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 k(cat) and k(cat)/K(m). 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 Mg(2+)-binding aspartate-rich domain, (80)DDLFD, resulted in reduction in the k(cat)/K(m) 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 k(cat)/K(m) 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 beta-caryophyllene (9) (9%). Finally, the F77Y mutant had a k(cat)/K(m) which was reduced by 20-fold compared to that of the wild-type synthase.
Seemann
Myriam
M
Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.
Zhai
Guangzhi
G
de Kraker
Jan-Willem
JW
Paschall
Chiana M
CM
Christianson
David W
DW
Cane
David E
DE
D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D019753:Intramolecular Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology
D001224:Aspartic Acid; D001216:Asparagine; D019753:Intramolecular Lyases; C048278:farnesylpyrophosphate cyclase