SmCB1) gene. We observed 80% reduction in transcript level 72 h after virus exposure and complete silencing of enzyme activity in transduced worms. This is the first report using this technology in any helminth parasite. It will facilitate the evaluation of potential drug targets and biochemical pathways for novel interventions in schistosomes.