Pentalenene Synthase: Analysis of Active Site Residues by Site-Directed Mutagenesis

Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in kcat and kcat/Km. By contrast, single H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2). Mutation to glutamate of each of the three aspartate residues in the Mg2+-binding aspartate-rich domain, 80DDLFD, resulted in reduction in the kcat/Km for farnesyl diphosphate and formation of varying proportions of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant had a kcat/Km which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of pentalenene (2) (91%) and the aberrant cyclization product ß-caryophyllene (9) (9%). Finally, the F77Y mutant had a kcat/Km which was reduced by 20-fold compared to that of the wild-type synthase

Questionable policy for large carnivore hunting

Terrestrial large carnivores have great ecological, economic and cultural importance, but are in global decline due to habitat loss, prey depletion, poaching, retributive killing and regulated hunting. While regulated carnivore hunting potentially reduces conflict with humans and livestock, increases social tolerance and provides revenue for conservation, it can also drive population declines. Some policies regulating carnivore hunting acknowledge and address negative effects on demography and population dynamics, but others do not. Using wolves as an example, we identify four aspects of hunting policy that do not align well with ecological theory and data, and suggest resolutions for these problems that have broad relevance to exploited carnivore populations